Focus on…ubiquitin-related tumor suppressors

Publication year: 2012
Source:FEBS Letters, Volume 586, Issue 11

Wilhelm Just

Posted on 27 May 2012 | 1:14 am

CRSBP-1/LYVE-1 ligands stimulate contraction of the CRSBP-1-associated ER network in lymphatic endothelial cells

Publication year: 2012
Source:FEBS Letters, Volume 586, Issue 10

Wei-Hsien Hou, I-Hua Liua, Shuan Shian Huang, Jung San Huang

CRSBP-l/LYVE-1 ligands (PDGF-BB, VEGF-A¹⁶⁵ and hyaluronic acid) have been shown to induce opening of lymphatic intercellular junctions in vitro and in vivo by stimulating contraction of lymphatic endothelial cells (LECs). The mechanism by which CRSBP-1 ligands stimulate contraction of LECs is not understood. Here we demonstrate that CRSBP-1 is localized to the plasma membrane as well as intracellular fibrillar structures in LECs, including primary human dermal LECs and SVEC4-10 cells. CRSBP-1-associated fibrillar structures are identical to the ER network as evidenced by the co-localization of CRSBP-1 and BiP in these cells. CRSBP-1 ligands stimulate contraction of the ER network in a CRSBP-1-dependent and paclitaxel (a microtubule-stabilizing agent)-sensitive manner. These results suggest that ligand-stimulated ER contraction is associated with ligand-stimulated contraction in LECs.

Highlights

Posted on 27 May 2012 | 1:14 am

Systemic VHL gene functions and the VHL disease

Publication year: 2012
Source:FEBS Letters, Volume 586, Issue 11

Hannah L. Bader, Tien Hsu

The von Hippel-Lindau tumor suppressor gene (VHL) is best known as an E3 ubiquitin ligase that negatively regulates the hypoxia inducible factor (HIF). VHL mutations are the genetic defects underlying several human diseases including polycythemia, familial VHL tumor syndrome and sporadic renal cell carcinoma. VHL mutations can lead to cell-autonomous phenotypes in the tumor cells. However, non-tumor cell-autonomous functions of VHL have also been noted. VHL tumor-derived cytokines can promote inflammation and induce mobilization of endothelial progenitor cells. Up-regulation of HIF caused by VHL loss-of-function mutants, including heterozygotes, has been shown to increase the activities of hematopoietic stem cells, endothelial cells and myeloid cells. As such, systemic functions of VHL likely play important roles in the development of VHL disease.

Posted on 27 May 2012 | 1:14 am

THAP11, a novel binding protein of PCBP1, negatively regulates CD44 alternative splicing and cell invasion in a human hepatoma cell line

Publication year: 2012
Source:FEBS Letters, Volume 586, Issue 10

Wen-Xi Lian, Rong-Hua Yin, Xiang-Zhen Kong, Tong Zhang, Xian-Hong Huang, Wei-Wei Zheng, Yang Yang, Yi-Qun Zhan, Wang-Xiang Xu, Miao Yu, Chang-Hui Ge, Jun-Tang Guo, Chang-Yan Li, Xiao-Ming Yang

THAP11 is an essential factor involved in ES cell pluripotency and cell growth. Here, we identified THAP11 as a novel physiological binding partner of PCBP1. In HepG2 cells, THAP11 overexpression inhibited CD44 v6 expression and cell invasion. However, when deleting the binding domain with PCBP1 or endogenous PCBP1 was knocked down, THAP11 failed to inhibit CD44 v6 expression, indicating that THAP11 regulates CD44 v6 expression through interacting with PCBP1. In HCC patients, the expression of THAP11 mRNA significantly correlated with PCBP1 mRNA expression. Our results suggest a novel role of THAP11 in CD44 alternative splicing and hepatoma invasion. Structured summary of protein interactions: THAP11 physically interacts with PCBP1 by anti bait coimmunoprecipitation (View interaction) THAP11 physically interacts with PCBP1 by anti tag coimmunoprecipitation (View Interaction: 1, 2) THAP11 and PCBP1 colocalize by fluorescence microscopy (View interaction)

Highlights

Posted on 27 May 2012 | 1:14 am

The human phosphatase interactome: An intricate family portrait

Publication year: 2012
Source:FEBS Letters

Francesca Sacco, Livia Perfetto, Luisa Castagnoli, Gianni Cesareni

The concerted activities of kinases and phosphatases modulate the phosphorylation levels of proteins, lipids and carbohydrates in eukaryotic cells. Despite considerable effort, we are still missing a holistic picture representing, at a proteome level, the functional relationships between kinases, phosphatases and their substrates. Here we focus on phosphatases and we review and integrate the available information that helps to place the members of the protein phosphatase superfamilies into the human protein interaction network. In addition we show how protein interaction domains and motifs, either covalently linked to the phosphatase domain or in regulatory/adaptor subunits, play a prominent role in substrate selection.

Posted on 27 May 2012 | 1:14 am

Galactinol synthase across evolutionary diverse taxa: Functional preference for higher plants?

Publication year: 2012
Source:FEBS Letters, Volume 586, Issue 10

Sonali Sengupta, Sritama Mukherjee, Sabiha Parween, Arun Lahiri Majumder

Galactinol synthase (GolS), a GT8 family glycosyltransferase, synthesizes galactinol and raffinose series of oligosaccharides (RFOs). Identification and analysis of conserved domains in GTs among evolutionarily diverse taxa, structure prediction by homology modeling and determination of substrate binding pocket followed by phylogenetic analysis of GolS sequences establish presence of functional GolS predominantly in higher plants, fungi having the closest possible ancestral sequences. Evolutionary preference for a functional GolS expression in higher plants might have arisen in response to the need for galactinol and RFO synthesis to combat abiotic stress, in contrast to other organisms lacking functional GolS for such functions.

Highlights

Posted on 27 May 2012 | 1:14 am

Role of CSN5/JAB1 in Wnt/?-catenin activation in colorectal cancer cells

Publication year: 2012
Source:FEBS Letters, Volume 586, Issue 11

Anke K. Schütz, Thomas Hennes, Sandra Jumpertz, Simone Fuchs, Jürgen Bernhagen

CSN5/JAB1 is a critical subunit of the COP9 signalosome (CSN) and is overexpressed in many human cancers, but little is known about the role of CSN5 in colorectal cancer (CRC). To explore the functional role of CSN5 in colorectal tumorigenesis, we applied siRNA technology to silence CSN5 in HeLa, SW480, HCT116, HT29, and CaCo2 cells. CSN5 knock-down led to reduced ?-catenin and phospho-?-catenin levels and this was paralleled by reduced CRC cell proliferation and reduced apoptosis rates, whereas the short-term ?-catenin protein stability was enhanced by CSN5 knock-down in SW480 cells. Together, these data implicate the CSN in the pathogenesis of CRC via regulation of the Wnt/?-catenin pathway.

Highlights

Posted on 27 May 2012 | 1:14 am

Inhibition of bacterial adhesion to live human cells: Activity and cytotoxicity of synthetic mannosides

Publication year: 2012
Source:FEBS Letters, Volume 586, Issue 10

Mirja Hartmann, Heike Papavlassopoulos, Vijayanand Chandrasekaran, Carsten Grabosch, Femke Beiroth, Thisbe K. Lindhorst, Claudia Röhl

Bacterial adhesion to glycosylated surfaces is a key issue in human health and disease. Inhibition of bacterial adhesion by suitable carbohydrates could lead to an anti-adhesion therapy as a novel approach against bacterial infections. A selection of five ?-mannosides has been evaluated as inhibitors of bacterial adhesion to the polysaccharide mannan, as well as to the surface of live human HT-29 cells. Cell toxicity studies were performed to identify the therapeutic window for a potential in vivo-application of the tested carbohydrates. A previously published mannosidic squaric acid diamide was shown to be exceptionally effective as inhibitor of the bacterial lectin FimH.

Highlights

Posted on 27 May 2012 | 1:14 am

Matrix-dependent perturbation of TGF? signaling and disease

Publication year: 2012
Source:FEBS Letters

Jefferson J. Doyle, Elizabeth E. Gerber, Harry C. Dietz

Transforming growth factor beta (TGF?) is a multipotent cytokine that is sequestered in the extracellular matrix (ECM) through interactions with a number of ECM proteins. The ECM serves to concentrate latent TGF? at sites of intended function, to influence the bioavailability and/or function of TGF? activators, and perhaps to regulate the intrinsic performance of cell surface effectors of TGF? signal propagation. The downstream consequences of TGF? signaling cascades in turn provide feedback modulation of the ECM. This review covers recent examples of how genetic mutations in constituents of the ECM or TGF? signaling cascade result in altered ECM homeostasis, cellular performance and ultimately disease, with an emphasis on emerging therapeutic strategies that seek to capitalize on this refined mechanistic understanding.

Posted on 27 May 2012 | 1:14 am

Dimer interface rearrangement of the 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase rat liver isoenzyme by cAMP-dependent Ser-32 phosphorylation

Publication year: 2012
Source:FEBS Letters, Volume 586, Issue 10

Sara Langer, David A. Okar, Julia Schultz, Sigurd Lenzen, Simone Baltrusch

The bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) is a key regulator of carbohydrate metabolism in liver. The goal of this study was to elucidate the regulatory role of Ser-32 phosphorylation on the kinase domain mediated dimerization of PFK-2/FBPase-2. Fluorescence-based mammalian two-hybrid and sensitized emission fluorescence resonance energy transfer analyses in cells revealed preferential binding within homodimers in contrast to heterodimers. Using isolated proteins a close proximity of two PFK-2/FBPase-2 monomers was only detectable in the phosphorylated enzyme dimer. Thus, a flexible kinase interaction mode exists, suggesting dimer conformation mediated coupling of hormonal and posttranslational enzyme regulation to the metabolic response in liver. Structured summary of protein interactions PFK-2/FBPase-2 physically interacts with PFK-2/FBPase-2 by fluorescent resonance energy transfer (View Interaction: 1, 2) PFK-2/FBPase-2 physically interacts with PFK-2/FBPase-2 by two hybrid (View interaction)

Highlights

Posted on 27 May 2012 | 1:14 am

HIF-1? protein is upregulated in HIF-2? depleted cells via enhanced translation

Publication year: 2012
Source:FEBS Letters, Volume 586, Issue 11

Kathrin Schulz, Larissa Milke, Daniela Rübsamen, Heidi Menrad, Tobias Schmid, Bernhard Brüne

The hypoxia-inducible factors HIF-1 and HIF-2 are primarily regulated via stabilization of their respective ?-subunits under hypoxic conditions. Previously, compensatory upregulation of one HIF-?-subunit upon depletion of the other ?-subunit was described, yet the underlying mechanism remained elusive. Here we provide evidence that enhanced HIF-1? protein expression in HIF-2? knockdown (k/d) cells neither results from elevated HIF-1? mRNA expression, nor from increased HIF-1? protein stability. Instead, we identify enhanced HIF-1? translation as molecular mechanism. Moreover, we found elevated levels of the RNA-binding protein HuR and provide evidence that HuR is critical for the compensatory HIF-1? regulation in HIF-2? k/d cells.

Highlights

Posted on 27 May 2012 | 1:14 am

Extensions of PSD-95/discs large/ZO-1 (PDZ) domains influence lipid binding and membrane targeting of syntenin-1

Publication year: 2012
Source:FEBS Letters, Volume 586, Issue 10

Anna Maria Wawrzyniak, Elke Vermeiren, Pascale Zimmermann, Ylva Ivarsson

Syntenin-1 is a PDZ protein involved in receptor recycling and clustering. Its two PDZ domains interact with various receptors and phosphoinositides, and are flanked by N- and C-terminal regions. Here, we report the identification of an autoinhibitory peptide stretch in the N-terminus that might be regulated by phosphorylation. We further establish that basic residues in the C-terminal region mediate electrostatic interactions with reconstituted liposomes and contribute to the plasma membrane targeting. Our study adds new components to the multi-dentate membrane targeting mechanism and highlights the role of N- and C-terminal PDZ extensions in the regulation of syntenin-1 plasma membrane localization. Structured summary of protein interactions PDZ1-PDZ2 and peptide bind by fluorescence technology (View Interaction: 1, 2, 3, 4).

Highlights

Posted on 27 May 2012 | 1:14 am

PHD finger of the SUMO ligase Siz/PIAS family in rice reveals specific binding for methylated histone H3 at lysine 4 and arginine 2

Publication year: 2012
Source:FEBS Letters

Heisaburo Shindo, Rintaro Suzuki, Wataru Tsuchiya, Misako Taichi, Yuji Nishiuchi, Toshimasa Yamazaki

We determined the three-dimensional structure of the PHD finger of the rice Siz/PIAS-type SUMO ligase, OsSiz1, by NMR spectroscopy and investigated binding ability for a variety of methylated histone H3 tails, showing that OsSiz1–PHD primarily recognizes dimethylated Arg2 of the histone H3 and that methylations at Arg2 and Lys4 reveal synergy effect on binding to OsSiz1–PHD. The K4 cage of OsSiz1–PHD for trimethylated Lys4 of H3K4me3 was similar to that of the BPTF–PHD finger, while the R2 pocket for Arg2 was different. It is intriguing that the PHD module of Siz/PIAS plays an important role, with collaboration with the DNA binding domain SAP, in gene regulation through SUMOylation of a variety of effectors associated with the methylated arginine-riched chromatin domains.

Highlights

Posted on 27 May 2012 | 1:14 am

MiR-26 controls LXR-dependent cholesterol efflux by targeting ABCA1 and ARL7

Publication year: 2012
Source:FEBS Letters, Volume 586, Issue 10

Dongsheng Sun, Jun Zhang, Jianhong Xie, Wei Wei, Mantao Chen, Xiang Zhao

Cellular cholesterol levels are tightly regulated and represent a balance of cholesterol uptake, endogenous synthesis and efflux. Although the classic transcriptional regulations of cholesterol metabolism by liver X receptors (LXRs) have been well studied, the potential effects of LXR-responsive microRNAs (miRNAs) still need to be unveiled. Here, we describe that miR-26, an LXR-suppressed miRNA, inhibits the expression of the ATP-binding cassette transporter A1 (ABCA1) and ADP-ribosylation factor-like 7 (ARL7), two LXR target genes which play critical roles in cholesterol efflux. These findings have not only figured out an alternative mechanism for LXR regulation, but also provided a potential therapeutic target for cholesterol metabolic disorders.

Highlights

Posted on 27 May 2012 | 1:14 am

Anammox organism KSU-1 expresses a NirK-type copper-containing nitrite reductase instead of a NirS-type with cytochrome cd1

Publication year: 2012
Source:FEBS Letters, Volume 586, Issue 11

Daisuke Hira, Hidehiro Toh, Catharina T. Migita, Hiroki Okubo, Takashi Nishiyama, Masahira Hattori, Kenji Furukawa, Takao Fujii

Anaerobic ammonium oxidation (anammox) and denitrification are two distinct microbial reactions relevant to the global nitrogen cycle. The proposed initial step of the anammox reactions, reduction of nitrite to nitric oxide, has been postulated to be identical to that in denitrification catalyzed by the dissimilatory nitrite reductase of the cytochrome cd 1-type. Here, we characterized the copper-containing nitrite reductase homolog encoded by nirK detected in the genome of an anammox bacterium strain KSU-1. We hypothesize that this NirK-type nitrite reductase, rather than a nitrite reductase of the cytochrome cd 1-type (NirS), is likely to catalyze nitrite reduction in anammox organism KSU-1.

Highlights

Posted on 27 May 2012 | 1:14 am

Suppression of cellular invasion by glybenclamide through inhibited secretion of platelet-derived growth factor in ovarian clear cell carcinoma ES-2 cells

Publication year: 2012
Source:FEBS Letters, Volume 586, Issue 10

Tamami Yasukagawa, Yuki Niwa, Siro Simizu, Kazuo Umezawa

It has been demonstrated that potassium channels (K⁺ channels) play significant roles in some malignant phenotypes. Here, we provide the first evidence that treatment with glybenclamide, an ATP-sensitive K⁺ channel blocker, inhibited cell migration in an ovarian clear cell carcinoma cell line, ES-2. Treatment with glybenclamide or knockdown by siRNA targeted against K⁺ channel subunits demonstrated the suppression of ovarian cancer cell invasion, which occurred via inhibition of PDGF-AA secretion. Therefore, our findings suggest that K⁺ channel blockers may be useful chemotherapeutic drugs for blocking the invasiveness of ovarian cancers.

Highlights

Posted on 27 May 2012 | 1:14 am

Editorial Board

Publication year: 2012
Source:FEBS Letters, Volume 586, Issue 10

Posted on 27 May 2012 | 1:14 am

Rat CYP2D2, not 2D1, is functionally conserved with human CYP2D6 in endogenous morphine formation

Publication year: 2012
Source:FEBS Letters

Nadja Grobe, Toni M. Kutchan, Meinhart H. Zenk

The assumption that CYP2D1 is the corresponding rat cytochrome to human CYP2D6 has been revisited using recombinant proteins in direct enzyme assays. CYP2D1 and 2D2 were incubated with known CYP2D6 substrates, the three morphine precursors thebaine, codeine and (R)-reticuline. Mass spectrometric analysis showed that rat CYP2D2, not 2D1, catalyzed the 3-O-demethylation reaction of thebaine and codeine. In addition, CYP2D2 incubated with (R)-reticuline generated four products corytuberine, pallidine, salutaridine and isoboldine while rat CYP2D1 was completely inactive. This intramolecular phenol-coupling reaction follows the same mechanism as observed for CYP2D6. Michaelis-Menten kinetic parameters revealed high catalytic efficiencies for rat CYP2D2. These findings suggest a critical evaluation of other commonly accepted, however untested, CYP2D1 substrates.

Highlights

Posted on 27 May 2012 | 1:14 am

Inducing phase-locking and chaos in cellular oscillators by modulating the driving stimuli

Publication year: 2012
Source:FEBS Letters, Volume 586, Issue 11

Mogens H. Jensen, Sandeep Krishna

Inflammatory responses in eucaryotic cells are often associated with oscillations in the nuclear-cytoplasmic translocation of the transcription factor NF-kB. In most laboratory realizations, the oscillations are triggered by a cytokine stimulus. We use a mathematical model to show that an oscillatory external stimulus can synchronize the NF-kB oscillations into states where the ratios of the internal to external frequency are close to rational numbers. We predict a response diagram of the TNF-driven NF-kB system which exhibits bands of synchronization known as “Arnold tongues”. We suggest that when the amplitude of the external stimulus exceeds a certain threshold, chaotic dynamics of the nuclear NF-kB concentration may occur. This behavior seems independent of the shape of the external oscillation and the non-linearities transducing this signal.

Highlights

Posted on 27 May 2012 | 1:14 am

P4-ATPase ATP8A2 acts in synergy with CDC50A to enhance neurite outgrowth

Publication year: 2012
Source:FEBS Letters

Qin Xu, Guo-Ying Yang, Na Liu, Peng Xu, Yue-Lei Chen, Zheng Zhou, Zhen-ge Luo, Xiaoyan Ding

P4-ATPases are lipid flippases that transport phospholipids across cellular membranes, playing vital roles in cell function. In humans, the disruption of the P4-ATPase ATP8A2 gene causes a severe neurological phenotype. Here, we found that Atp8a2 mRNA was highly expressed in PC12 cells, hippocampal neurons and the brain. Overexpression of ATP8A2 increased the length of neurite outgrowth in NGF-induced PC12 cells and in primary cultures of rat hippocampal neurons. Inducing the loss of function of CDC50A in hippocampal neurons via RNA interference reduced neurite outgrowth, and the co-overexpression of CDC50A and ATP8A2 in PC12 cells enhanced NGF-induced neurite outgrowth. These results indicate that ATP8A2, acting in synergy with CDC50A, performs an important role in neurite outgrowth in neurons.

Highlights

Posted on 27 May 2012 | 1:14 am

Mdm2 and MdmX partner to regulate p53

Publication year: 2012
Source:FEBS Letters, Volume 586, Issue 10

Xinjiang Wang, Xuejun Jiang

Mdm2 regulates the stability, translation, subcellular localization and transcriptional activity of p53 protein. Mdm2-dependent p53 inhibition is essential in regulating p53 activity during embryonic development and in adult tissues. MdmX, an Mdm2 homolog, is also essential for p53 inhibition in vivo. Recent advances in the field from biochemical and genetic studies have revealed an essential role for the MdmX RING domain in Mdm2-dependent p53 polyubiquitination and degradation. Mdm2 on its own is a monoubiquitin E3 ligase for p53, but is converted to a p53 polyubiquitin E3 ligase by MdmX through their RING–RING domain interactions. MdmX acts as an activator as well as a substrate of Mdm2/MdmX E3 complex. The insufficiency of Mdm2 for p53 polyubiquitination also demands other p53 E3 ligases or E4 factors be incorporated into the p53 degradation arena. Deubiquitinases nullify the effects of E3 actions and reverse the ubiquitination process, which permits a diverse and dynamic pattern of p53 stability control. Unsurprisingly, stress signals target MdmX to disengage the p53/Mdm2 feedback loop for timely and appropriate p53 responses to these stresses.

Highlights

Posted on 27 May 2012 | 1:14 am

Structural recognition mechanisms between human Src Homology domain 3 (SH3) and ALG-2-Interacting protein X (Alix)

Publication year: 2012
Source:FEBS Letters

Xiaoli Shi, Stephane Betzi, Adrien Lugari, Sandrine Opi, Audrey Restouin, Isabelle Parrot, Jean Martinez, Pascale Zimmermann, Patrick Lecine, Mingdong Huang, Stefan T. Arold, Yves Collette, Xavier Morelli

The functions of Src family kinases are tightly regulated through Src Homology (SH) domain-mediated protein–protein interactions. We previously reported the biophysical characteristics of the Apoptosis-Linked Gene 2-Interacting protein X (Alix) in complex with the Haemopoietic cell kinase (Hck) SH3 domain. In the current study, we have combined ITC, NMR, SAXS and molecular modeling to determine a 3D model of the complex. We demonstrate that Hck SH3 recognizes an extended linear prolin-rich region of Alix. This particular binding mode enables Hck SH3 to sense a specific non-canonical residue situated in the SH3 RT-loop of the kinase. The resulting model helps clarify the mechanistic insights of Alix-Hck interaction. Structured summary of protein interactions Hck physically interacts with SAM68 by two hybrid (View interaction)FynR96I physically interacts with Alix by two hybrid (View interaction)Hck binds to Alix by pull down (View interaction)Fyn physically interacts with SAM68 by two hybrid (View interaction)Hck and Alix bind by nuclear magnetic resonance (View interaction)FynR96I and Alix bind by isothermal titration calorimetry (View Interaction: 1, 2)FynR96I and Alix bind by nuclear magnetic resonance (View interaction)FynR96I binds to Alix by pull down (View interaction)Hck physically interacts with Alix by two hybrid (View interaction)FynR96I and Alix bind by x ray scattering (View interaction)Hck physically interacts with NEF by two hybrid (View interaction)FynR96I physically interacts with NEF by two hybrid (View interaction)

Highlights

Posted on 27 May 2012 | 1:14 am

Novel amphiphilic compounds effectively inactivate the vaccinia virus

Publication year: 2012
Source:FEBS Letters, Volume 586, Issue 11

A.A. Fedorova, E.P. Goncharova, E.I. Ryabchikova, V.V. Vlasov, M.A. Zenkova

Recent studies demonstrated the ability of artificial ribonucleases (aRNases, small organic RNA cleaving compounds) to inactivate RNA-viruses via the synergetic effect of viral RNA cleavage and disruption of viral envelope [1,2]. Herein, we describe the antiviral activity of aRNases against DNA-containing vaccinia virus: screening of aRNases of various structures revealed that amphiphilic compounds built of positively charged 1,4-diazabicyclo[2.2.2] octane substituted at the bridge nitrogen atoms with aliphatic residues efficiently inactivate this virus. The first stage was the destruction of viral membrane and structure of surface proteins (electron microscopy data). Thus, 1,4-diazabicyclo[2.2.2] octane-based aRNases are novel universal agents inactivating both RNA- and DNA-containing viruses.

Highlights

Posted on 27 May 2012 | 1:14 am

The NRF2-related interactome and regulome contain multifunctional proteins and fine-tuned autoregulatory loops

Publication year: 2012
Source:FEBS Letters

Diána Papp, Katalin Lenti, Dezs? Módos, Dávid Fazekas, Zoltán Dúl, Dénes Türei, László Földvári-Nagy, Ruth Nussinov, Péter Csermely, Tamás Korcsmáros

NRF2 is a well-known, master transcription factor (TF) of oxidative and xenobiotic stress responses. Recent studies uncovered an even wider regulatory role of NRF2 influencing carcinogenesis, inflammation and neurodegeneration. Prompted by these advances here we present a systems-level resource for NRF2 interactome and regulome that includes 289 protein-protein, 7469 TF-DNA and 85 miRNA interactions. As systems-level examples of NRF2-related signaling we identified regulatory loops of NRF2 interacting proteins (e.g., JNK1 and CBP) and a fine-tuned regulatory system, where 35 TFs regulated by NRF2 influence 63 miRNAs that down-regulate NRF2. The presented network and the uncovered regulatory loops may facilitate the development of efficient, NRF2-based therapeutic agents.

Highlights

Posted on 27 May 2012 | 1:14 am

Merlin, a multi-suppressor from cell membrane to the nucleus

Publication year: 2012
Source:FEBS Letters, Volume 586, Issue 10

Lu Zhou, C. Oliver Hanemann

Recent evidence suggests that the neurofibromatosis type 2 (NF2) gene encoded protein merlin suppresses mitogenic signalling not only at the cell membrane but also in the nucleus. At the membrane, merlin inhibits signalling by integrins and tyrosine receptor kinases (RTKs) and the activation of downstream pathways, including the Ras/Raf/MEK/ERK, FAK/Src, PI3K/AKT, Rac/PAK/JNK, mTORC1, and Wnt/?-catenin pathways. In the nucleus, merlin suppresses the E3 ubiquitin ligase CRL4^DCAF1 to inhibit proliferation. Gene expression analysis suggested that CRL4^DCAF1 could also regulate the expression of integrins and RTKs. In this review, we explore the links between merlin function at the membrane and in the nucleus, and discuss the potential of targeting the master regulator CRL4 ^DCAF1 to treat NF2 and other merlin-deficient tumours.

Posted on 27 May 2012 | 1:14 am

Mechanistic details of the DNA recognition by the Dnmt1 DNA methyltransferase

Publication year: 2012
Source:FEBS Letters

Pavel Bashtrykov, Sergey Ragozin, Albert Jeltsch

A recently solved Dnmt1-DNA crystal structure revealed several enzyme-DNA contacts and large structural rearrangements of the DNA at the target site, including the flipping of the non-target strand base of the base pair flanking the CpG site and formation of a non-canonical base pair between the non-target strand Gua and the flanking base pair. Here, we show that the contacts of the enzyme to the target base and the Gua:5mC base pair that are observed in the structure are very important for catalytic activity. The contacts to the non target strand Gua are not important since its exchange by Ade stimulated activity. Except target base flipping, we could not find evidence that the DNA rearrangements have a functional role.

Highlights

Posted on 27 May 2012 | 1:14 am

Induction of epithelial-mesenchymal transition with O-glycosylated oncofetal fibronectin

Publication year: 2012
Source:FEBS Letters

Yao Ding, Kirill Gelfenbeyn, Leonardo Freire-de-Lima, Kazuko Handa, Sen-itiroh Hakomori

Epithelial-mesenchymal transition (EMT) has been shown to play a key role in embryogenesis and cancer progression. We previously found that fibronectin (FN) carrying O-GalNAc at a specific site is selectively expressed in cancer and fetal cells/tissues, and termed oncofetal FN (onfFN). Here, we show that (i) a newly-established monoclonal antibody against FN lacking the O-GalNAc, termed normalFN (norFN), is useful for isolation of onfFN, (ii) onfFN, but not norFN, can induce EMT in human lung carcinoma cells, (iii) onfFN has a synergistic effect with transforming growth factor (TGF)?1 in EMT induction.

Highlights

Posted on 27 May 2012 | 1:14 am

Pirh2 RING-finger E3 ubiquitin ligase: Its role in tumorigenesis and cancer therapy

Publication year: 2012
Source:FEBS Letters, Volume 586, Issue 10

Yong-Sam Jung, Yingjuan Qian, Xinbin Chen

The ubiquitin-dependent proteasome system plays a critical role in many cellular processes and pathogenesis of various human diseases, including cancer. Although there are a large number of E3 ubiquitin ligases, the majority are RING-finger type E3s. Pirh2, a target of p53 transcription factor, contains a highly conserved C3H2C3 type RING domain. Importantly, Pirh2 was found to regulate a group of key factors dedicated to the DNA damage response, such as p53, p73, PolH, and c-Myc. Interestingly, Pirh2 was upregulated or downregulated in different types of cancers. These suggest that Pirh2 is implicated in either promoting or suppressing tumor progression in a tissue-dependent manner. This review will focus on the major findings in these studies and discuss the potential to explore Pirh2 as a cancer therapeutic target.

Posted on 27 May 2012 | 1:14 am

Insights in small Heat Shock Protein/client interaction by combined protection analysis of two different client proteins

Publication year: 2012
Source:FEBS Letters

Benjamin D. Eisenhardt, Christoph Forreiter

sHSPs interact with clients under denaturing conditions. CPH1?2, a truncated version of cyanobacterial phytochrome CPH1, was introduced as a new reporter (client). Comparative analyses of At17.8 and At17.6B as cytosolic class I sHSP representatives demonstrated the advantages of a chromophore-bearing photoreversible protein as new client for analyzing sHSP holdase function in addition to malate dehydrogenase (MDH). The tested sHSPs protected both clients in similar ways but with different efficiencies. Bis-ANS binding studies with sHSPs suggested that the bis-ANS binding is dependent on interactions between different sHSPs and MDH under denaturing temperatures. Structured summary of protein interactions CPH1?2 and CPH1?2 bind by molecular sieving (View interaction)CPH1?2 and HSP17.8 bind by molecular sieving (View interaction)CPH1?2 and HSP17.6 bind by molecular sieving (View interaction)MDH and HSP17.8 bind by molecular sieving (View interaction)MDH and HSP17.6B bind by molecular sieving (View interaction)

Posted on 27 May 2012 | 1:14 am

Active site phosphoryl groups in the biphosphorylated phosphotransferase complex reveal dynamics in a millisecond time scale

Publication year: 2012
Source:FEBS Letters, Volume 586, Issue 10

Tae-Kyung Yu, Young-Joo Yun, Ko On Lee, Kyung Jun Ahn, Jeong-Yong Suh

The N-terminal domain of Enzyme I (EIN) and phosphocarrier HPr can form a biphosphorylated complex when they are both phosphorylated by excess cellular phosphoenolpyruvate. Here we show that the electrostatic repulsion between the phosphoryl groups in the biphosphorylated complex results in characteristic dynamics at the active site in a millisecond time scale. The dynamics is localized to phospho-His15 and the stabilizing backbone amide groups of HPr, and does not impact on the phospho-His189 of EIN. The dynamics occurs with the k ex of ?500s^?1 which compares to the phosphoryl transfer rate of ?850s^?1 between EIN and HPr. The conformational dynamics in HPr may be important for its phosphotransfer reactions with multiple partner proteins. Structured summary of protein interactions EIN and HPr bind by nuclear magnetic resonance (View Interaction).

Highlights

Posted on 27 May 2012 | 1:14 am

Nemo-like kinase promotes etoposide-induced apoptosis of male germ cell-derived GC-1 cells in vitro

Publication year: 2012
Source:FEBS Letters, Volume 586, Issue 10

Xiaowen Cheng, Junbo Liang, Yu Teng, Jun Fu, Shiying Miao, Shudong Zong, Linfang Wang

Spermatogenesis is an extremely intricate process that is tightly regulated and orchestrated by a series of well-coordinated gene expression programmes. Nemo-like kinase (NLK) is an evolutionarily conserved serine/threonine kinase that functions in a wide variety of developmental events. Nevertheless, the function of NLK in spermatogenesis has not been investigated. In this study, we found that the distribution of NLK in mice exhibited a dynamic change during testicular development and gradually became concentrated in the acrosomes of elongated spermatids. NLK overexpression promoted etoposide-induced apoptosis of male germ cell-derived GC-1 cells, while knockdown of NLK by RNA interference (RNAi) attenuated etoposide-induced apoptosis. Our findings suggest that NLK plays an important role in etoposide-induced germ cell apoptosis and may be associated with spermatogenesis.

Highlights

Posted on 27 May 2012 | 1:14 am

Conformational changes upon ligand binding in the essential class II fumarase Rv1098c from Mycobacterium tuberculosis

Publication year: 2012
Source:FEBS Letters, Volume 586, Issue 11

Ariel E. Mechaly, Ahmed Haouz, Isabelle Miras, Nathalie Barilone, Patrick Weber, William Shepard, Pedro M. Alzari, Marco Bellinzoni

rv1098c, an essential gene in Mycobacterium tuberculosis, codes for a class II fumarase. We describe here the crystal structure of Rv1098c in complex with l-malate, fumarate or the competitive inhibitor meso-tartrate. The models reveal that substrate binding promotes the closure of the active site through conformational changes involving the catalytic SS-loop and the C-terminal domain, which likely represents a general feature of this enzyme superfamily. Analysis of ligand–enzyme interactions as well as site-directed mutagenesis suggest Ser318 as one of the two acid–base catalysts. Structured summary of protein interactions Rv1098c and Rv1098c bind by X-ray crystallography (View interaction)

Highlights

Posted on 27 May 2012 | 1:14 am

?-Actinin4 nuclear translocation mediates gonadotropin-releasing hormone stimulation of follicle-stimulating hormone ?-subunit gene transcription in L?T2 cells

Publication year: 2012
Source:FEBS Letters, Volume 586, Issue 10

Han Yu, Zhengjun Li, Dipanjana Ghosh, Teck Kwang Lim, Yuehui He, Qingsong Lin

Gonadotropin-releasing hormone (GnRH) regulates the synthesis and secretion of follicle-stimulating hormone (FSH) by stimulating the transcription of Fsh? gene. Our iTRAQ quantitative proteomics result showed that the abundance of ?-actinin4 (ACTN4) increased in the nuclei of L?T2 cells upon GnRH induction. Using RNA interference, reverse transcription and real-time PCR, luciferase and transient transfection assays, we proved that ACTN4 is involved in the regulation of mouse Fsh? gene (mFsh?) transcription and its C-terminal calmodulin (CaM)-like domain is crucial for this process. Our study suggests that ACTN4 nuclear translocation mediates GnRH stimulation of mFsh? gene transcription.

Highlights

Posted on 27 May 2012 | 1:14 am

FOCUS ON… Ubiquitin-related tumor suppressors

Publication year: 2012
Source:FEBS Letters, Volume 586, Issue 10

Wilhelm W. Just

Posted on 27 May 2012 | 1:14 am

Genomic analysis of ICEVchBan8: An atypical genetic element in Vibrio cholerae

Publication year: 2012
Source:FEBS Letters, Volume 586, Issue 11

Elisa Taviani, Matteo Spagnoletti, Daniela Ceccarelli, Bradd J. Haley, Nur A. Hasan, Arlene Chen, Mauro M. Colombo, Anwar Huq, Rita R. Colwell

Genomic islands (GIs) and integrative conjugative elements (ICEs) are major players in bacterial evolution since they encode genes involved in adaptive functions of medical or environmental importance. Here we performed the genomic analysis of ICEVchBan8, an unusual ICE found in the genome of a clinical non-toxigenic Vibrio cholerae O37 isolate. ICEVchBan8 shares most of its genetic structure with SXT/R391 ICEs. However, this ICE codes for a different integration/excision module is located at a different insertion site, and part of its genetic cargo shows homology to other pathogenicity islands of V. cholerae.

Highlights

Posted on 27 May 2012 | 1:14 am

X-linked inhibitor of apoptosis protein mediates neddylation by itself but does not function as a NEDD8–E3 ligase for caspase-7

Publication year: 2012
Source:FEBS Letters, Volume 586, Issue 11

Taiki Nagano, Toshiaki Hashimoto, Akio Nakashima, Ushio Kikkawa, Shinji Kamada

X-linked inhibitor of apoptosis protein (XIAP) is a potent antagonist of caspases, and functions as a ubiquitin–E3 ligase by itself and for caspases. Recently, NEDD8, a ubiquitin-like modifier, has been suggested to be used for modification of caspase-7 mediated by XIAP. However, it is not clear whether caspase-7 is a bona fide target for NEDD8. Here we showed that no neddylation of caspase-7 but that of XIAP itself was observed under the conditions in which caspase-7 was modified with ubiquitin. These results reveal that XIAP does not function as a NEDD8–E3 ligase for caspase-7 in vivo. Structured summary of protein interactions NEDD8 physically interacts with Caspase-7 by pull down ( View interaction ) XIAP physically interacts with NEDD8 by anti-bait coimmunoprecipitation ( View interaction )

Highlights

Posted on 27 May 2012 | 1:14 am

Catalytic promiscuity in Pseudomonas aeruginosa arylsulfatase as an example of chemistry-driven protein evolution

Publication year: 2012
Source:FEBS Letters, Volume 586, Issue 11

Jinghui Luo, Bert van Loo, Shina C.L. Kamerlin

In recent years, it has become increasingly clear that many enzymes are catalytically “promiscuous”. This can provide a springboard for protein evolution, allowing enzymes to acquire novel functionality without compromising their native activities. We present here a detailed study of Pseudomonas aeruginosa arylsulfatase (PAS), which catalyzes the hydrolysis of a number of chemically distinct substrates, with proficiencies comparable to that towards its native reaction. We demonstrate that the main driving force for the promiscuity is the ability to exploit the electrostatic preorganization of the active site for the native substrate, providing an example of chemistry-driven protein evolution.

Highlights

Posted on 27 May 2012 | 1:14 am

Tumor suppressor functions of FBW7 in cancer development and progression

Publication year: 2012
Source:FEBS Letters, Volume 586, Issue 10

Zhiwei Wang, Hiroyuki Inuzuka, Jiateng Zhong, Lixin Wan, Hidefumi Fukushima, Fazlul H. Sarkar, Wenyi Wei

FBW7 (F-box and WD repeat domain-containing 7) has been characterized as an onco-suppressor protein in human cancers. Recent studies have also shown that FBW7 exerts its anti-tumor function primarily by promoting the degradation of various oncoproteins, through which FBW7 regulates cellular proliferation, differentiation and causes genetic instability. In this review, we will discuss the role of FBW7 downstream substrates and how dysregulation of Fbw7-mediated proteolysis of these substrates contributes to tumorigenesis. Additionally, we will also summarize the currently available various Fbw7-knockout mouse models that support Fbw7 as a tumor suppressor gene in the development and progression of human malignancies.

Highlights

Posted on 27 May 2012 | 1:14 am

Cyclic AMP-specific phosphodiesterase, PDE8A1, is activated by protein kinase A-mediated phosphorylation

Publication year: 2012
Source:FEBS Letters, Volume 586, Issue 11

Kim M. Brown, Louisa C.Y. Lee, Jane E. Findlay, Jonathan P. Day, George S. Baillie

The cyclic AMP-specific phosphodiesterase PDE8 has been shown to play a pivotal role in important processes such as steroidogenesis, T cell adhesion, regulation of heart beat and chemotaxis. However, no information exists on how the activity of this enzyme is regulated. We show that under elevated cAMP conditions, PKA acts to phosphorylate PDE8A on serine 359 and this action serves to enhance the activity of the enzyme. This is the first indication that PDE8 activity can be modulated by a kinase, and we propose that this mechanism forms a feedback loop that results in the restoration of basal cAMP levels.

Highlights

Posted on 27 May 2012 | 1:14 am

Live imaging of active fluorophore labelled Wnt proteins

Publication year: 2012
Source:FEBS Letters, Volume 586, Issue 11

Tatjana Holzer, Katrin Liffers, Karolin Rahm, Benjamin Trageser, Suat Özbek, Dietmar Gradl

For almost 30years, Wnt proteins have been known as key regulators of many developmental decisions, including the formation of the embryonic axes, patterning of the CNS, limb bud outgrowth and segment polarity. However, live cell imaging of active Wnt proteins was rarely reported. Here, we have generated a Wnt2b–EGFP fusion protein that retains functionality in bona fide Wnt activity assays, although the secreted protein is rapidly cleaved by extracellular proteases. We can show with this new tool that Wnt2b–EGFP moves along the microtubules of Wnt producing cells and that this directed movement is essential for the secretion of active Wnt protein.

Highlights

Posted on 27 May 2012 | 1:14 am

Ubiquitination and phosphorylation of Beclin 1 and its binding partners: Tuning class III phosphatidylinositol 3-kinase activity and tumor suppression

Publication year: 2012
Source:FEBS Letters, Volume 586, Issue 11

Hilde Abrahamsen, Harald Stenmark, Harald W. Platta

The class III phosphatidylinositol 3-kinase (PI3K-III) complex and its phosphorylated lipid product phosphatidylinositol 3-phosphate (PtdIns3P) control the three topologically related membrane-involution processes autophagy, endocytosis, and cytokinesis. The activity of the catalytic unit of PI3K-III complex, the Vacuolar sorting protein 34 (VPS34), depends on the membrane targeting unit Vacuolar sorting protein 15 (VPS15), and the tumor suppressor protein Beclin 1. It is established that the overall activity of VPS34 is positively regulated by Beclin 1, whose positive influence is further controlled through the association with a set of Beclin1 interacting components, which stimulate or inhibit VPS34. The interaction between Beclin 1 and Beclin 1-associated components are controllable and is regulated by phosphorylation in a context-dependent manner. Here, we focus on an emerging concept whereby the activity of the PI3K-III complex is controlled by ubiquitination of Beclin 1 or Beclin 1-associated molecules. In summary, at least three different ubiquitin ligases can affect the positive regulatory function of Beclin 1 towards VPS34, suggesting that ubiquitination is an important and physiologically relevant event in tuning the tumor suppressor function of Beclin 1.

Posted on 27 May 2012 | 1:14 am

The enigmatic role of H2Bub1 in cancer

Publication year: 2012
Source:FEBS Letters, Volume 586, Issue 11

Steven A. Johnsen

The post-translational modification of histone proteins plays an important role in controlling cell fate by directing essentially all DNA-associated nuclear processes. Misregulation and mutation of histone modifying enzymes is a hallmark of tumorigenesis. However, how these different epigenetic modifications lead to tumor initiation and/or progression remains poorly understood. Recent studies have uncovered a potential tumor suppressor role for histone H2B monoubiquitination (H2Bub1). Like many other histone modifications, H2Bub1 has diverse functions and plays roles both in transcriptional activation and repression as well as in controlling mRNA processing and directing DNA repair processes. Notably, H2Bub1 has been linked to transcriptional elongation and is preferentially found in the transcribed region of active genes. Its activity is intimately connected to active transcription and the transcriptional elongation regulatory protein cyclin-dependent kinase-9 (CDK9) and the facilitates chromatin transcription (FACT) complex. This review provides an overview of the current understanding of H2Bub1 function in mammalian systems with a particular emphasis on its role in cancer and potential options for exploiting this knowledge for the treatment of cancer.

Posted on 27 May 2012 | 1:14 am

Trypanosomes contain two highly different isoforms of peroxin PEX13 involved in glycosome biogenesis

Publication year: 2012
Source:FEBS Letters

Ana Brennand, Daniel J. Rigden, Paul A.M. Michels

We previously identified the peroxin PEX13 in Trypanosoma brucei. Although lacking some features considered typical of PEX13s, it appeared functional in the biogenesis of glycosomes, the peroxisome-like organelles of trypanosomatids. Here we report the identification of a very different trypanosomatid PEX13, not containing the commonly encountered PEX13 SH3 domain but having other typical features. It is readily detected with the jackhmmer database search program, but not with PSI-BLAST. This is the first time different PEX13 isoforms are reported in a single organism. We show that this PEX13.2, like the PEX13.1 previously described, is associated with glycosomes and that its depletion by RNA interference affects the biogenesis of the organelles and viability of trypanosomes. The features considered typical of PEX13s are discussed.

Posted on 27 May 2012 | 1:14 am

Spatiotemporal resolution of Ca2+ signaling events by real time imaging of single B cells

Publication year: 2012
Source:FEBS Letters, Volume 586, Issue 10

Stephan Junek, Michael Engelke, Detlev Schild, Jürgen Wienands

Antigen-induced B cell activation requires mobilization of the Ca²⁺ second messenger. This process is associated with the subcellular relocalization of signal effector proteins of the B cell antigen receptor such as the adaptor protein SLP65. Here we describe a broadly applicable live cell imaging method to simultaneously visualize intracellular Ca²⁺ flux profiles and the translocation of cytosolic signaling proteins to the plasma membrane in real time. Our approach delineated the kinetic hierarchy of Ca²⁺ signaling events in B cells and revealed a timely ordered contribution of various organelles to the overall Ca²⁺ signal. The developed experimental setup provides a useful tool to resolve the spatiotemporal signaling dynamics in various receptor signaling systems.

Highlights

Posted on 27 May 2012 | 1:14 am

Editorial Board

Publication year: 2012
Source:FEBS Letters, Volume 586, Issue 11

Posted on 27 May 2012 | 1:14 am

4?-Hydroxyflavanone suppresses activation of sterol regulatory element-binding proteins and de novo lipid synthesis

Publication year: 2012
Source:FEBS Letters

Shingo Miyata, Jun Inoue, Makoto Shimizu, Sato Ryuichiro

Sterol regulatory element-binding proteins (SREBPs) are major transcription factors that regulate the expression of genes involved in fatty acid and cholesterol biosynthesis. Here we show that 4?-hydroxyflavanone (4?-HF) impairs the fatty acid synthase promoter activity and reduces the activation of SREBPs and their target gene expression in human hepatoma Huh-7 cells. Moreover, 4?-HF suppresses de novo fatty acid and cholesterol synthesis. This study identifies 4?-HF as an inhibitor of SREBP maturation and lipid synthesis, and provides evidence that 4?-HF may have major potential as a pharmaceutical preparation against hepatic steatosis and dyslipidemia.

Highlights

Posted on 27 May 2012 | 1:14 am

Ubiquitination and deubiquitination of REST and its roles in cancers

Publication year: 2012
Source:FEBS Letters, Volume 586, Issue 11

Zhi Huang, Shideng Bao

REST/NRSF (the RE-1 silencing transcription factor or neuron-restrictive silencer factor) was originally identified as a transcriptional repressor of a number of neuronal-specific genes in neural stem cells and non-neuronal cells. REST functions as a master regulator in the maintenance of neural stem cells. During tumorigenesis, REST shows opposing roles in different type of cells. In human epithelial cancers such as colon cancer, REST acts as a tumor suppressor. In contrast, REST plays an oncogenic role in the development of brain tumors and other cancers. Abnormal upregulation of REST has been found in medulloblastoma, neuroblastoma and glioblastoma (GBM). Recent studies in GBMs suggest that REST exerts its oncogenic function by maintaining self-renewal potential of glioma stem cells (GSCs).

Posted on 27 May 2012 | 1:14 am

Functional analysis of the conserved histidine residue of Bamboo mosaic virus capping enzyme in the activity for the formation of the covalent enzyme-m7GMP intermediate

Publication year: 2012
Source:FEBS Letters

Hua-Yang Lin, Chiao-Yuan Yu, Yau-Heiu Hsu, Menghsiao Meng

The alphavirus-like mRNA capping enzyme of Bamboo mosaic virus (BaMV) exhibits an AdoMet-dependent guanylyltransferase activity by which the methyl group of AdoMet is transferred to GTP, leading to the formation of m⁷GTP, and the m⁷GMP moiety is next transferred to the 5 end of ppRNA via a covalent enzyme-m⁷GMP intermediate. The function of the conserved H68 of the BaMV capping enzyme in the intermediate formation was analyzed by mutagenesis in this study. The nature of the bond linking the enzyme and m⁷GMP was changed in the H68C mutant protein, strongly suggesting that H68 covalently binds to m⁷GMP in the intermediate.

Posted on 27 May 2012 | 1:14 am

NIRF/UHRF2 occupies a central position in the cell cycle network and allows coupling with the epigenetic landscape

Publication year: 2012
Source:FEBS Letters, Volume 586, Issue 11

Tsutomu Mori, Daisuke D. Ikeda, Yoshiki Yamaguchi, Motoko Unoki

As predicted by systems biology, a paradigm shift will emerge through the integration of information about different layers of cellular processes. The cell cycle network is at the heart of the cellular computing system, and orchestrates versatile cellular functions. The NIRF/UHRF2 ubiquitin ligase is an “intermodular hub” that occupies a central position in the network, and facilitates coordination among the cell cycle machinery, the ubiquitin–proteasome system, and the epigenetic system. NIRF interacts with cyclins, CDKs, p53, pRB, PCNA, HDAC1, DNMTs, G9a, methylated histone H3 lysine 9, and methylated DNA. NIRF ubiquitinates cyclins D1 and E1, and induces G1 arrest. The NIRF gene is frequently lost in tumors and is a candidate tumor suppressor, while its paralog, the UHRF1 gene, is hardly altered. Thus, investigations of NIRF are essential to understand the entire biological systems. Through integration of the enormous information flows, NIRF may contribute to the coupling between the cell cycle network and the epigenetic landscape. We propose the new paradigm that NIRF produces the extreme diversity in the network wiring that helps the diversity of Waddington’s canals.

Posted on 27 May 2012 | 1:14 am

The murine Gcap14 gene encodes a novel microtubule binding and bundling protein

Publication year: 2012
Source:FEBS Letters, Volume 586, Issue 10

Hitomi Hosono, Nao Yamaguchi, Kenzi Oshima, Tsukasa Matsuda, Daita Nadano

Microtubules form flexible fibers, which are utilized in cell proliferation and differentiation. Although the flexibility of microtubules was shown to be regulated by various microtubule-associated proteins, this regulation is still far from complete understanding. Here, we report a new potential regulator of microtubules in mammals. Gcap14 colocalizes with microtubules in mammalian cells transfected with Gcap14 expression vector. Association of Gcap14 with microtubules was confirmed by biochemical subcellular fractionation. Recombinant Gcap14 protein cosedimented with pure microtubules, indicating a direct binding between the two. Furthermore, recombinant Gcap14 was shown to have the ability of inducing microtubule bundling in vitro. Structured summary of protein interactions Gcap14 physically interacts with Gcap14 by anti tag coimmunoprecipitation (View Interaction: 1, 2)

Highlights

Posted on 27 May 2012 | 1:14 am

Glycan structures and intrageneric variations of venom acidic phospholipases A2 from Tropidolaemus pitvipers

Most of the phospholipases A2 (PLA2s) variants isolated so far from snake venoms are non-glycosylated enzymes. We herein purified an active glycosylated PLA2 and an inactive non-glycosylated Lys49-like PLA2 from two geographic venom samples of Tropidolaemus. The PLA2 variants from the two samples have rather different N-terminal sequences, implying that the samples were probably derived from two species (T. subannulatus and T. wagleri). The active PLA2s from Sulawesi and Sumatra venoms were designated as Tsu-E6 and Twa-E6, respectively, due to the presence of their conserved Glu6 residue. Tsu-E6 inhibited ADP-induced aggregation of mouse and human platelets. Twa-E6, however, stimulated the aggregation of mouse platelets but inhibited the aggregation of human platelets. Both of the PLA2s were found to be glycosylated at Asn14. Using MALDI-TOF analysis, the released glycans were shown to be complex type oligosaccharides without sialylation. This is the first glycan structure of the snake venom PLA2 solved. Furthermore, the enzymatic removal of glycans from both the PLA2s did not significantly alter their effects on lipid hydrolysis and platelet aggregation. The thermostability of glycosylated Twa-E6 was also found to be as good as those of other homologous PLA2s. The presence of these oligosaccharides on the PLA2s warrants further analyses which may provide useful insights into the functional regulation of these biomolecules.

Posted on 26 May 2012 | 5:19 pm

Understanding metabolic alterations in cancer cells: a promising new/old approach to eradicate cancer.

A key task for cancer researchers is to understand the differences between normal cells and cancer cells, with the idea of finding a therapeutic window that would exclusively target cancer cells. Alterations in several aspects of cell metabolism have recently been re-discovered as one of the main features of most cancer cells.

Posted on 26 May 2012 | 5:17 pm

On the biomechanics of stem cell niche formation in the gut: Modelling growing organoids

In vitro culture of intestinal tissue has been tried for decades. Only recently Sato and co-workers succeeded in establishing long term intestinal culture demonstrating that cells expressing the Lgr5 gene can give rise to organoids with crypt-like domains similar to those found in vivo. In these cultures Paneth cells provide essential signals in supporting stem cell function.We have recently developed an individual cell-based computational model of the intestinal tissue. The model is capable of quantitatively reproducing a comprehensive set of experimental data on intestinal cell organization. Here, we present a significant extension of this model which allows simulating intestinal organoid formation in silico. For this purpose we introduce a flexible basal membrane which assigns a bending modulus to the organoid surface. This membrane can be re-organized by cells attached to it depending on the cells’ differentiation status. Accordingly, the morphology of the epithelium is self-organized.We hypothesize that local tissue curvature is a key regulatory factor regarding stem cell organization in the intestinal tissue by controlling Paneth cell specification. In simulation studies our model does closely resemble the spatio-temporal organization of intestinal organoids. According to our results proliferation-induced shape fluctuations are sufficient to induce crypt-like domains, and spontaneous tissue curvature induced by Paneth cells can control cell number ratios. Thereby, stem cell expansion in an organoid sensitively depends on its biomechanics.We encourage a number of experiments that will enable new insights into mechano-transduction in the intestine and suggest model extensions in the field of gland formation.

Posted on 25 May 2012 | 4:55 pm

Systematic analysis and comparison of nucleotide-binding site disease resistance genes in maize

Nucleotide-binding site (NBS) disease resistance genes play an integral role in defending plants from a range of pathogens and insect pests. Consequently, a number of recent studies have focused on NBS-encoding genes in molecular disease resistance breeding programmes for several important plant species. Little information, however, has been reported with an emphasis on systematic analysis and a comparison of NBS-encoding genes in maize. In the present study, 109 NBS-encoding genes were identified based on the complete genome sequence of maize (Zea mays cv. B73), classified as four different subgroups, and then characterized according to chromosomal locations, gene duplications, structural diversity and conserved protein motifs. Subsequent phylogenetic comparisons indicated that several maize NBS-encoding genes possessed high similarity to function-known NBS-encoding genes, and revealed the evolutionary relationships of NBS-encoding genes in maize comparede to those in other model plants. Analyses of the physical locations and duplications of NBS-encoding genes showed that gene duplication events of disease resistance genes were lower in maize than in other model plants, which may have led to an increase in the functional diversity of the maize NBS-encoding genes. Various expression patterns of maize NBS-encoding genes in different tissues were observed using an expressed-sequence tags database and, alternatively, after southern leaf blight infection or the application of exogenous salicylic acid. The results reported in the present study contribute to an improved understanding of the NBS-encoding gene family in maize.

Posted on 24 May 2012 | 5:38 pm

The cofactor preference of glucose-6-phosphate dehydrogenase from Escherichia coli– modeling the physiological production of reduced cofactors

In Escherichia coli, the pentose phosphate pathway is one of the main sources of NADPH. The first enzyme of the pathway, glucose-6-phosphate dehydrogenase (G6PDH), is generally considered an exclusive NADPH producer, but a rigorous assessment of cofactor preference has yet to be reported. In this work, the specificity constants for NADP and NAD for G6PDH were determined using a pure enzyme preparation. Absence of the phosphate group on the cofactor leads to a 410-fold reduction in the performance of the enzyme. Furthermore, the contribution of the phosphate group to binding of the transition state to the active site was calculated to be 3.6 kcal·mol?1. In order to estimate the main kinetic parameters for NAD(P) and NAD(P)H, we used the classical initial-rates approach, together with an analysis of reaction time courses. To achieve this, we developed a new analytical solution to the integrated Michaelis–Menten equation by including the effect of competitive product inhibition using the ?-function. With reference to relevant kinetic parameters and intracellular metabolite concentrations reported by others, we modeled the sensitivity of reduced cofactor production by G6PDH as a function of the redox ratios of NAD/NADH (rRNAD) and NADP/NADPH (rRNADP). Our analysis shows that NADPH production sharply increases within the range of thermodynamically feasible values of rRNADP, but NADH production remains low within the range feasible for rRNAD. Nevertheless, we show that certain combinations of rRNADP and rRNAD sustain greater levels of NADH production over NADPH.

Posted on 24 May 2012 | 5:38 pm

Distinct biological activities of C3 and ADP-ribosyltransferase-deficient C3-E174Q.

Background:? Low molecular weight GTP-binding proteins of the Rho family control the organization of the actin cytoskeleton in eukaryotic cells. Dramatic reorganization of the actin cytoskeleton is caused by C3 exoenzyme derived from Clostridium botulinum (C3), based on ADP-ribosylation of RhoA / B / C. In addition, wild type as well as ADP-ribosyltransferase-deficient C3-E174Q induce axonal outgrowth of primary murine hippocampal neurons and prevents growth cone collapse indicating a non-enzymatic mode of action.Methodology / Principal Findings: In this study, we compared the effects of C3-E174Q and wild type C3 in the murine hippocampal cell line HT22. Treatment of HT22 cells with C3 resulted in Rho ADP-ribosylation and cell rounding. The ADP-ribosyltransferase-deficient mutant C3-E174Q did induce neither Rho ADP-ribosylation nor morphological changes. C3 as well as C3-E174Q treatment resulted in a growth arrest, reduced expression of cyclin D levels, and increased expression of RhoB, a negative regulator of cell cycle progression. Serum starvation induced apoptosis in HT22 cells, as detected in terms of increased expression of caspase-9 and Bax. Specifically C3 (not C3-E174Q) protected serum-starvated HT22 cells from apoptosis.Conclusions / Significance: This is the first study, dissecting ADP-ribosyltransferase (ART)-dependent from ART-independent effects of C3. While morphological changes and anti-apoptotic activity, strictly depends on ART activity, the anti-proliferative effects turned out to be independent of ART activity.

Posted on 23 May 2012 | 5:01 pm

Lipid metabolism in cancer

Lipids form a diverse group of water insoluble molecules that include triacylglycerides, phosphoglycerides, sterols and sphingolipids. They play several important roles at the cellular and organismal level. Fatty acids are the major building blocks for the synthesis of triacylglycerides, which are mainly used for energy storage. Phosphoglycerides, together with sterols and sphingolipids, represent the major structural components of biological membranes. Lipids can also have important roles in signalling, functioning as second messengers and hormones. There is increasing evidence that cancer cells show specific alterations in different aspects of lipid metabolism. These alterations can affect the availability of structural lipids for the synthesis of membranes, the synthesis and degradation of lipids that contribute to energy homeostasis and the abundance of lipids with signalling functions. Changes in lipid metabolism can affect numerous cellular processes including cell growth, proliferation, differentiation and motility. This review will examine some of the alterations in lipid metabolism that have been reported in cancer, both at the cellular and the organismal level, and discuss how they contribute to different aspects of tumourigenesis.

Posted on 23 May 2012 | 5:01 pm

Metabolite profiling of plasma and urine from rats with TNBS-induced acute colitis using UPLC-ESI-QTOF-MS-based metabonomics – a pilot study

The incidence of inflammatory bowel disease, a relapsing intestinal condition whose precise etiology is still unclear, has continually increased over recent years. Metabolic profiling is an effective method with high sample throughput that can detect and identify potential biomarkers, and thus may be useful in investigating the pathogenesis of inflammatory bowel disease. In this study, using a metabonomics approach, a pilot study based on ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOF-MS) was performed to characterize the metabolic profile of plasma and urine samples of rats with experimental colitis induced by 2,4,6-trinitrobenzene sulfonic acid. Acquired metabolic profile data were processed by multivariate data analysis for differentiation and screening of potential biomarkers. Five metabolites were identified in urine: two tryptophan metabolites [4-(2-aminophenyl)-2,4-dioxobutanoic acid and 4,6-cihydroxyquinoline], two gut microbial metabolites (phenyl-acetylglycine and p-cresol glucuronide), and the bile acid 12?-hydroxy-3-oxocholadienic acid. Seven metabolites were identified in plasma: three members of the bile acid/alcohol group (cholic acid, 12?-hydroxy-3-oxocholadienic acid and cholestane-3,7,12,24,25-pentol) and four lysophosphatidylcholines [LysoPC(20:4), LysoPC(16:0), LysoPC(18:1) and LysoPC(18:0)]. These metabolites are associated with damage of the intestinal barrier function, microbiota homeostasis, immune modulation and the inflammatory response, and play important roles in the pathogenesis of inflammatory bowel disease. Our results positively support application of the metabonomic approach in study of the pathophysiological mechanism of inflammatory bowel disease.

Posted on 22 May 2012 | 6:48 pm

Automatic generation of causal networks linking growth factor stimuli to functional cell state changes

Despite the increasing number of growth factor-related signalling networks, their lack of logical and causal connection to factual changes in cell states frequently impairs the functional interpretation of microarray data. We present a novel method enabling the automatic inference of causal multi-layer networks from such data, allowing the functional interpretation of growth factor stimulation experiments using pathway databases. Our environment of evaluation was hepatocyte growth factor-stimulated cell migration and proliferation in a keratinocyte-fibroblast co-culture. The network for this system was obtained by applying the steps: (a) automatic integration of the comprehensive set of all known cellular networks from the Pathway Interaction Database into a master structure; (b) retrieval of an active-network from the master structure, where the network edges that connect nodes with an absent mRNA level were excluded; and (c) reduction of the active-network complexity to a causal subnetwork from a set of seed nodes specific for the microarray experiment. The seed nodes comprised the receptors stimulated in the experiment, the consequently differentially expressed genes, and the expected cell states. The resulting network shows how well-known players, in the context of hepatocyte growth factor stimulation, are mechanistically linked in a pathway triggering functional cell state changes. Using bioquali, we checked and validated the consistency of the network with respect to microarray data by computational simulation. The network has properties that can be classified into different functional layers because it not only shows signal processing down to the transcriptional level, but also the modulation of the network structure by the preceeding stimulation. The software for generating computable objects from the Pathway Interaction Database database, as well as the generated networks, are freely available at: http://www.tiga.uni-hd.de/supplements/inferringFromPID.html.

Posted on 22 May 2012 | 6:47 pm

Site-directed mutagenesis and spectral studies suggest a putative role of FurA from Anabaena sp. PCC 7120 as a heme sensor protein

The transcriptional repressor Fur (ferric uptake regulator) is one of the most important switches regulating prokaryotic iron metabolism. Cyanobacterial FurA binds heme in the micromolar concentration range and this interaction negatively affects its in vitro DNA binding ability in a concentration-dependent manner. Using site-directed mutagenesis along with difference absorption UV–visible, circular dichroism and electronic paramagnetic resonance spectroscopies, we further analyse the nature of heme binding in FurA. Our data point to Cys141, within a Cys-Pro motif, as an axial ligand of the Fe(III) high-spin heme. In the Fe(II) oxidation state, the heme shows low-spin form with an electronic absorption spectrum typical of six-coordinated low-spin heme proteins with a Soret absorption maximum blue-shifted by 25 nm in relation to typical low-spin thiolate-ligated Fe(II) heme proteins. Moreover, the ferrous C141S mutant shows Soret, ? and ? bands almost identical to those observed for ferrous wild-type heme–FurA, indicating that the heme in ferrous C141S is in the same six-coordinated heme ligation as the ferrous native form. Therefore, Cys141 is not a ligand of the Fe(II) heme centre, suggesting a redox-dependent ligand switch undergone by this regulator. Our results indicate that the binding of heme to FurA exhibits the same physicochemical features as previously described for heme sensor proteins.

Posted on 22 May 2012 | 12:45 pm

Antifreeze protein gene amplification facilitated niche exploitation and speciation in wolffish

During winter, the coastal waters of Newfoundland can be considered a ‘freeze risk ecozone’ for teleost fishes, where the shallower habitats pose a high (and the deeper habitats a low) risk of freezing. Atlantic (Anarhichas lupus) and spotted (Anarhichas minor) wolffish, which inhabit these waters, reside at opposite ends of this ecozone, with the Atlantic wolffish being the species facing the greatest risk, because of its shallower niche. In order to resist freezing, this species secretes five times the level of antifreeze protein (AFP) activity into the plasma than does the spotted wolffish. The main basis for this interspecific difference in AFP levels is gene dosage, as the Atlantic wolffish has approximately three times as many AFP gene copies as the spotted wolffish. In addition, AFP transcript levels in liver (the primary source of circulating AFPs) are several times higher in the Atlantic wolffish. One explanation for the difference in gene dosage and transcript levels is the presence of tandemly arrayed repeats in the latter, which make up two-thirds of its AFP gene pool. Such repeats are not present in the spotted wolffish. The available evidence indicates that the two species diverged from a common ancestor at a time when the ebb and flow of northern glaciations would have resulted in the emergence of shallow water ‘freeze risk ecozones’. The results of this study suggest that the duplication/amplification of AFP genes in a subpopulation of ancestral wolffish would have facilitated the exploitation of this high-risk habitat, resulting in the divergence and evolution of modern-day Atlantic and spotted wolffish species.Database Nucleotide sequence data are available in the GenBank database under the accession numbers AWG1-4, JQ040521, JQ040515, JQ040516, JQ040517, AWE1-3, JQ040522, JQ040523, JQ040524, SWG1-3, JQ040518, JQ040519, and JQ040520

Posted on 22 May 2012 | 12:45 pm

Nelfinavir inhibits regulated intramembrane proteolysis of sterol regulatory element binding protein-1 and activating transcription factor 6 in castration-resistant prostate cancer

Nelfinavir induces apoptosis in liposarcoma by inhibiting site-2 protease (S2P) activity, which leads to suppression of regulated intramembrane proteolysis. We postulate similar effects in castration-resistant prostate cancer because it exhibits a lipogenic phenotype. Nelfinavir inhibited androgen receptor activation in androgen-sensitive prostate cancer and the nuclear translocation of the fusion proteins sterol regulatory element binding protein-1 (SREBP-1)–enhanced green fluorescence protein (EGFP) and activating transcription factor 6 (ATF6)–EGFP in castration-resistant prostate cancer cells, viewed under confocal microscopy. Nelfinavir and site-1 protease (S1P) and S2P small interfering RNAs (siRNAs) reduced the proliferation of castration-resistant prostate cancer and induced apoptosis, which was opposed by autophagy. Inhibition of autophagy with hydroxychloroquine was additive to the apoptotic effect of nelfinavir. Western blotting of S1P and S2P siRNA knockdown and/or nelfinavir-treated cells confirmed the accumulation of precursor SREBP-1 and ATF6. 3,4-Dichloroisocoumarin, an S1P inhibitor, did not affect SREBP-1 processing. In contrast, 1,10-phenanthroline, an S2P inhibitor, reproduced the nelfinavir-treated molecular and biological phenotype. Nelfinavir-mediated inhibition of regulated intramembrane proteolysis led to the accumulation of unprocessed SREBP-1 and ATF6. This resulted in sequential endoplasmic reticulum stress, inhibition of the unfolded protein response, reduced fatty acid synthase expression and apoptosis, which was countered by autophagy. Inhibition of autophagy was at least additive to this pro-apoptotic effect. These findings provide new insights into nelfinavir-induced endoplasmic reticulum stress and cancer cell death, and lead us to propose investigating its clinical activity in castration-resistant prostate cancer. This report validates S2P as a therapeutic target in castration-resistant prostate cancer.

Posted on 21 May 2012 | 7:22 pm

Distinct sulfonation activities in resveratrol-sensitive and resveratrol-insensitive human glioblastoma cells

Glioblastoma multiforme (GBM) cells show different responses to resveratrol, for unknown reasons. Our data from human medulloblastoma cells and primary cultures of rat brain cells revealed an inverse correlation of sulfonation activity with resveratrol sensitivities, providing a clue to the underlying mechanisms of the variable sensitivities of GBM cells to resveratrol. In this study, we found that U251 cells were sensitive and LN229 cells were insensitive to resveratrol. Thus, these two cell lines were taken as comparable models for elucidating the influence of sulfonation activities on resveratrol sensitivity. HPLC showed identical resveratrol metabolic patterns in both cell lines. LC/MS and high-resolution mass MS analyses further demonstrated that resveratrol monosulfate generated by sulfotransferases (SULTs) was the major metabolite of human GBM cells. The levels of brain-associated SULT (SULT1A1, SULT1C2, and SULT4A1) expression in U251 cells were lower than those in LN229 cells, suggesting the inverse relationship of SULT-mediated sulfonation activity with high intracellular resveratrol bioavailability and resveratrol sensitivity of human GBM cells. Furthermore, immunohistochemical staining revealed reductions in expression of the three brain-associated SULTs in 72.8%, 47.5% and 66.3% of astrocytomas, respectively. Therefore, the levels of brain-associated SULTs and sulfonation activity mediated by them could be important parameters for evaluating the potential response of human GBM cells to resveratrol, and may have value in the personalized treatment of GBMs with resveratrol.Database Nucleotide sequence data for SULT1A1, SULT1C2 and SULT4A1 are available in the GenBank database under the accession numbers BC110887, BC005353, and BC028171.

Posted on 21 May 2012 | 7:21 pm

PROSO II – a new method for protein solubility prediction

Many fields of science and industry depend on efficient production of active protein using heterologous expression in Escherichia coli. The solubility of proteins upon expression is dependent on their amino acid sequence. Prediction of solubility from sequence is therefore highly valuable. We present a novel machine-learning-based model called PROSO II which makes use of new classification methods and growth in experimental data to improve coverage and accuracy of solubility predictions. The classification algorithm is organized as a two-layered structure in which the output of a primary Parzen window model for sequence similarity and a logistic regression classifier of amino acid k-mer composition serve as input for a second-level logistic regression classifier. Compared with previously published research our model is trained on five times more data than used by any other method before (82 000 proteins). When tested on a separate holdout set not used at any point of method development our server attained the best results in comparison with other currently available methods: accuracy 75.4%, Matthew’s correlation coefficient 0.39, sensitivity 0.731, specificity 0.759, gain (soluble) 2.263. In summary, due to utilization of cutting edge machine learning technologies combined with the largest currently available experimental data set the PROSO II server constitutes a substantial improvement in protein solubility predictions. PROSO II is available at http://mips.helmholtz-muenchen.de/prosoII.

Posted on 21 May 2012 | 7:21 pm

NMR study of ligand release from asialoglycoprotein receptor under solution conditions in early endosomes

Asialoglycoprotein receptor (ASGP-R) is an endocytic C-type lectin receptor in hepatocytes that clears plasma glycoconjugates containing a terminal galactose or N-acetylgalactosamine. The carbohydrate recognition domain (CRD) of ASGP-R has three Ca2+ binding sites (Sites 1, 2, and 3), with Ca2+ at Site 2 being directly involved in ligand binding. Following endocytosis, the ligands are released from ASGP-R in endosomes to allow receptor recycling to the cell membrane. Although dissociation of the receptor-ligand complex is mediated by the acidic environment within the mature endosomes, many of these complexes also dissociate in early time of endocytosis, where pH is approximately neutral. To investigate the mechanism of ligand release from ASGP-R in early endosomes, we examined the binding mode of Ca2+ and ligands to ASGP-R CRD by NMR. We demonstrate that Sites 1 and 2 of ASGP-R are high affinity Ca2+ binding sites, Site 3 is low affinity, and that Ca2+ ions bind to Sites 1 and 2 cooperatively. The pH and Ca2+ concentration dependences of Ca2+-binding states indicated that early endosome conditions favor apo-ASGP-R CRD, allowing ligand release. Our results elucidated that cooperative binding mode of Ca2+ makes it possible for ASGP-R to be more sensitive to Ca2+ concentrations in early endosomes, and plays an important role in the efficient release of ligand from ASGP-R. In our proposed mechanism, ASGP-R can rapidly release Ca2+ and its ligand even at nearly neutral pH. Sequence comparisons of endocytic C-type lectin receptors suggest that this mechanism is common in their family.

Posted on 21 May 2012 | 3:29 pm

Identification of a potential Tumor Differentiation Factor receptor candidate in prostate cancer cells

Tumor differentiation factor (TDF) is a pituitary protein that is secreted into the blood stream and has an endocrine function. TDF and TDF-P1, a 20 amino acid peptide selected from the open reading frame of TDF, induce differentiation in human breast and prostate cancer cells, but not in other cells. TDF protein has no known mechanism of action. In our recent study, we identified HSP70 proteins as TDF-Rs in breast cancer cells. Therefore, we sought to investigate whether TDF-R candidates from prostate cancer cells are the same as those identified in breast cancer cells. Here, we used TDF-P1 to purify the potential TDF receptor (TDF-R) candidates by affinity purification chromatography (AP) from DU145 and PC3 steroid-resistant prostate cancer cells, LNCaP steroid-responsive prostate cancer cells and from non-prostate NG108 neuroblastoma and BLK CL.4 fibroblast-like cells. We identified the purified proteins by mass spectrometry (MS) and validated them by Western blotting (WB), immunofluorescence microscopy (IF), immunoaffinity purification chromatography (IAP), and structural biology. We identified seven candidate proteins, of which three were from the 70 kDa Heat Shock Protein (HSP70) family. These three proteins were validated as potential TDF-R candidates in LNCaP steroid-responsive- and in DU145 and PC3 steroid-resistant prostate cancer cells, but not in NG108 neuroblastoma and BLK CL.4 fibroblast-like cells. Our previous study and current study suggest that GRP78 and perhaps HSP70 proteins are strong TDF-R candidates and further suggest that TDF interacts with its receptors exclusively in breast and prostate cells inducing cell differentiation through a novel, steroid-independent pathway.

Posted on 21 May 2012 | 3:26 pm

N-Glycosylation promotes the cell surface expression of Kv1.3 potassium channels

The voltage-gated potassium channel Kv1.3 plays an essential role in modulating membrane excitability in many cell types. Kv1.3 is a heavily glycosylated membrane protein. Two successive N-glycosylation consensus sites, N228NS and N229ST, are present on the S1-S2 linker of rat Kv1.3. Our data suggest that Kv1.3 contains only one N-glycan and it is predominantly attached to N229 in the S1-S2 extracellular linker. Preventing N-glycosylation of Kv1.3 significantly decreased its surface protein level and surface conductance density level, which were ?49% and ?46% the level of wild type, respectively. Supplementation of GlcNAc, L-fucose, or Neu5Ac to the culture medium promoted Kv1.3 surface protein expression, whereas supplementation of D-glucose, D-mannose, or D-galactose did not. Among the three effective monosaccharides/derivatives, adding GlcNAc appeared to reduce sialic acid content and increase the degree of branching in the N-glycan of Kv1.3, suggesting that the N-glycan structure and composition have changed. Furthermore, the cell surface half-live of the Kv1.3 surface protein was increased upon GlcNAc supplementation, indicating that it had decreased internalization. The GlcNAc effect appears to apply mainly to membrane proteins containing complex type N-glycans. Thus, N-glycosylation promotes Kv1.3 cell surface expression; supplementation of GlcNAc increased Kv1.3 surface protein level and decreased its internalization, presumably by a combined effect of decreased branch size and increased branching of the N-glycan.

Posted on 21 May 2012 | 3:25 pm

Protein Ser/?Thr phosphatases – the ugly ducklings of cell signalling

This review traces the historical origins and conceptual developments leading to the current state of knowledge of the three superfamilies of protein Ser/Thr phosphatases. ‘PR enzyme’ was identified as an enzyme that inactivates glycogen phosphorylase, although it took 10 years before this ugly duckling was recognized for its true identity as a protein Ser/Thr phosphatase. Ethanol denaturation for purification in the 1970s yielded a phosphatase that exhibited broad specificity, which was resolved into type-1 and type-2 phosphatases in the 1980s. More recent developments show that regulation and specificity are achieved through assembly of multisubunit holoenzymes, transient phosphorylation and the action of inhibitor proteins. Still not widely appreciated, there are hundreds of discrete protein Ser/?Thr phosphatases available to counteract protein kinases, offering potential therapeutic targets. Signalling networks and modelling schemes need to incorporate the full gamut of protein Ser/?Thr phosphatases and their interconnections.

Posted on 21 May 2012 | 1:23 pm

Curcumin inhibits proliferation and invasion of osteosarcoma cells through inactivation of Notch-1 signaling

The Notch signaling pathway plays critical roles in human cancers, including osteosarcoma, suggesting that the discovery of specific agents targeting Notch would be extremely valuable for osteosarcoma. Curcumin, a naturally occurring phenolic compound found in curcuma longa, has been shown to inhibit proliferation and induce apoptosis of osteosarcoma cells in vitro and tumor growth in xenotransplant or orthotransplant models. However, the precise molecular mechanisms by which curcumin exerts its antitumor activity remain unclear. Here we used multiple molecular approaches, such as the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, the invasion assay, gene transfection, real-time RT-PCR, western blot and gelatin zymography, to investigate whether the downregulation of Notch-1 contributes to curcumin-induced inhibition of proliferation and invasion in osteosarcoma cells. The results showed that curcumin caused marked inhibition of osteosarcoma cell growth and G2/M phase cell cycle arrest. This was associated with concomitant attenuation of Notch-1 and downregulation of its downstream genes, such as matrix metalloproteinases, resulting in the inhibition of osteosarcoma cell invasion through Matrigel. We also found that specific downregulation of Notch-1 via small-interfering RNA prior to curcumin treatment resulted in enhanced inhibition of cell growth and invasion. These results suggest that antitumor activity of curcumin is mediated through a novel mechanism involving inactivation of the Notch-1 signaling pathway. Our data provide the first evidence that the downregulation of Notch-1 by curcumin may be an effective approach for the treatment of osteosarcoma.

Posted on 21 May 2012 | 1:19 pm

CcpA forms complexes with CodY and RpoA in Bacillus subtilis

The Bacillus subtilis catabolite control protein A (CcpA) is a global transcriptional regulator that is controlled by interactions with the phosphoproteins histidine-containing protein (HPr)Ser46P and the catabolite responsive HPr (Crh)Ser46P and with low molecular weight effectors, depending on the availability of preferred carbon sources such as glucose. Distinct point mutations in CcpA abolish the regulation of some but not all target genes, suggesting additional interactions of CcpA. Therefore, in vivo crosslinking and MS were applied to identify CcpA complexes active in repression and activation. To compensate for an excess of promoters only repressed by CcpA, this experiment was accomplished with cells using multiple copies of the activated ackA promoter. Among the identified proteins HPr, RNA polymerase subunits and the global regulator transcriptional pleiotropic repressor (CodY) were observed. Bacterial two-hybrid assays combining each RNA polymerase subunit with CcpA localized CcpA binding at the ?-subunit of the RNA polymerase (RpoA). In vivo crosslinking combined with immunoblot analyses revealed CcpA–RpoA complexes in cultures with or without glucose, whereas CcpA–HPr and CcpA–CodY complexes occurred only or predominantly in cultures with glucose. Surface plasmon resonance analyses confirmed the binding of CcpA to the N-terminal domain (?NTD) and C-terminal domain (?CTD) of RpoA, as well as to CodY. Furthermore, interactions of CodY with the ?NTD and the ?CTD were detected by surface plasmon resonance. The KD values of complexes of CcpA or CodY with the ?NTD or the ?CTD are in the range 5–8 ?m. CcpA and CodY form a loose complex with a KD of 60 ?m. These data were combined to propose a model for a transcription initiation complex at the ackA promoter.Structured digital abstract•?A list of the large number of protein-protein interactions described in this article is available via the MINT article ID MINT-8367262

Posted on 21 May 2012 | 1:19 pm

Characterization of the protein ubiquitination response induced by Doxorubicin

Doxorubicin is commonly considered to exert its anti-tumor activity by triggering apoptosis of cancer cells through DNA damage. Recent reports have shown that Doxorubicin elicits a marked heat shock response, and that either inhibition or silencing of heat shock proteins enhance the Doxorubicin apoptotic effect in neuroblastoma cells. In order to investigate whether Doxorubicin may also act through protein modification, we performed a proteomic analysis of ubiquitinated proteins. Here we show that nanomolar Doxorubicin treatment of neuroblastoma cells caused: (a) dose-dependent over-ubiquitination of a specific set of proteins in the absence of measurable inhibition of proteasome, (b) protein ubiquination patterns similar to those with Bortezomib, a proteasome inhibitor, (c) depletion and loss of activity of ubiquitinated enzymes such as lactate dehydrogenase and ?-enolase, and (d) a decrease in HSP27 solubility, probably a consequence of its binding to denatured proteins. These data strongly reinforce the hypothesis that Doxorubicin may also exert its effect by damaging proteins.

Posted on 21 May 2012 | 1:16 pm

Laser-flash photolysis indicates that internal electron transfer is triggered by proton uptake by Alcaligenes xylosoxidans copper-dependent nitrite reductase

Enzyme-catalysed electron transfer reactions are often controlled by protein motions and coupled to chemical change such as proton transfer. We have investigated the nature of this control in the blue copper-dependent nitrite reductase from Alcaligenes xylosoxidans (AxNiR). Inter-Cu electron transfer from the T1Cu site to the T2Cu catalytic site in AxNiR occurs via a proton-coupled electron transfer mechanism. Here we have studied the kinetics of both electron and proton transfer independently using laser-flash photolysis for native AxNiR and its proton-channel mutant N90S. In native AxNiR, both inter-Cu electron transfer and proton transfer exhibit similar rates, and show an unusual dependence on the nitrite concentration. An initial decrease in the observed rates at low nitrite concentrations is followed by an increase in the observed rates at high nitrite concentrations (> 5 mm). In N90S, in which the T1Cu reduction potential is elevated by 60 mV, no inter-Cu electron transfer or proton transfer was observed in the absence of nitrite. Only in the presence of nitrite were both processes detected, with similar [nitrite] dependence, but the nitrite dependence was different compared with native enzyme. The substrate dependence in N90S was similar to that observed in steady-state assays, suggesting that this substitution resulted in proton-coupled electron transfer becoming rate-limiting. A pH perturbation experiment with native AxNiR revealed that protonation triggers inter-Cu electron transfer and generation of NO. Our results show a strong coupling of inter-Cu electron transfer and proton transfer for both native AxNiR and N90S, and provide novel insights into the controlled delivery of electrons and protons to the substrate-utilization T2Cu active site of AxNiR.

Posted on 21 May 2012 | 1:14 pm

Distinct localization of T cell Agrin during antigen presentation – evidence for the expression of Agrin receptor(s) in antigen-presenting cells

Agrin is over-expressed by activated and autoimmune T cells, and synergizes with the T cell receptor (TCR) to augment cell activation. In the present study, we show that Agrin accumulates to distinct areas of the plasma membrane and that cell activation causes its redistribution. During antigen presentation, Agrin primarily accumulates to the periphery of the mature immunological synapse, mostly in lamellipodia-like protrusions that wrap around the antigen-presenting cell and, conversely, anti-Agrin sera induced a significant redistribution of TCR at the plasma membrane. We also provide evidence for the expression of Agrin receptors in peripheral blood monocytes, dendritic cells and a fraction of B cells. Interestingly, interferon-? treatment, which induces the expression of Agrin in T cells, also augmented Agrin binding to monocytes. Stimulation of monocytes with recombinant Agrin induced the clustering of surface receptors, including major histocompatibility complex class II, activation of intracellular signalling cascades, as well as enhanced dsRNA-induced expression of pro-inflammatory cytokines interleukin-6 and tumour necrosis factor-?. Collectively, these results confirm the location of Agrin at the immunological synapse between T cells and antigen-presenting cells and justify further characterization of its receptors in the immune system.

Posted on 21 May 2012 | 1:03 pm

Structural features of the C-terminus from the human neurokinin-1 receptor

The neurokinin-1 receptor (NK1R) is a G-protein coupled receptor found in the central and peripheral nervous systems of vertebrates, and is responsible for many physiological processes. The C-terminus domain seems to be essential for coupling to the corresponding G-protein and ?-arrestin, and is important for receptor desensitization, internalization and recycling. We have focused our study on expression of the human NK1R (hNK1R) C-terminus in Escherichia coli, and its purification and characterization, in order to elucidate its structural properties. CD and Fourier transform infrared spectroscopy showed that the hNK1R C-terminus, rather than having a random structure, has well-defined secondary-structure patterns. The presence of three tyrosine residues in the primary sequence of the hNK1R C-terminus facilitated the use of UV and fluorescence spectroscopy techniques which revealed tyrosine fluorescence and UV absorption at anomalous wavelengths. In their entirety, the results show that the hNK1R C-terminus has clearly defined secondary (25%?-helix, 27% unordered structure and 48%?-sheets and ?-turns) and tertiary structures which, it is believed, are tightly related to its multiple functions.

Posted on 21 May 2012 | 1:03 pm

The evolutionary relationship of the transcriptionally active fabp11a (intronless) and fabp11b genes of medaka with fabp11 genes of other teleost fishes

Here we describe the structure of the fatty acid-binding protein 11a and 11b genes (fabp11a and fabp11b) in medaka, and their evolutionary relationship to fabp11 genes from other teleost fishes. Initial studies indicated that the medaka fabp11a gene is intronless, but the fabp11b gene consists of four exons separated by three introns, a genomic organization that is characteristic of most members of the intracellular lipid-binding protein family. Based on genomic sequence, we conclude that the intronless fabp11a gene most likely arose as a result of reverse transcription of its mRNA transcript into cDNA followed by integration into chromosomal DNA. The ancestral intron-containing fabp11a gene was presumably lost from the medaka genome. The duplicated fabp11 genes extant in medaka encode polypeptides of 134 amino acids, which share highest sequence identity and similarity, and cluster in a distinct phylogenetic clade, with their orthologs in other teleost fishes. The fabp11a and fabp11b genes in medaka are therefore orthologs of the fabp11a and fabp11b genes, respectively, of other teleost fishes. No conserved gene synteny was found between medaka fabp11a and fabp11a genes from other teleost fishes, supporting our suggestion as to how this intronless gene arose. However, conserved gene synteny was evident between medaka fabp11b and fabp11b genes from other teleost fishes. The tissue-specific distribution of transcripts for medaka and zebrafish fabp11a and fabp11b genes revealed acquisition of a new function(s) in various tissues by the medaka fabp11b gene, which may explain the retention of sister duplicates of fabp11 in the medaka genome.

Posted on 21 May 2012 | 1:00 pm

Human HAD Phosphatases: Structure, Mechanism, and Roles in Health and Disease

Phosphatases of the haloacid dehalogenase (HAD) superfamily of hydrolases are an ancient and very large class of enzymes that have evolved to dephosphorylate a wide range of low- and high molecular weight substrates with often exquisite specificities. HAD phosphatases constitute approximately one fifth of all human phosphatase catalytic subunits. While the overall sequence similarity between HAD phosphatases is generally very low, family members can be identified based on the presence of a characteristic Rossmann-like fold and the active site sequence DxDx(V/T). HAD phosphatases employ an aspartate residue as a nucleophile in a magnesium-dependent phosphoaspartyl transferase reaction. Although there is genetic evidence demonstrating a causal involvement of some HAD phosphatases in diseases such as cancer, cardiovascular, metabolic and neurological disorders, the physiological roles of many of these enzymes are still poorly understood. In this review, we discuss the structure and evolution of human HAD phosphatases, and summarize their known functions in health and disease.

Posted on 21 May 2012 | 9:28 am

Tight-binding inhibitors efficiently inactivate both reaction centers of monomeric Plasmodium falciparum glyoxalase 1

Glucose consumption and therefore methylglyoxal production of human erythrocytes increase significantly upon infection with malaria parasites. The glyoxalase systems of the host-parasite unit cope with this metabolic challenge by catalyzing the removal of harmful methylglyoxal. Thus, glyoxalase 1 from the malaria parasite Plasmodium falciparum (PfGlo1) could be a promising drug target. However, the enzyme has two different active sites and their simultaneous inactivation has been considered to be a challenging task. Here we describe the inactivation of PfGlo1 by two glyoxalase-specific tight-binding inhibitors with nanomolar Kiapp values and non-competitive inhibition patterns. The inhibitors do not discriminate between the high-affinity and the high-activity conformations of PfGlo1 but seem to stabilize or trigger a conformational change in analogy with the substrate. In summary, we have characterized the most potent inhibitors of PfGlo1 known to date.

Posted on 19 May 2012 | 5:45 pm

Sustained glycolytic oscillations in individual, isolated yeast cells

Yeast glycolytic oscillations have been studied since the 1950s in cell free extracts and in intact cells. Thus far, for intact cells sustained oscillations have only been observed at the population level, i.e. for synchronized cultures at high biomass concentrations. Using optical tweezers to position yeast cells in a microuidic chamber, we were able to observe sustained oscillations in individual, isolated cells. With a detailed kinetic model for the cellular reactions we could simulate the heterogeneity in the response of the individual cells assuming small di_erences in a single internal parameter. This is the _rst time that sustained limit cycle oscillations are shown in isolated yeast cells.

Posted on 18 May 2012 | 6:00 pm

Targeted delivery into motor nerve terminals of inhibitors for SNARE-cleaving proteases via liposomes coupled to an atoxic botulinum neurotoxin

A targeted drug carrier (TDC) is described for transferring functional proteins or peptides into motor nerve terminals, a pivotal locus for therapeutics to treat neuromuscular disorders. It exploits the pronounced selectivity of botulinum neurotoxin type B (BoNT/B) for interacting with acceptors on these cholinergic nerve endings and becoming internalized. The gene encoding an innocuous BoNT/B protease-inactive mutant (BoTIM) was fused to that for core streptavidin, expressed in E. coli and the purified protein conjugated to surface-biotinylated liposomes. Such decorated liposomes, loaded with fluorescein as traceable cargo, acquired pronounced specificity for motor nerve terminals in isolated mouse hemi-diaphragms and facilitated the intra-neuronal transfer of the fluor, as revealed by confocal microscopy. Delivery of the protease light chain of BoNT/A via this TDC accelerated the onset of neuromuscular paralysis, indicative of improved translocation of this enzyme into the pre-synaptic cytosol with subsequent proteolytic inactivation of SNAP-25, an exocytotic SNARE protein essential for neurotransmitter release. BoTIM-coupled liposomes loaded with peptide inhibitors of proteases yielded considerable attenuation of the neuroparalytic effects of BoNT/A or /F due to their cytosolic transfer, the first in situ demonstration of the ability of designer anti-proteases to suppress the symptoms of botulism ex-vivo. Delivery of the /A inhibitor by liposomes targeted with the full-length BoTIM proved more effective than that mediated by its C-terminal neuro-acceptor binding domain. This demonstrated versatility of TDC for non-viral cargo transfer into cholinergic nerve endings has unveiled its potential for direct delivery of functional targets into motor nerve endings.

Posted on 18 May 2012 | 6:00 pm

Tetramerisation of human guanylate binding protein 1 is mediated by coiled coil formation of the C-terminal ?-helices

The human guanylate binding protein 1 (hGBP1) is a large GTP binding protein belonging to the dynamin family a common feature of which is nucleotide dependent assembly to homotypic oligomers. Assembly leads to stimulation of GTPase activity which in the case of dynamin is responsible for scission of vesicles from membranes. By yeast two hybrid and biochemical experiments we address intermolecular interactions between all subdomains of hGBP1 and identify the C-terminal subdomain, ?12/13, as a new interaction site for self-assembly. ?12/13 represents a stable subdomain of hGBP1 as shown by circular dichroism spectroscopy. In addition to contacts between GTPase domains, leading to dimer formation, the interaction between two ?12/13 subdomains in the course of GTP hydrolysis, results in tetramer formation of the protein. With the help of circular dichroism spectroscopy we can show coiled coil formation of two ?12/13 subdomains and concentration dependent measurements allow estimating a value for the dissociation constant of 7.3 ?M. We suggest GTP hydrolysis driven release of the ?12/13 subdomain making it available for coiled coil formation. Furthermore, we can demonstrate the biological relevance of hGBP1 tetramer formation in living cells by chemical crosslink experiments.

Posted on 18 May 2012 | 5:59 pm

Disulfide bonds: protein folding and subcellular protein trafficking

The study of disulfide-bond-containing proteins has permitted an advance in our understanding of the mechanism(s) by which a majority of secretory and membrane-bound proteins acquire their biologically functional folded forms. This covalent linkage has been exploited by a number of research laboratories to harness or trap intermediates populating the folding trajectories of biopolymers. The resulting body of gathered in vitro data demonstrates, than in general, there is a common event underscoring the maturation of disulfide-bond-containing proteins. This commonality is the existence of a competition between a physical, conformational folding reaction and, a chemical, thiol-disulfide exchange reaction during fold acquisition. The competition, in turn, impacts the fate of the polypeptide in that of it being secreted or retrotranslocated. The role of a host of subcellular factors including protein disulfide isomerase that influences this critical spatio-temporal juncture of the fold maturation process is discussed. Finally, the impact of this competition on the onset of neurodegenerative disorders is elaborated upon.

Posted on 17 May 2012 | 7:40 pm

Exploring the sequence-function relationship in transcriptional regulation by the lac O1 operator

Understanding how the binding of a transcription factor (TF) to an operator is influenced by the operator sequence is an ongoing quest. It facilitates discovery of alternate binding sites as well as tuning of transcriptional regulation. We investigated the behavior of the Escherichia coli Lac repressor (LacI) protein with a large set of lac O1 operator variants. The 114 variants examined contained an average of 2.9 (range = 0 to 4) mutations at the positions -4, -2, +2, and +4 in the minimally required 17 bp operator. The relative affinity of LacI for the operators was examined by quantifying the expression of a gfp reporter gene and by Rosetta structural modeling. The combinations of mutations in the operator sequence created a wide range of regulatory behaviors. We observed more than an order of magnitude variations in GFP fluorescent signal among the operator variants under both the uninduced and induced conditions. We found that a single nucleotide change can result in as much as six- and 12-fold changes in uninduced and induced GFP signals, respectively. Among the four positions mutated, we found that a nucleotide G at position -4 is strongly correlated with strong repression. In Rosetta modeling, we found significant correlation between the calculated binding energy and the experimentally observed transcription repression strength for many operators. However, exceptions were also observed, underscoring the necessity for further improvement in biophysical models of protein-DNA interactions.

Posted on 17 May 2012 | 7:38 pm

Subcellular localization and role of Ran1 in Tetrahymena thermophila amitotic macronucleus

Amitosis, a direct method of cell division is common in ciliated protozoan, fungi and some animal and plant cells. During amitosis, intranuclear microtubules are reorganized into specified arrays which assist in separation of nucleus, despite lack of a bipolar spindle. However, the regulation of amitosis is not understood. Here, we focused on the localization and role of mitotic spindle assembly regulator: Ran GTPase (Ran1) in macronuclear amitosis in binucleated protozoan Tetrahymena thermophila. HA-tagged Ran1 was localized in the macronucleus throughout the cell cycle of Tetrahymena during vegetative growth, and the accessory factor binding domains of Ran1 contributed to its macronuclear localization. Incomplete somatic knockout of RAN1 resulted in aberrant intramacronuclear microtubule array formation, missegregation of macronuclear chromosomes and ultimately blocked macronuclei proliferation. When the Ran1 cycle was perturbed by overexpression of Ran1T25N (GDP-bound Ran1-mimetic) or Ran1Q70L (GTP-bound Ran1-mimetic), intramacronuclear microtubule assembly was inhibited or multi-micronucleate cells formed. These results suggest that Ran GTPase pathway is involved in assembly of a specialized intramacronuclear microtubule network and coordinates amitotic progression in Tetrahymena.

Posted on 17 May 2012 | 7:37 pm

Significant stabilization of ribonuclease A by additive effects

Among the strategies that employ genetic engineering to stabilize proteins, the introduction of disulfide bonds has proven to be a very potential approach. As, however, the replacement of amino acid residues by cysteines and the subsequent formation of the covalent bond can result in a severe deformation of the parental protein structure, the stabilization effect is strongly context dependent. Alternatively, the introduction of charged amino acid residues at the surface, which may result in the formation of extra ionic interactions or hydrogen bonds, provide propitious means for protein stabilization. The generation of an extra disulfide bond between residues 4 and 118 in ribonuclease A had resulted in a stabilization by 6 °C or 7 kJ mol-1, which was mainly caused by a deceleration of the unfolding reaction [Pecher, P. & Arnold, U. (2009) Biophys. Chem., 141, 21-28]. Here, Asp83 was replaced by Glu resulting in a comparable stabilization. Moreover, combination of both mutations led to an additive effect and the resulting ribonuclease A variant (Tm ? 76°C, ?G°? 53 kJ mol-1) is the most stable ribonuclease A variant described so far. The analysis of the crystal structure of A4C/D83E/V118C-ribonuclease A reveals the formation of a salt bridge between the ?-carboxyl group of Glu83 and the ?-amino group of Lys104.

Posted on 17 May 2012 | 7:32 pm

Author index

Posted on 15 May 2012 | 3:05 pm

A highly conserved tryptophan in the N-terminal variable domain regulates disulfide bond formation and oligomeric assembly of adiponectin

Adiponectin is a collageneous adipokine with direct anti-diabetic and anti-atherogenic properties. It can assume an ensemble of oligomeric states e.g. trimers, hexamers and octadecamers, each being involved in distinct signaling pathways relevant to adiponectin’s diverse biological function in metabolism, immunity, inflammation and cellular homeostasis. Assembly of the active variants principally the octadecameric high-molecualr weight (HMW) form is achieved via the tightly controlled oxidation of cysteine 39 located in the adiponectin hyper-variable domain (AHD, residues 18 to 44) between the signal sequence and the collagen-like domain. We show that mutation of a highly conserved tryptophan (W42A) in AHD profoundly affects assembly by trapping full-length adiponectin in the oxidized trimeric or hexameric states with a concomitant major reduction in the HMW form. Our biophysical measurements on synthesized analogues of the AHD, suggests that the aberrant oligomer distribution can be explained based on the fact that the proximity of W42 to C39 causes a reduction in the rate of C39 oxidation, an effect that to our knowledge has not been documented before. At the biological level, the perturbed oligomer distribution of full-length mutant adiponectin leads to a major reduction in the AMPK activation in endothelial cells and liver tissues.

Posted on 14 May 2012 | 11:02 pm

N-terminal phosphorylation of PP2A/B?2 regulates translocation to mitochondria, Drp1 dephosphorylation, and neuronal survival

The neuron-specific B?2 regulatory subunit of protein phosphatase 2A (PP2A), a product of the spinocerebellar ataxia type 12 disease gene PPP2R2B, recruits heterotrimeric PP2A to the outer mitochondrial membrane (OMM) through its N-terminal mitochondrial targeting sequence. OMM-localized PP2A/B?2 induces mitochondrial fragmentation, thereby increasing susceptibility to neuronal insults. Here, we report that PP2A/B?2 activates the mitochondrial fission enzyme dynamin-related protein 1 (Drp1) by dephosphorylating Ser656, a highly conserved inhibitory phosphorylation site targeted by the neuroprotective PKA/AKAP1 kinase complex. We further show that translocation of PP2A/B?2 to mitochondria is regulated by phosphorylation of B?2 at three N-terminal Ser residues. Phosphomimetic substitution of Ser20-22 renders B?2 cytosolic, blocks Drp1 dephosphorylation and mitochondrial fragmentation, and abolishes the ability of B?2 overexpression to induce apoptosis in cultured hippocampal neurons. Ala substitution of Ser20-22 to prevent phosphorylation has the opposite effect, promoting association of B?2 with mitochondria, Drp1 dephosphorylation, mitochondrial fission, and neuronal death. OMM translocation of B?2 can be attenuated by mutation of residues in close proximity to the catalytic site, but only if Ser20-22 are available for phosphorylation, suggesting that PP2A/B?2 autodephosphorylation is necessary for OMM association, likely by uncovering the net positive charge of the mitochondrial targeting sequence. These results reveal another layer of complexity in the regulation of the mitochondrial fission/fusion equilibrium and its physiological and pathophysiological consequences in the nervous system.

Posted on 14 May 2012 | 11:02 pm

New insights into red-ox response modulation in Fanconi’s anemia cells by hydrogen peroxide and glutathione depletors.

Fanconi’s anemia (FA) patients face severe pathological consequences. Bone marrow (BM) failure, the major cause of death in FA accounting for as much as 80-90% of FA mortality, appear significantly linked to excessive apoptosis of hematopoietic cells induced by oxidative stress. However as 20-25% of the FA patients develop malignancies of myeloid origin. A surviving strategy for bone marrow and hematopoietic cells under selective pressure has evidently taken place. This study reports how lymphoblastoid cell lines derived from two FA patients display significant resistance to oxidative stress induced by treatments with H2O2 and different glutathione (GSH) inhibitors which induce ROS, GSH depletion and mitochondrial membrane depolarization (MMD). Among the various GSH inhibitors employed FA cells appear peculiarly resistant to Menadione (5 ?M) and Ethacrynic acid (ETA, 50 ?M), two drugs which specifically target mitochondria. Even after pre-treatments with Buthionine Sulfoximine (BSO), a GSH synthesis inhibitor which induces enhanced ROS induction, FA cells maintain significant resistance to these stressors. These data suggest that the resistance to oxidative stress and the altered mitochondrial and metabolic functionality found in the FA mutant cells used in this study may define a survival strategy adopted in FA cells undergoing transformation. The study of red-ox and mitochondria regulation in FA may be of help in the diagnosis of the disease and in the care of the patients.

Posted on 12 May 2012 | 5:35 pm

Disulfide-coupled protein folding: Looking back, Looking forward

A half century has passed since Anfinsen reported the first refolding study of ribonuclease A and established the central dogma of protein folding, namely ‘the amino acid sequence of a protein contains all the information required for the protein to fold into the native tertiary structure’.

Posted on 12 May 2012 | 5:33 pm

Laforin is Required for the Functional Activation of Malin in Endoplasmic Reticulum Stress Resistance in Neuronal Cells

Mutations in either EPM2A, the gene encoding a dual-specificity phosphatase named laforin, or NHLRC1, the gene encoding an E3 ubiquitin ligase named malin, cause Lafora disease (LD) in humans. LD is a fatal neurological disorder characterized by progressive myoclonus epilepsy, severe neurological deterioration, and accumulation of poorly branched glycogen inclusions, called Lafora bodies (LBs) or polyglucosan bodies (PGBs), within the cell cytoplasm. The molecular mechanism underlying the neuropathogenesis of LD remains unknown. Here we present data demonstrating that in the cells expressing low levels of laforin protein, overexpressed malin and its LD-causing missense mutants are stably polyubiquitinated. Malin and malin mutants form ubiquitin-positive aggregates in or around the nuclei of the cells in which they are expressed. Neither wild type (WT) malin nor its mutants elicit endoplasmic reticulum (ER) stress, although the mutants exaggerate the response to ER stress. Overexpressed laforin impairs the polyubiquitination of malin and recruits malin to PGBs. The recruitment and activities of laforin and malin are both required for the PGB disruption. Consistently, targeted deletion of laforin in brain cells from Epm2a knockout (KO) mice increases polyubiquitinated proteins. Knockdown of Epm2a or Nhlrc1 in neuronal Neuro2a cells shows that they cooperate to allow cells to resist ER stress and apoptosis. These results reveal that a functional laforin-malin complex plays a critical role in destroying LB and relieving ER stress, implying that a causative pathogenic mechanism underlies their deficiency in LD.

Posted on 12 May 2012 | 5:33 pm

Methods to monitor classical protein-tyrosine phosphatase oxidation

Reactive oxygen species (ROS), particularly H2O2, act as intracellular second messengers in many signaling pathways. Protein-tyrosine phosphatases (PTPs) are now believed to be important targets of ROS. PTPs contain a conserved catalytic cysteine with an unusually low pKa. This property allows PTPs to execute nucleophilic attack on substrate phosphotyrosyl residues, but also renders them highly susceptible to oxidation. Reversible oxidation, which inactivates PTPs, is emerging as an important cellular regulatory mechanism and might contribute to human diseases, including cancer. Given their potential toxicity, it seems likely that ROS generation is highly controlled within cells to restrict oxidation to those PTPs that must be inactivated for signaling to proceed. Thus, identifying ROS-inactivated PTPs could be tantamount to finding the PTP(s) that critically regulate a specific signaling pathway. This article provides an overview of the methods currently available to identify and quantify PTP oxidation and outlines future challenges in redox signaling.

Posted on 12 May 2012 | 5:32 pm

Bivalent binding drives the formation of the Grb2–Gab1 signaling complex in a noncooperative manner

Although the growth factor receptor binder 2 (Grb2)–Grb2-associated binder (Gab)1 macromolecular complex mediates a multitude of cellular signaling cascades, the molecular basis of its assembly has hitherto remained largely elusive. Herein, using an array of biophysical techniques, we show that, whereas Grb2 exists in a monomer–dimer equilibrium, the proline-rich (PR) domain of Gab1 is a monomer in solution. Of particular interest is the observation that although the PR domain appears to be structurally disordered, it nonetheless adopts a more or less compact conformation reminiscent of natively folded globular proteins. Importantly, the structurally flexible conformation of the PR domain appears to facilitate the binding of Gab1 to Grb2 with a 1 : 2 stoichiometry. More specifically, the formation of the Grb2–Gab1 signaling complex is driven via a bivalent interaction through the binding of the C-terminal homology 3 (cSH3) domain within each monomer of Grb2 homodimer to two distinct RXXK motifs, herein designated G1 and G2, located within the PR domain of Gab1. Strikingly, in spite of the key role of bivalency in driving this macromolecular assembly, the cSH3 domains bind to the G1 and G2 motifs in an independent manner with zero cooperativity. Taken together, our findings shed new light on the physicochemical forces driving the assembly of a key macromolecular signaling complex that is relevant to cellular health and disease.Structured digital abstract•?GRB2and GRB2bind by light scattering (View interaction)•?GAB1and GRB2bind by isothermal titration calorimetry (View Interaction: 1, 2, 3)

Posted on 9 May 2012 | 12:17 pm

Functional characterization of UvrD helicases from Haemophilus influenzae and Helicobacter pylori

Haemophilus influenzae and Helicobacter pylori are major bacterial pathogens that face high levels of genotoxic stress within their host. UvrD, a ubiquitous bacterial helicase that plays important roles in multiple DNA metabolic pathways, is essential for genome stability and might, therefore, be crucial in bacterial physiology and pathogenesis. In this study, the functional characterization of UvrD helicase from Haemophilus influenzae and Helicobacter pylori is reported. UvrD from Haemophilus influenzae (HiUvrD) and Helicobacter pylori (HpUvrD) exhibit strong single-stranded DNA-specific ATPase and 3?–5? helicase activities. Mutation of highly conserved arginine (R288) in HiUvrD and glutamate (E206) in HpUvrD abrogated their activities. Both the proteins were able to bind and unwind a variety of DNA structures including duplexes with strand discontinuities and branches, three- and four-way junctions that underpin their role in DNA replication, repair and recombination. HiUvrD required a minimum of 12 nucleotides, whereas HpUvrD preferred 20 or more nucleotides of 3?-single-stranded DNA tail for efficient unwinding of duplex DNA. Interestingly, HpUvrD was able to hydrolyze and utilize GTP for its helicase activity although not as effectively as ATP, which has not been reported to date for UvrD characterized from other organisms. HiUvrD and HpUvrD were found to exist predominantly as monomers in solution together with multimeric forms. Noticeably, deletion of distal C-terminal 48 amino acid residues disrupted the oligomerization of HiUvrD, whereas deletion of 63 amino acids from C-terminus of HpUvrD had no effect on its oligomerization. This study presents the characteristic features and comparative analysis of Haemophilus influenzae and Helicobacter pylori UvrD, and constitutes the basis for understanding the role of UvrD in the biology and virulence of these pathogens.Structured digital abstract•?HiUvrD and HiUvrD interact by molecular sieving (View interaction)•?HpUvrD and HpUvrD interact by molecular sieving (View interaction)•?HiUvrD and HiUvrD interact by cross-linking study (View interaction)•?HpUvrD and HpUvrD interact by cross-linking study (View interaction)

Posted on 9 May 2012 | 12:16 pm

Adenosine A2A receptor activation and hyaluronan fragment inhibition reduce inflammation in mouse articular chondrocytes stimulated with interleukin-1?

Small hyaluronan (HA) fragments produced from native HA during inflammation contribute greatly to cell injury in many pathologies. HA oligosaccharides increase proinflammatory cytokine levels by activating both CD44 and toll-like receptor (TLR)-4. Stimulation of CD44 and TLR-4 then activates nuclear factor-?B, which induces the production of proinflammatory cytokines. The adenosine 2A receptor (A2AR) is also involved in several inflammation pathologies, and the nucleoside adenosine acts as a potent endogenous inhibitor of inflammation in various tissues by interacting with this receptor. The aim of this study was to investigate the effects of an HA-blocking peptide that inhibits the proinflammatory action of HA oligosaccharides produced during inflammation, together with a specific A2AR agonist in a model of normal mouse articular chondrocytes stimulated with interleukin (IL)-1?. IL-1? stimulation significantly increased mRNA expression and the related protein production of TLR-4, TLR-2, CD44 and A2AR in articular chondrocytes. The induced nuclear factor-?B activation was also associated with increased levels of inflammatory cytokines, including tumor necrosis factor-? and IL-6, and other inflammatory mediators, such as matrix metalloprotease-13 and inducible nitric oxide synthase. Treatment of chondrocytes with the HA-blocking peptide Pep-1 and/or a specific A2AR agonist (CGS-21680) significantly reduced all of the inflammatory parameters upregulated by IL-1?. These results suggest that the inflammatory response may be reduced either by blocking oligosaccharides from HA degradation or by A2AR stimulation.

Posted on 9 May 2012 | 12:13 pm

A S-adenosylmethionine methyltransferase-like domain within the essential, Fe-S-containing yeast protein Dre2

Yeast Dre2 is an essential Fe-S cluster-containing protein that has been implicated in cytosolic Fe-S protein biogenesis and in cell death regulation in response to oxidative stress. Its absence in yeast can be complemented by the human homologous antiapoptotic protein cytokine-induced apoptosis inhibitor 1 (also known as anamorsin), suggesting at least one common function. Using complementary techniques, we have investigated the biochemical and biophysical properties of Dre2. We show that it contains an N-terminal domain whose structure in solution consists of a stable well-structured monomer with an overall typical S-adenosylmethionine methyltransferase fold lacking two ?-helices and a ?-strand. The highly conserved C-terminus of Dre2, containing two Fe-S clusters, influences the flexibility of the N-terminal domain. We discuss the hypotheses that the activity of the N-terminal domain could be modulated by the redox activity of Fe-S clusters containing the C-terminus domain in vivo.Database Structural data have been deposited in the Protein Data Bank under accession number 2KM1.

Posted on 9 May 2012 | 12:13 pm

BioV Suite – a collection of programs for the study of transport protein evolution

The Bio-V Suite is a collection of Python scripts designed specifically for bioinformatic research regarding transport protein evolution. The Bio-V Suite contains nine powerful programs for Unix-based environments, each of which can be run as a standalone tool or be accessed in a programmatic manner. These programs and their functions are as follows. The transmembrane alpha-helical statistical prediction tool (TMStats) generates topological statistics for transport proteins. The Global Sequence Alignment Tool (GSAT) performs shuffle-based binary alignments and is fully scalable. It can cross-compare two FASTA files or individual sequences. Protocol1 performs remote PSI-Blast searches and filters redundant/similar sequences and annotates them. Protocol2 finds homologues between FASTA lists and generates graphical reports. The Targeted Smith–Waterman Search (TSSearch) uses a rapid search algorithm to find distant homologues in FASTA files in a heuristic manner. SSearch is the exhaustive version of TSSearch. Genome-Blast (GBlast) will identify potential transport proteins in any genome/proteome file or find similar transport protein homologues between two different genomes/proteomes before generating a graphical report. AncientRep (AR) will find putative transmembrane repeat units using a list of homologues. DefineFamily (DF) will generate a FASTA list to represent an entire Transporter Classification family. These nine programs are tabulated with descriptions of their capabilities in Table 1.1 ?Programs developed for studies of transport protein evolution but useful for the analysis of other protein (and nucleic acid) classes. ProgramTask performedTMStatsCreates graphical charts to represent topological statistics within any TC hierarchy or FASTA fileGSATProvides a simple, shuffle-based global alignment tool for detecting distant homologiesProtocol1Performs remote PSI-BLAST searches with iterations using the NCBI NR databaseProtocol2Compares two different families of transport proteins to find similarities. It uses TSSearch and SSearch and generates easy-to-read graphical reportsTSSearchUses a heuristic local search algorithm for rapid comparison of two different FASTA files and find similarities between themSSearchUses a simple, exhaustive shuffle-based local search algorithm to compare two different FASTA files and find their similaritiesGBlastProvides a search tool designed to identify potential transporters within entire genomes/proteomes. It generates easy-to-read graphical reportsAncientRepProvides a horizontal or vertical search approach to find transmembrane repeat units within a list of homologuesDefineFamilyGenerates a FASTA list to represent any TC familyThe Bio-V Suite is a collection of python scripts designed specifically for bioinformatic research regarding transport protein evolution. The Bio-V Suite contains nine powerful programs for Unix-based environments, each of which can be run as a standalone tool or be accessed in a programmatic fashion.

Posted on 8 May 2012 | 2:58 pm

The major facilitator superfamily (MFS) revisited

The major facilitator superfamily (MFS) is the largest known superfamily of secondary carriers found in the biosphere. It is ubiquitously distributed throughout virtually all currently recognized organismal phyla. This superfamily currently (2012) consists of 74 families, each of which is usually concerned with the transport of a certain type of substrate. Many of these families, defined phylogenetically, do not include even a single member that is functionally characterized. In this article, we probe the evolutionary origins of these transporters, providing evidence that they arose from a single 2-transmembrane segment (TMS) hairpin structure that triplicated to give a 6-TMS unit that duplicated to a 12-TMS protein, the most frequent topological type of these permeases. We globally examine MFS protein topologies, focusing on exceptional proteins that deviate from the norm. Nine distantly related families appear to have members with 14 TMSs in which the extra two are usually centrally localized between the two 6-TMS repeat units. They probably have arisen by intragenic duplication of an adjacent hairpin. This alternative topology probably arose multiple times during MFS evolution. Convincing evidence for MFS permeases with fewer than 12 TMSs was not forthcoming, leading to the suggestion that all 12 TMSs are required for optimal function. Some homologs appear to have 13, 14, 15 or 16 TMSs, and the probable locations of the extra TMSs were identified. A few MFS permeases are fused to other functional domains or are fully duplicated to give 24-TMS proteins with dual functions. Finally, the MFS families with no known function were subjected to genomic context analyses leading to functional predictions.In this article we probe the evolutionary origins of the major facilitator superfamily transporters, providing evidence that they arose from a single 2 TMS hairpin structure that triplicated to give a 6 TMS unit that duplicated to a 12 TMS protein, the most frequent topological type of these permeases.

Posted on 8 May 2012 | 2:58 pm

Effect of positive feedback loops on the robustness of oscillations in the network of cyclin-dependent kinases driving the mammalian cell cycle

The transitions between the G1, S, G2 and M phases of the mammalian cell cycle are driven by a network of cyclin-dependent kinases (Cdks), whose sequential activation is regulated by intertwined negative and positive feedback loops. We previously proposed a detailed computational model for the Cdk network, and showed that this network is capable of temporal self-organization in the form of sustained oscillations, which govern ordered progression through the successive phases of the cell cycle [Gérard and Goldbeter (2009) Proc Natl Acad Sci USA106, 21643–21648]. We subsequently proposed a skeleton model for the cell cycle that retains the core regulatory mechanisms of the detailed model [Gérard and Goldbeter (2011) Interface Focus1, 24–35]. Here we extend this skeleton model by incorporating Cdk regulation through phosphorylation/dephosphorylation and by including the positive feedback loops that underlie the dynamics of the G1/S and G2/M transitions via phosphatase Cdc25 and via phosphatase Cdc25 and kinase Wee1, respectively. We determine the effects of these positive feedback loops and ultrasensitivity in phosphorylation/dephosphorylation on the dynamics of the Cdk network. The multiplicity of positive feedback loops as well as the existence of ultrasensitivity promote the occurrence of bistability and increase the amplitude of the oscillations in the various cyclin/Cdk complexes. By resorting to stochastic simulations, we further show that the presence of multiple, redundant positive feedback loops in the G2/M transition of the cell cycle markedly enhances the robustness of the Cdk oscillations with respect to molecular noise.

Posted on 8 May 2012 | 2:57 pm

Functional analysis of the Notch ligand Jagged1 missense mutant proteins underlying Alagille syndrome

Heterozygous mutations in the JAG1 gene, encoding Notch ligand Jagged1, cause Alagille syndrome (ALGS). As most of the mutations are nonsense or frameshift mutations producing inactive truncated proteins, haplo-insufficiency is considered the major pathogenic mechanism of ALGS. However, the molecular mechanisms by which the missense mutations cause ALGS remain unclear. Here we analyzed the functional properties of four ALGS missense mutant proteins, P163L, R184H, G386R and C714Y, using transfected mammalian cells. P163L and R184H showed Notch-binding activities similar to that of the wild-type when assessed by immunoprecipitation. However, their trans-activation and cis-inhibition activities were almost completely impaired. These mutant proteins localized mainly to the endoplasmic reticulum (ER), suggesting that the mutations induced improper protein folding. Furthermore, the mutant proteins bound more strongly to the ER chaperone proteins calnexin and calreticulin than the wild-type did. C714Y also localized to the ER, but possessed significant trans-activation activity and lacked enhanced binding to the chaperones, indicating a less severe phenotype. The properties of G386R were the same as those of the wild-type. Dominant-negative effects were not detected for any mutant protein. These results indicate that accumulation in the ER and binding to the chaperones correlate with the impaired signal-transduction activities of the missense mutant proteins, which may contribute to the pathogenic mechanism of ALGS. Our findings, which suggest the requirement for cell-surface localization of Jagged1 for cis-inhibition activities, also provide important information for understanding the molecular basis of Notch-signaling pathways.Structured digital abstract•?Jagged-1physically interacts with Calreticulin and Calnexin by anti tag coimmunoprecipitation (View Interaction: 1, 2)•?Jagged-1physically interacts with NOTCH3 by anti tag coimmunoprecipitation (View interaction)

Posted on 8 May 2012 | 2:42 pm

HSP60 is transported through the secretory pathway of 3-MCA-induced fibrosarcoma tumour cells and undergoes N-glycosylation

There is increasing evidence localizes the mitochondrial chaperone heat shock protein (HSP)60, outside the cell, where it mediates interactions between immune cells and other body tissues. However, the mechanisms by which HSP60 is secreted into the extracellular environment are not fully understood. Recent studies have shown that HSP60 is actively released by a nonconventional secretion mechanism, the lipid raft–exosome pathway. In the present study, we show for the first time that HSP60, produced by 3-methylcholantrene-induced fibrosarcoma tumour cells, is secreted through the conventional endoplasmic reticulum–Golgi secretory pathway. Confocal microscopy using anti-TGN38 and anti-HSP60 antibodies together with monensin, a Golgi transport inhibitor, demonstrated the relocation of HSP60 to the Golgi of malignant cells but not primary fibroblast cells subjected to heat shock or fibroblast cell lines. Transmission electron microscopy, flow cytometry and cell fractionation of cell treated with brefeldin A, an inhibitor of endoplasmic reticulum to Golgi protein transport, further indicated that HSP60 is present both in the endoplasmic reticulum and the Golgi complex of malignant cells. We found a single mRNA with a mitochondrial targeting sequence encoding for HSP60 in the malignant cells but two HSP60 translation products, namely the native unmodified protein and a protein post-translationally modified by N-glycosylation. The N-glycans observed were composed of high-mannose structures and bi-, tri- and tetra-antennary complex type structures occupying sites of the three potential glycosylation sites present on HSP60. Accordingly, we propose that HSP60 in malignant cells is transported through the endoplasmic reticulum–Golgi secretion pathway, where it acquires N-glycans, and thus can affect the immunological properties of the proteins in the tumour microenvironment.Structured digital abstract•?Grp94 and Hsp60 colocalize by cosedimentation through density gradient (View interaction)

Posted on 8 May 2012 | 2:42 pm

The membrane anchor of penicillin-binding protein PBP2a from Streptococcus pneumoniae influences peptidoglycan chain length

The pneumococcus is an important Gram-positive pathogen, which shows increasing resistance to antibiotics, including ?-lactams that target peptidoglycan assembly. Understanding cell-wall synthesis, at the molecular and cellular level, is essential for the prospect of combating drug resistance. As a first step towards reconstituting pneumococcal cell-wall assembly in vitro, we present the characterization of the glycosyltransferase activity of penicillin-binding protein (PBP)2a from Streptococcus pneumoniae. Recombinant full-length membrane-anchored PBP2a was purified by ion-exchange chromatography. The glycosyltransferase activity of this enzyme was found to differ from that of a truncated periplasmic form. The full-length protein with its cytoplasmic and transmembrane segment synthesizes longer glycan chains than the shorter form. The transpeptidase active site was functional, as shown by its reactivity towards bocillin and the catalysis of the hydrolysis of a thiol-ester substrate analogue. However, PBP2a did not cross-link the peptide stems of glycan chains in vitro. The absence of transpeptidase activity indicates that an essential component is missing from the in vitro system.Structured digital abstract•?PBP2a and PBP2a bind by cross-linking study (View interaction)Towards reconstituting pneumococcal cell wall assembly in vitro, we show here the glycosyltransferase activity of PBP2a from Streptococcus pneumoniae. Full-length PBP2a with its cytoplasmic and transmembrane segment was found to synthesize longer glycan chains than the periplasmic form. The transpeptidase active site was functional, as it reacted towards bocillin, but did not cross-link the peptides of glycan chains in vitro.

Posted on 8 May 2012 | 2:38 pm

Placenta- versus bone marrow-derived mesenchymal cells for the repair of segmental bone defects in a rabbit model

Tissue engineered bones (TEBs) constructed with bone marrow-derived mesenchymal stem cells (BMSCs) seeded on biomaterial scaffolds have achieved good results for bone defect repair in both animal experiments and clinical trials. This, however, has been limited by the source and quantity of BMSCs. We hereby explored TEBs constructed by placenta-derived mesenchymal stem cells (PMSCs), and compared its effect for the repair of critical-sized segmental osteoperiosteal defects with TEBs constructed with BMSCs. PMSCs were isolated from rabbit placenta by gradient centrifugation and in vitro monolayer culturing, and BMSCs were isolated from hind limb bone marrow of newborn rabbit. Primary cultured PMSCs and BMSCs were uniformly in a spindle shape. Immunocytochemistry indicated that both types of cells are positive for CD44 and CD105, and negative for CD34 and CD40L, confirming that they are mesenchymal stem cells. BrdU-labeled PMSCs and BMSCs were respectively co-cultured with bio-derived bone materials to construct TEBs in vitro. Critical-sized segmental osteoperiosteal defects of radii were created in 24 rabbits by surgery. And the defects were repaired with TEBs constructed with PMSCs and BMSCs. The results showed that TEBs constructed by both PMSCs and BMSCs could repair the osteoperiosteal defects in a ‘multipoint’ manner. Measurement of radiography, histological, immunohistochemical, alkaline phosphatase activity, osteocalcin assaying, and biomechanical properties have found no significant difference between the two groups at 2, 4, 8, 12 weeks after the transplantation (P > 0.05). Taken together, our results indicate that PMSCs have similar biological characteristics and osteogenic capacity with BMSCs, and can be used as a new source of seeding cells for TEBs.

Posted on 7 May 2012 | 6:05 pm

Protein-tyrosine phosphatase PTPRJ is negatively regulated by microRNA-328

Expression of PTPRJ, which is a ubiquitous receptor-type protein tyrosine phosphatase, is significantly reduced in a vast majority of human epithelial cancers and cancer cell lines (i.e. colon, lung, thyroid, mammary, and pancreatic tumours). A possible role for microRNAs (miRNAs) in the negative regulation of PTPRJ expression has never been investigated. In this study, we show that overexpression of miR-328 decreases PTPRJ expression in HeLa and SKBr3 cells. Further investigations demonstrate that miR-328 acts directly on the 3? untranslated region (3?UTR) of PTPRJ, resulting in reduced mRNA levels. Luciferase assay and site-specific mutagenesis were used to identify a functional miRNA response element (MRE) in the 3?UTR of PTPRJ. Expression of miR-328 significantly enhances cell proliferation in HeLa and SKBr3 cells, similar to the effects of down-regulation of PTPRJ with siRNA. Additionally, in HeLa cells, the proliferative effect of miR-328 was not observed when PTPRJ was silenced with siRNA; conversely, restoration of PTPRJ expression in miR-328-overexpressing cells abolished the proliferative activity of miR-328. In conclusion, we report the identification of miR-328 as an important player in the regulation of PTPRJ expression, and we propose that the interaction of miR-328 with PTPRJ is responsible for miR-328-dependent increase of epithelial cell proliferation.

Posted on 7 May 2012 | 6:04 pm

The Regulatory Circuits for Hysteretic Switching in Cellular Signal Transduction Pathways

Hysteresis can be found in many physical systems, and a hysteretic switch has been used for various mechanical and electrical systems. Such a hysteretic switch can be created using a single positive feedback circuit, as often used in engineering systems. It is, however, intriguing that various cellular signaling systems use coupled positive feedback circuits to implement the hysteretic switch. A question then arises about the advantage of using coupled positive feedback circuits instead of simple isolated positive feedback for an apparently equivalent hysteretic switch. Through mathematical simulations, we determined that cellular systems with coupled positive feedback circuits show enhanced hysteretic switching, and can thereby make a more reliable decision under noisy signaling. As most intracellular processes are accompanied with intrinsic noises, important cellular decisions such as differentiation and apoptosis are required to be highly robust to such noises. The coupled positive feedback circuits might be evolutionarily acquired to make a correct cell fate decision through enhanced hysteretic switching under noisy cellular environments.

Posted on 7 May 2012 | 5:56 pm

Ischemia/reperfusion-induced myosin light chain 1 phosphorylation increases its degradation by matrix metalloproteinase-2

Degradation of myosin light chain 1 (MLC1) by matrix metalloproteinase-2 (MMP-2) during myocardial ischemia/reperfusion (I/R) injury has been established. However, the exact mechanisms controlling this process remain unknown. I/R increases the phosphorylation of MLC1, but the consequences of this modification are not known. We hypothesized that phosphorylation of MLC1 plays an important role in its degradation by MMP-2. To examine this, isolated perfused rat hearts were subjected to 20 min global ischemia followed by 30 min of aerobic reperfusion. I/R increased phosphorylation of MLC1 (as measured by mass spectrometry). If hearts were subjected to I/R in the presence of ML-7 (a myosin light chain kinase (MLCK) inhibitor) or doxycycline (a MMP inhibitor) an improved recovery of contractile function was seen compared to aerobic hearts and MLC1 was protected from degradation. Enzyme kinetic studies revealed an increased affinity of MMP-2 for the phosphorylated form of MLC1 compared to non-phosphorylated MLC1. We conclude that MLC1 phosphorylation is important mechanism controlling the intracellular action of MMP-2 and promoting the degradation of MLC1. These results further support previous findings implicating posttranslational modifications of contractile proteins as a key factor in the pathology of cardiac dysfunction during and following ischemia.

Posted on 7 May 2012 | 5:55 pm

A modelling–experimental approach reveals insulin receptor substrate (IRS)-dependent regulation of adenosine monosphosphate-dependent kinase (AMPK) by insulin

Mammalian target of rapamycin (mTOR) kinase responds to growth factors, nutrients and cellular energy status and is a central controller of cellular growth. mTOR exists in two multiprotein complexes that are embedded into a complex signalling network. Adenosine monophosphate-dependent kinase (AMPK) is activated by energy deprivation and shuts off adenosine 5?-triphosphate (ATP)-consuming anabolic processes, in part via the inactivation of mTORC1. Surprisingly, we observed that AMPK not only responds to energy deprivation but can also be activated by insulin, and is further induced in mTORC1-deficient cells. We have recently modelled the mTOR network, covering both mTOR complexes and their insulin and nutrient inputs. In the present study we extended the network by an AMPK module to generate the to date most comprehensive data-driven dynamic AMPK-mTOR network model. In order to define the intersection via which AMPK is activated by the insulin network, we compared simulations for six different hypothetical model structures to our observed AMPK dynamics. Hypotheses ranking suggested that the most probable intersection between insulin and AMPK was the insulin receptor substrate (IRS) and that the effects of canonical IRS downstream cues on AMPK would be mediated via an mTORC1-driven negative-feedback loop. We tested these predictions experimentally in multiple set-ups, where we inhibited or induced players along the insulin–mTORC1 signalling axis and observed AMPK induction or inhibition. We confirmed the identified model and therefore report a novel connection within the insulin–mTOR–AMPK network: we conclude that AMPK is positively regulated by IRS and can be inhibited via the negative-feedback loop.

Posted on 3 May 2012 | 12:28 pm

Human MUS81 complexes stimulate flap endonuclease 1

The yeast heterodimeric Mus81-Mms4 complex possesses a structure-specific endonuclease activity that is critical for the restart of stalled replication forks and removal of toxic recombination intermediates. Previously, we reported that Mus81-Mms4 and Rad27 (yeast FEN1, another structure-specific endonuclease) mutually stimulated their nuclease activity. In this report, we investigated the interactions between human FEN1 and MUS81-EME1/EME2, the human homologs of yeast Mus81-Mms4 complex. We found that both MUS81-EME1 and MUS81-EME2 increased the activity of FEN1, while FEN1 did not stimulate the activity of MUS81-EME1/EME2. The MUS81 subunit alone and its N-terminal half were able to bind to FEN1 and stimulate its endonuclease activity. A truncated FEN1 fragment devoid of the C-terminal region that still retained its catalytic activity was not stimulated by MUS81. Michaelis-Menten kinetic analysis revealed that MUS81 increased the interaction between FEN1 and its substrates, resulting in an increased turnover rate. We also showed that following DNA damage in human cells, FEN1 is co-localized with MUS81. These findings indicate that the human proteins and the yeast homologs act similarly, except that the human FEN1 does not stimulate the nuclease activities of MUS81-EME1 or MUS81-EME2. Thus, the mammalian MUS81 complexes and FEN1 collaborate to remove various flap structures that arise during many DNA transactions including Okazaki fragment processing.

Posted on 2 May 2012 | 5:22 pm

The RING-type ubiquitin ligases Pex2p, Pex10p and Pex12p form a heteromeric complex that displays enhanced activity in an ubiquitin conjugating enzyme-selective manner

The RING finger peroxins Pex2p, Pex10p and Pex12p are central components of the peroxisomal matrix protein import machinery. The RING  domain enables each of these proteins to exhibit ubiquitin-protein ligase activity, which has been linked to ubiquitin-dependent regulation of the peroxisomal import receptor Pex5p. The RING peroxins are considered to form a heteromeric complex in vivo, although the elucidation of the structural assembly, as well as the functional interplay of the RING domains, has remained elusive. Using in vitro approaches, we show that the RING  domains form a heteromeric complex with Pex10p(RING) as a central component that directly binds the Pex2p(RING) and Pex12p(RING). The RING domains proved to function as heteromeric pairs that display an Pex10p-dependent enhanced ligase activity in an ubiquitin conjugating enzyme-selective manner.Structured digital abstract•?Pex10p binds to Pex2p by pull down (Viewinteraction)•?Pex12p physically interacts with Pex10p and Pex2p by pull down (View Interaction: 1, 2)•?Pex10p binds to Pex12p by pull down (View Interaction: 1, 2)We have analyzed the RING-domains of the peroxisomal ubiquitin-ligases Pex2p, Pex10p and Pex12p. We find that the domains form a hetero-trimeric complex in vitro, with Pex10p(RING) acting as central binding component. Using ligase-assays, we demonstrate that Pex10p(RING) specifically enhances the ubiquitination-activity of the E3/E2-enzyme pairs Pex2p(RING)/Ubc4p as well as Pex12p(RING)/Pex4p. The data suggest that the RING-peroxins function as heteromeric pairs in an E2-selective manner.

Posted on 2 May 2012 | 5:05 pm

Studies of H3K4me3 demethylation by KDM5B/Jarid1B/PLU1 reveals strong substrate recognition in vitro and identifies 2,4-pyridine-dicarboxylic acid as an in vitro and in cell inhibitor

Dynamic methylations and demethylations of histone lysine residues are important for gene regulation and are facilitated by histone methyltransferases and histone demethylases (HDMs). KDM5B/Jarid1B/PLU1 is an H3K4me3/me2-specific lysine demethylase belonging to the JmjC domain-containing family of histone demethylases (JHDMs). Several studies have linked KDM5B to breast, prostate and skin cancer, highlighting its potential as a drug target. However, most inhibitor studies have focused on other JHDMs, and inhibitors for KDM5B remain to be explored. Here, we report the expression, purification and characterization of the catalytic core of recombinant KDM5B (ccKDM5B, residues 1-769). We show that ccKDM5B, recombinantly expressed in insect cells, demethylates H3K4me3 and H3K4me2 in vitro. The kinetic characterization showed that ccKDM5B has an apparent Michaelis constant (Kmapp) value of 0.5 ?m for its trimethylated substrate H3(1-15)K4me3, a considerably increased apparent substrate affinity than reported for related HDMs. Despite the presence of a PHD domain, the catalytic activity was not affected by additional methylation at the H3K9 position, suggesting that in vitro chromatin cross-talk between H3K4 and H3K9 does not occur for ccKDM5B. Inhibition studies of ccKDM5B showed both in vitro and in cell inhibition of ccKDM5B by 2,4-pyridinedicarboxylic acid (2,4-PDCA) with a potency similar to that reported for the HDM KDM4C. Structure-guided sequence alignment indicated that the binding mode of 2,4-PDCA is conserved between KDM4A/C and KDM5B.KDM5B/Jarid1B/PLU1 is a lysine demethylase that through its linkage to breast, prostate and skin cancer, represents a potential drug target. The catalytic core of recombinant KDM5B showed in vitro demethylase activity toward H3K4me3/me2 with a Kmapp value of 0.5 ?m for its tri-methylated substrate H3(1-15)K4me3. Inhibition studies showed both in vitro and in cell inhibition of KDM5B by 2,4-pyridinedicarboxylic acid.

Posted on 2 May 2012 | 5:05 pm

Downregulated miR-214 contributes to intrahepatic cholangiocarcinoma metastasis by targeting Twist

microRNAs (miRNAs) play an important role in many human diseases, including cancer metastasis. However, the mechanisms by which miRNAs regulate intrahepatic cholangiocarcinoma metastasis remain poorly understood. In the current study we assayed the expression level of miR-214 in intrahepatic cholangiocarcinoma tissues by real-time PCR, and defined the target gene and biologically functional effect by luciferase reporter assay and western blot analysis. We found that the miR-214 levels were remarkably decreased in metastatic intrahepatic cholangiocarcinoma tissues compared to non-metastatic tissues. Inhibition of miR-214 levels by its inhibitor promoted metastasis of human intrahepatic cholangiocarcinoma cell. We further demonstrated that downregulated miR-214 increased the levels of the EMT-associated genes Twist, and decreased E-cadherin levels. We confirmed that downregulated miR-214 promoted EMT by directly targeting Twist gene. These results suggest an important role of miR-214 in regulating metastasis of intrahepatic cholangiocarcinoma and implicate the potential application of miR-214 in intrahepatic cholangiocarcinoma therapy.

Posted on 27 April 2012 | 5:45 pm

Biochemical and pharmacological study of N-linked glycosylation of the human serotonin 5-HT7(a) receptor

The 5-hydroxytryptamine (5-HT)7(a) receptor is a G-protein-coupled receptor critically involved in human psychiatric and neurological disorders. In the present study, we evaluate the presence and the functional role of N-glycosylation of the human 5-HT7 receptor. Western blot analysis of HEK293T cells transiently expressing the 5-HT7(a) receptor in the presence of tunicamycin gave rise to a band shift, indicating the existence of an N-glycosylated form of the 5-HT7(a) receptor. To further investigate this, we mutated the two predicted N-glycosylation sites (N5Q and N66Q) and compared the molecular mass of the immunoreactive bands with those of the wild-type receptor, indicating that both asparagines were N-glycosylated. The mutant receptors had the same binding affinity for [3H]5-CT and the same potency and efficacy with regard to 5-HT-induced activation of adenylyl cyclase. However, there was a reduction in maximal ligand binding for the single and double mutants compared to the wild-type receptor. Next, membrane labelling and immunocytochemical studies demonstrated that the N-glycosylation mutants were expressed at the cell surface. We conclude that N-glycosylation is not important for cell surface expression of the 5-HT7 receptor.We show the presence of N-glycosylation of the human serotonin 5-HT7 receptor. Next, functionality was studied using receptor mutants. Receptor glycosylation does not influence (a) cell surface expression of the receptor, (b) binding of the agonist 5-CT or (c) the potency and efficacy with regard to 5-HT-induced activation of adenylyl cyclase.

Posted on 25 April 2012 | 4:09 pm

A model of dirigent proteins derived from structural and functional similarities with allene oxide cyclase and lipocalins

Dirigent proteins impart stereoselectivity on the phenoxy radical-coupling reaction, yielding optically active lignans from two molecules of coniferyl alcohol. By an unknown mechanism, they direct the coupling of two phenoxy radicals toward the formation of optically active (+)- or (?)-pinoresinol. We show here that the dirigent protein AtDIR6 from Arabidopsis thaliana is a homodimeric all-beta protein in the superfamily of calycins. Based on its homology with calycins, the structure of AtDIR6 was modeled using allene oxide cyclase as template. The structural model of AtDIR6 was supported experimentally by confirmation of a predicted disulfide bridge and by the characterization of two N-linked glycans at the solvent-exposed protein surface. The model shows AtDIR6 as an eight-stranded antiparallel ?-barrel with a central hydrophobic cavity for substrate binding, suggesting that dirigent proteins evolved from hydrophobic ligand-binding proteins. The data are fully consistent with the current view of the dirigent protein mode of action, according to which each subunit of the homodimer captures one of the substrate radicals and orients them in a way that precludes undesired reaction channels, thus favoring the formation of the optically pure coupling product.Structured digital abstract?•?AtDIR6 and AtDIR6 bind by cross-linking study (View interaction)•?AtDIR6 and AtDIR6 bind by molecular sieving (View interaction)Dirigent proteins (DIRs) control stereoselectivity of phenoxy radical coupling reactions in plant secondary metabolism. They evolved from binding proteins for hydrophobic ligands in the calycin superfamily. The structure of AtDIR6 from Arabidopsis thaliana was modeled as an antiparallel ?-barrel with a central hydrophobic cavity for substrate binding.

Posted on 25 April 2012 | 4:08 pm

The biogenesis of active protein phosphatase 2A holoenzymes: a tightly regulated process creating phosphatase specificity

Protein phosphatase type 2A (PP2A) enzymes constitute a large family of Ser/Thr phosphatases with multiple functions in cellular signaling and physiology. The composition of heterotrimeric PP2A holoenzymes, resulting from the combinatorial assembly of a catalytic C subunit, a structural A subunit, and regulatory B-type subunit, provides the essential determinants for substrate specificity, subcellular targeting, and fine-tuning of phosphatase activity, largely explaining why PP2A is functionally involved in so many diverse physiological processes, sometimes in seemingly opposing ways. In this review, we highlight how PP2A holoenzyme biogenesis and enzymatic activity are controlled by a sophisticatedly coordinated network of five PP2A modulators, consisting of ?4, phosphatase 2A phosphatase activator (PTPA), leucine carboxyl methyl transferase 1 (LCMT1), PP2A methyl esterase 1 (PME-1) and, potentially, target of rapamycin signaling pathway regulator-like 1 (TIPRL1), which serve to prevent promiscuous phosphatase activity until the holoenzyme is completely assembled. Likewise, these modulators may come into play when PP2A holoenzymes are disassembled following particular cellular stresses. Malfunctioning of these cellular control mechanisms contributes to human disease. The potential therapeutic benefits or pitfalls of interfering with these regulatory mechanisms will be briefly discussed.

Posted on 25 April 2012 | 4:08 pm

The plastidial glucan, water dikinase (GWD) catalyses multiple phosphotransfer reactions

The plant genome encodes at least two distinct and evolutionary conserved plastidial starch-related dikinases that phosphorylate a low percentage of glucosyl residues at the starch granule surface. Esterification of starch favours the transition of highly ordered ?-glucans to a less ordered state and thereby facilitates the cleavage of interglucose bonds by hydrolases. Metabolically most important is the phosphorylation at position C6, which is catalysed by the glucan, water dikinase (GWD). The reactions mediated by recombinant wild-type GWD from Arabidopsis thaliana (AtGWD) and from Solanum tuberosum (StGWD) were studied. Two mutated proteins lacking the conserved histidine residue that is indispensible for glucan phosphorylation were also included. The wild-type GWDs consume approximately 20% more ATP than is required for glucan phosphorylation. Similarly, although incapable of phosphorylating ?-glucans, the two mutated dikinase proteins are capable of degrading ATP. Thus, consumption of ATP and phosphorylation of ?-glucans are not strictly coupled processes but, to some extent, occur as independent phosphotransfer reactions. As revealed by incubation of the GWDs with [?-33P]ATP, the consumption of ATP includes the transfer of the ?-phosphate group to the GWD protein but this autophosphorylation does not require the conserved histidine residue. Thus, the GWD proteins possess two vicinal phosphorylation sites, both of which are transiently phosphorylated. Following autophosphorylation at both sites, native dikinases flexibly use various terminal phosphate acceptors, such as water, ?-glucans, AMP and ADP. A model is presented describing the complex phosphotransfer reactions of GWDs as affected by the availability of the various acceptors.Structured digital abstract•?StGWD autophosphorylates by protein kinase assay (View interaction)•?AtGWD autophosphorylates by protein kinase assay (View interaction)The plant genome encodes for plastidial starch-related dikinases which phosphorylate a low percentage of glucosyl residues of starch. Metabolically most important is the phosphorylation at position C6 which is catalyzed by the glucan, water dikinase (GWD). The reactions mediated by GWD and two mutated GWD proteins were analyzed. A model is presented describing the complex phosphotransfer reactions of GWD.

Posted on 25 April 2012 | 4:04 pm

MicroRNA-199a targets CD44 to suppress the tumorigenicity and multidrug resistance of ovarian cancer-initiating cells

In ovarian cancer, CD44+/CD117+ stem cells, also known as cancer-initiating cells (CICs), are highly proliferative, have a low degree of differentiation, and are resistant to chemotherapeutics. Therefore, the CD44+/CD117+ subpopulation is thought to be an important target for novel therapeutic strategies. In this study, we investigated the role of microRNA-199a (miR-199a) in ovarian cancer stem cells. Luciferase reporter gene assays confirmed that miR-199a targets CD44 via an miR-199a-binding site in the 3?-UTR. CD44+/CD117+ ovarian CICs were enriched from human primary ovarian tumor tissues and confirmed by flow cytometric sorting. miR-199a was cloned and transfected into ovarian CICs. CD44 mRNA and protein expression was significantly decreased in miR-199a-transfected ovarian CICs as compared with miR-199a mutant-transfected and untransfected cells. Cell cycle analysis, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide proliferation assays, the colony formation assay and the transwell migration assay indicated that miR-199a significantly affected cell cycle regulation and suppressed the proliferation and invasive capacity of ovarian CICs in vitro. miR-199a significantly increased the chemosensitivity of ovarian CICs to cisplatin, pacitaxel, and adriamycin, and reduced mRNA expression of the multidrug resistance gene ABCG2 as compared with miR-199a mutant-transfected and untransfected cells. The expression of stemness markers was also significantly reduced in miR-199a-transfected CICs as compared with miR-199a mutant-transfected and untransfected ovarian cells. Furthermore, xenograft experiments confirmed that miR-199a suppressed the growth of xenograft tumors formed by ovarian CICs in vivo. Thus, expression of endogenous mature miR-199a may prevent tumorigenesis in human ovarian cancer by regulating expression of its target gene CD44.The experiments confirmed that miR-199a significantly affected cell cycle regulation and suppressed the proliferation and invasive capacity of ovarian CICs, and significantly increased the chemosensitivity of ovarian CICs to chemotherapeutic drugs in vitro. Furthermore, xenograft experiments confirmed that miR-199a suppressed the growth of xenograft tumors formed by ovarian CICs in vivo. Thus, miR-199a may prevent tumorigenesis in human ovarian cancer.

Posted on 24 April 2012 | 11:36 am

Regulating naïve and memory CD8 T cell homeostasis – a role for protein tyrosine phosphatases

A complex network of signalling events coordinate the differentiation, activation and maintenance of T lymphocytes. Tyrosine phosphorylation and dephosphorylation by protein tyrosine kinases and protein tyrosine phosphatases (PTPs) respectively, are critical for the activation and propagation of these signalling cascades. Intriguingly, the removal of tyrosyl phosphate moieties from phosphorylated proteins by phosphatases can contribute to both the positive and negative regulation of signalling events. The complex and diverse roles of individual PTP family members in immune cells is evident by the range of immune disorders caused by PTP deficiencies. Central to several such immune disorders is the disturbance of T cell homeostasis, as characterized by aberrant cell growth, survival and activation. The survival and homeostatic proliferation of naïve and memory CD8 T cells is primarily regulated by signalling events downstream of the T cell receptor complex and common ? chain cytokine receptors, events frequently targeted by PTP activity. We review the primary PTPs involved in CD8 T cell homeostasis, focusing on the signalling nodes that they target. In addition, because the mechanisms that co-ordinate PTP activity are only partially understood, we discuss currently proposed models of regulation and highlight unanswered questions.

Posted on 24 April 2012 | 11:35 am

Structural role of the active-site metal in the conformation of Trypanosoma brucei phosphoglycerate mutase

Phosphoglycerate mutases (PGAMs) participate in both the glycolytic and the gluconeogenic pathways in reversible isomerization of 3-phosphoglycerate and 2-phosphoglycerate. PGAMs are members of two distinct protein families: enzymes that are dependent on or independent of the 2,3-bisphosphoglycerate cofactor. We determined the X-ray structure of the monomeric Trypanosoma brucei independent PGAM (TbiPGAM) in its apoenzyme form, and confirmed this observation by small angle X-ray scattering data. Comparing the TbiPGAM structure with the Leishmania mexicana independent PGAM structure, previously reported with a phosphoglycerate molecule bound to the active site, revealed the domain movement resulting from active site occupation. The structure reported here shows the interaction between Asp319 and the metal bound to the active site, and its contribution to the domain movement. Substitution of the metal-binding residue Asp319 by Ala resulted in complete loss of independent PGAM activity, and showed for the first time its involvement in the enzyme’s function. As TbiPGAM is an attractive molecular target for drug development, the apoenzyme conformation described here provides opportunities for its use in structure-based drug design approaches.Database Structural data for the Trypanosoma brucei 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (iPGAM) has been deposited with the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank under code 3NVL.Structured digital abstract•?TbiPGAM and TbiPGAM bind by x-raycrystallography (View interaction)Phosphoglycerate mutase participates in the glycolytic and gluconeogenic pathways. We determined the X-ray structure of Trypanosoma brucei phosphoglycerate mutase in its apo-form. The structure, in an open conformation, reveals the domain movement resulting from active site occupation. Furthermore, substitution of the metal-binding residue Asp319 resulted in the complete loss of enzyme activity and confirmed its contribution in enzyme function.

Posted on 24 April 2012 | 11:35 am

Mapping the surface-exposed regions of papaya mosaic virus nanoparticles

In general, the structure of the papaya mosaic virus (PapMV) and other members of the potexviruses is poorly understood. Production of PapMV coat proteins in a bacterial expression system and their self-assembly in vitro into nanoparticles is a very useful tool to study the structure of this virus. Using recombinant PapMV nanoparticles that are similar in shape and appearance to the plant virus, we evaluated surface-exposed regions by two different methods, immunoblot assay and chemical modification with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or diethyl-pyrocarbonate followed by mass spectrometry. Three regions were targeted by the two techniques. The N- and C-termini were shown to be surfaced exposed as expected. However, the region 125–136 was revealed for the first time as the major surface-exposed region of the nanoparticles. The presence of linear peptides at the surface was finally confirmed using antibodies directed to those peptides. It is likely that region 125–136 plays a key role in the lifecycle of PapMV and other members of the potexvirus group.We have evaluated surface-exposed regions of PapMV by immunoblot assay and chemical modification followed by mass spectrometry. Three regions were targeted by both techniques, the N- and C-termini and region 125-136. The N and C terminus were expected to be found at the surface, but the region 125-136 was shown for the first time to be surface exposed on the CP of PapMV.

Posted on 24 April 2012 | 11:35 am

Amino acid screening based on structural modeling identifies critical residues for function, ion selectivity and structure of Arabidopsis MTP1

Arabidopsis thaliana MTP1 is a vacuolar membrane Zn2+/H+ antiporter of the cation diffusion facilitator family. Here we present a structure-function analysis of AtMTP1-mediated transport and remarkable Zn2+ selectivity through functional complementation tests of more than 50 mutant variants in metal-sensitive yeast strains. This was combined with homology modeling of AtMTP1 based on the crystal structure of the Escherichia coli broad-specificity divalent cation transporter YiiP. The Zn2+-binding sites of EcYiiP in the cytoplasmic C-terminus, and the pore formed by transmembrane helices TM2 and TM5, are conserved in AtMTP1. Although missing in EcYiiP, Cys31 and Cys36 in the extended N-terminal cytosolic domain of AtMTP1 are necessary for complementation of a Zn-sensitive yeast strain. On the cytosolic side of the active Zn2+-binding site inside the transmembrane pore, Ala substitution of either Asn258 in TM5 or Ser101 in TM2 non-selectively enhanced metal tolerance conferred by AtMTP1. Modeling predicts these residues to obstruct the movement of cytosolic Zn2+ into the intra-membrane Zn2+-binding site of AtMTP1. A conformational change in the directly preceding His-rich cytosolic loop could displace Asn258 and permit Zn2+ entry into the pore. This would allow the dynamic coupling of Zn2+ transport to the His-rich loop acting as selectivity filter or Zn2+ sensor. Individual mutations at diverse sites within AtMTP1 conferred Co and Cd tolerance to yeast, including deletions in N-terminal and His-rich intra-molecular cytosolic domains, and mutations of single residues flanking the transmembrane pore or participating in intra- or inter-molecular domain interactions, all of which are divergent from the non-selective EcYiiP.

Posted on 21 April 2012 | 5:45 pm

On the function of the various quinone species in e. Coli

The respiratory chain of Escherichia coli contains three quinones. Whereas the low-midpoint potential menaquinone and demethylmenaquinone are involved in anaerobic respiration, the high-midpoint potential ubiquinone is involved in aerobic- and nitrate respiration. Here, we report that demethylmenaquinone can play a role not only in TMAO-, DMSO- and fumarate-dependent respiration, but in aerobic respiration as well. Furthermore, we demonstrate that demethylmenaquinone can serve as an electron acceptor for the oxidation of succinate to fumarate, and that all three quinol oxidases of E. coli can accept electrons from this naphtoquinone derivative.

Posted on 21 April 2012 | 4:41 pm

Many of the functional differences between acetohydroxyacid synthase (AHAS) isozyme I and other AHASs are a result of the rapid formation and breakdown of the covalent acetolactate–thiamin diphosphate adduct in AHAS I

Acetohydroxy acid synthase (AHAS; EC 2.2.1.6) is a thiamin diphosphate (ThDP)-dependent decarboxylase-ligase that catalyzes the first common step in the biosynthesis of branched-chain amino acids. In the first stage of the reaction, pyruvate is decarboxylated and the reactive intermediate hydroxyethyl-ThDP carbanion/enamine is formed. In the second stage, the intermediate is ligated to another 2-ketoacid to form either acetolactate or acetohydroxybutyrate. AHAS isozyme I from Escherichia coli is unique among the AHAS isozymes in that it is not specific for 2-ketobutyrate (2-KB) over pyruvate as an acceptor substrate. It also appears to have a different mechanism for inhibition by valine than does AHAS III from E. coli. An investigation of this enzyme by directed mutagenesis and knowledge of detailed kinetics using the rapid mixing–quench NMR method or stopped-flow spectroscopy, as well as the use of alternative substrates, suggests that two residues determine most of the unique properties of AHAS I. Gln480 and Met476 in AHAS I replace the Trp and Leu residues conserved in other AHASs and lead to accelerated ligation and product release steps. This difference in kinetics accounts for the unique specificity, reversibility and allosteric response of AHAS I. The rate of decarboxylation of the initially formed 2-lactyl-ThDP intermediate is, in some AHAS I mutants, different for the alternative acceptors pyruvate and 2-KB, putting into question whether AHAS operates via a pure ping–pong mechanism. This finding might be compatible with a concerted mechanism (i.e. the formation of a ternary donor–acceptor:enzyme complex followed by covalent, ThDP-promoted catalysis with concerted decarboxylation–carboligation). It might alternatively be explained by an allosteric interaction between the multiple catalytic sites in AHAS.AHASs are ThDP-dependent decarboxylase-ligases. Decarboxylation of pyruvate forms hydroxyethyl-ThDP carbanion/enamine (HEThDP?) which is ligated to pyruvate or 2-ketobutyrate (2-KB) to form acetohydroxyacids. AHAS I is unique in being nonspecific for 2-KB as acceptor and in other properties. Mutagenesis and detailed kinetics suggest that many properties of AHAS I are due to accelerated ligation and product release. AHAS I Gln480 is a major contributor to this.

Posted on 20 April 2012 | 10:56 am

PTP1B and TCPTP – nonredundant phosphatases in insulin signaling and glucose homeostasis

Insulin resistance is a key pathological feature of type 2 diabetes and is characterized by defects in signaling by the insulin receptor (IR) protein tyrosine kinase. The inhibition of protein tyrosine phosphatases (PTPs) that antagonize IR signaling may provide a means for enhancing the insulin response and alleviating insulin resistance. The prototypic phosphotyrosine-specific phosphatase PTP1B dephosphorylates the IR and attenuates insulin signaling in muscle and liver. Mice that are deficient for PTP1B exhibit improved glucose homeostasis in diet and genetic models of insulin resistance and type 2 diabetes. The phosphatase TCPTP shares 72% catalytic domain sequence identity with PTP1B and has also been implicated in IR regulation. Despite their high degree of similarity, PTP1B and TCPTP act together in vitro and in vivo to regulate insulin signaling and glucose homeostasis. This review highlights their capacity to act specifically and nonredundantly in cellular signaling, describes their roles in IR regulation and glucose homeostasis, and discusses their potential as drug targets for the enhancement of IR phosphorylation and insulin sensitivity in type 2 diabetes.

Posted on 18 April 2012 | 11:26 am

PEP-1-heat shock protein 27 protects from neuronal damage in cells and in a Parkinson’s disease mouse model

Heat shock proteins (HSPs) are a highly conserved family of proteins that are induced in response to various environmental stressors including reactive oxygen species. HSP27 is a chaperone protein with the ability to increase cell survival in response to oxidative stress. Parkinson’s disease (PD) is a neurodegenerative disorder characterized by loss of dopaminergic neurons. Although the mechanism of PD remains unclear, oxidative stress is known to be important in its pathogenesis. This study investigated the protective effects of PEP-1-HSP27 on neuronal damage induced by 1-methyl-4-phenyl pyridinium (MPP+) in SH-SY5Y cells and in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model. PEP-1-HSP27 rapidly entered the cells and protected them against MPP+-induced toxicity by inhibiting the reactive oxygen species levels and DNA fragmentation. Furthermore, transduced PEP-1-HSP27 prevented dopaminergic neuronal cell death in the substantia nigra of MPTP-induced PD mouse models. These results demonstrate that PEP-1-HSP27 provides a potential strategy for therapeutic delivery against various diseases and is a potential tool for the treatment of PD.HSP27 is a chaperone protein with the ability to increase cell survival in response to oxidative stress which is known to be important in the pathogenesis of Parkinson’s disease (PD). Transduced PEP-1-HSP27 prevented dopaminergic neuronal cell death in the MPTP-induced PD mouse models. These results indicate that PEP-1-HSP27 is a potential therapeutic tool for the treatment of PD.

Posted on 16 April 2012 | 4:36 pm

Balance of human choline kinase isoforms is critical for cell cycle regulation

The enzyme choline kinase (CK), which catalyzes the phosphorylation of choline to phosphorylcholine in the presence of ATP, has an essential role in the biosynthesis of phosphatidylcholine, the major constituent of all mammalian cell membranes. CK is encoded by two separate genes expressing the three isoforms CK?1, CK?2 and CK? that are active as homodimeric or heterodimeric species. Metabolic changes observed in various cancer cell lines and tumors have been associated with differential and marked up-regulation of the CK? genes, and specific inhibition of CK? activity has been proposed as a potential anti-cancer strategy. As a result, less attention has been given to CK? and its interaction with CK?. With the aim of profiling the intracellular roles of CK? and CK?, we used RNA interference (RNAi) as a molecular approach to down-regulate the expression of CK in HeLa cells. Individual and simultaneous RNAi-based silencing of the CK ? and ? isoforms was achieved using different combinations of knockdown strategies. Efficient knockdown was confirmed by immunodetection using our isoform-specific antibodies and by quantitative real-time PCR. Our analyses of the phenotypic consequences of CK depletion showed the expected lethal effect of CK? knockdown. However, CK?- and CK? + CK?-silenced cells had no aberrant phenotype. Therefore, our results support the hypothesis that the balance of the ? and ? isoforms is critical for cancer cell survival. The suppression of the cancer cell killing effect of CK? silencing by simultaneous knockdown of both isoforms implies that a more effective CK-based anti-cancer strategy can be achieved by reducing cross-reactivity with CK?.Choline kinases are key enzymes for the synthesis of phospholipids that are essential constituents of cell membranes, and are involved in regulation of cell proliferation and oncogenic transformation. Down-regulation of the two choline kinase isoforms ? and ? in HeLa cells by RNA interference showed that single ? knock-down was lethal, whereas single ?- and double-silenced cells grew normally.

Posted on 16 April 2012 | 4:36 pm

Ceramide profiles in the brain of rats with diabetes induced by streptozotocin

Diabetes is associated with disturbances of brain activity and cognitive impairment. We hypothesize that ceramides may constitute an important contribution to diabetes-linked neuro-dysfunction. In our study we used rats injected with streptozotocin (STZ) as a model of severe hyperglycemia. Using the gas–liquid chromatography technique we found a significant increase of ceramide content in brains and a decrease in plasma of diabetic rats. The inhibitor of serine palmitoyltransferase, myriocin, reduced ceramide generation in hyperglycemic brains, although injected alone it exerted a paradoxical effect of ceramide upregulation. Myriocin had no impact on ceramide concentration in the plasma of either control or diabetic rats. The level of ceramide saturated fatty acids was elevated whereas the level of ceramide poly-unsaturated fatty acids was downregulated in brains of all experimental groups. The concentration of ceramide mono-unsaturated fatty acids remained unchanged. The pattern of individual ceramide species was altered depending on treatment. We noted an STZ-evoked increase of brain ceramide C16:0, C18:0 and C20:0 and a strong decline in ceramide C18:2 fatty acid levels. Some changes of brain ceramide pattern were modified by myriocin. We found a decreased amount of total ceramide-?-6 fatty acids in STZ-treated rat brains and no changes in ceramide-?-3 concentration. We conclude that ceramides may be important mediators of diabetes-accompanied brain dysfunction.STZ-induced diabetes in rats significantly increased the ceramide content in brains and decreased the plasma content. Myriocin, inhibitor of serine palmitoyltransferase, reduced ceramide concentration in the brain. We found elevated levels of ceramide-SAFA, down-regulation of ceramide-PUFA and decreased amounts of ceramide-?-6 fatty acids in diabetic brains. We conclude that ceramides may be the important mediators of diabetes-accompanied brain dysfunctions.

Posted on 16 April 2012 | 1:03 pm

An integrative model links multiple inputs and signaling pathways to the onset of DNA synthesis in hepatocytes

During liver regeneration, quiescent hepatocytes re-enter the cell cycle to proliferate and compensate for lost tissue. Multiple signals including hepatocyte growth factor, epidermal growth factor, tumor necrosis factor ?, interleukin-6, insulin and transforming growth factor ? orchestrate these responses and are integrated during the G1 phase of the cell cycle. To investigate how these inputs influence DNA synthesis as a measure for proliferation, we established a large-scale integrated logical model connecting multiple signaling pathways and the cell cycle. We constructed our model based upon established literature knowledge, and successively improved and validated its structure using hepatocyte-specific literature as well as experimental DNA synthesis data. Model analyses showed that activation of the mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways was sufficient and necessary for triggering DNA synthesis. In addition, we identified key species in these pathways that mediate DNA replication. Our model predicted oncogenic mutations that were compared with the COSMIC database, and proposed intervention targets to block hepatocyte growth factor-induced DNA synthesis, which we validated experimentally. Our integrative approach demonstrates that, despite the complexity and size of the underlying interlaced network, logical modeling enables an integrative understanding of signaling-controlled proliferation at the cellular level, and thus can provide intervention strategies for distinct perturbation scenarios at various regulatory levels.

Posted on 10 April 2012 | 6:47 pm

Post-transcriptional regulatory networks play a key role in noise reduction that is conserved from micro-organisms to mammals

RNA-binding proteins (RBPs) are core regulators of mRNA transcript stability and translation in prokaryotes and eukaryotes alike. Genome-wide studies in yeast have shown intriguing relationships between the expression dynamics of RBPs, the structure of post-transcriptional regulatory networks of RBP–mRNA binding interactions and noise reduction in post-transcriptionally regulated expression profiles. In the present study, we assembled and compared the genomic properties of RBPs and integrated transcriptional and post-transcriptional regulatory networks in four species: Escherichia coli, yeast, mouse and human. We found that RBPs are consistently regulated to have minimal levels of protein noise, that known noise-buffering network motifs are enriched in the integrated networks and that post-transcriptional feedback loops act as regulators of other regulators. These results support a general model where RBPs are the key regulators of stochastic noise-buffering in numerous downstream cellular processes. The currently available datasets do not allow clarification of whether post-transcriptional regulation by RBPs and by noncoding RNAs plays a similar or distinct role, although we found evidence that specific combinations of transcription factors, RBPs and micro-RNAs jointly regulate known disease pathways in humans, suggesting complementarity rather than redundancy between both modes of post-transcriptional regulation.

Posted on 10 April 2012 | 6:45 pm

Molecular mechanisms of the PRL phosphatases

The phosphatases of regenerating liver (PRLs) are an intriguing family of dual specificity phosphatases due to their oncogenicity. The three members are small, single domain enzymes. We provide an overview of the phosphatases of regenerating liver, compare them to related phosphatases, and review recent reports about each phosphatase. Finally, we discuss similarities and differences between the phosphatases of regenerating liver, focusing on their molecular mechanisms and signalling pathways.

Posted on 10 April 2012 | 6:41 pm

A chemical method for investigating disulfide-coupled peptide and protein folding

Investigations of protein folding have largely involved studies using disulfide-containing proteins since the disulfide-coupled folding of proteins permits the folding intermediates to be trapped and their conformations determined. In this decade, a combination of new biotechnical and chemical methodology has resulted in a remarkable acceleration in our understanding of the mechanism of disulfide-coupled protein folding. In particular, expressed protein ligation, a combination of native chemical ligation and an intein-based approach, permits specifically-labeled proteins to be easily produced for studies of protein folding using biophysical methods, such as nuclear magnetic resonance spectroscopy and X-ray crystallography. A method for the regioselective formation of the complex disulfide bonds using chemical procedures has also been established. This strategy is particularly relevant for the study of disulfide-coupled protein folding and provides us, not only with the native conformation, but also the kinetically-trapped topological isomer with the native disulfide bonds. In this minireview, recent developments and applications of biotechnical and chemical methods to investigations of disulfide-coupled peptide and protein folding are reviewed. In addition, chemical additives designed to accelerate correct protein folding and to avoid non-specific aggregation are also discussed.

Posted on 9 April 2012 | 10:20 am

Disulfide bond formation network in the biological kingdoms

Almost all organisms, from bacteria to humans, possess catalytic systems that promote disulfide bond formation-coupled protein folding, i.e., oxidative protein folding. These systems are necessary for the biosynthesis of many secretory and membrane proteins such as antibodies, MHC (major histocompatibility complex) molecules, growth factors and insulin. Over the last decade, structural studies have made striking progress in this field of research, identifying how oxidative systems operate in a specific and regulated manner to maintain redox and protein homeostasis within cells. Interestingly, more and more novel catalysts that promote disulfide bond formation have been discovered in mammals, suggesting that the oxidative protein folding network is even more complicated in higher eukaryotes than previously thought. This review highlights the physiological roles and molecular bases of the disulfide bond formation pathways that have evolved in the bacterial periplasm and the endoplasmic reticulum of fungi and mammals. Accumulating knowledge about disulfide bond formation networks widely distributed throughout the biological kingdom has significantly advanced our understanding of the cellular mechanisms dedicated to protein quality control.

Posted on 5 April 2012 | 8:35 pm

NAD+ biosynthesis and salvage – a phylogenetic perspective

NAD is best known as an electron carrier and a cosubstrate of various redox reactions. However, over the past 20 years, NAD+ has been shown to be a key signaling molecule that mediates post-translational protein modifications and serves as precursor of ADP-ribose-containing messenger molecules, which are involved in calcium mobilization. In contrast to its role as a redox carrier, NAD+-dependent signaling processes involve the release of nicotinamide (Nam) and require constant replenishment of cellular NAD+ pools. So far, very little is known about the evolution of NAD(P) synthesis in eukaryotes. In the present study, genes involved in NAD(P) metabolism in 45 species were identified and analyzed with regard to similarities and differences in NAD(P) synthesis. The results show that the Preiss–Handler pathway and NAD+ kinase are present in all organisms investigated, and thus seem to be ancestral routes. Additionally, two pathways exist that convert Nam to NAD+; we identified several species that have apparently functional copies of both biosynthetic routes, which have been thought to be mutually exclusive. Furthermore, our findings suggest the parallel phylogenetic appearance of Nam N-methyltransferase, Nam phosphoribosyl transferase, and poly-ADP-ribosyltransferases.

Posted on 4 April 2012 | 8:03 pm

The PP1 binding code: a molecular-lego strategy that governs specificity

Ser/Thr protein phosphatase 1 (PP1) is a single-domain hub protein with nearly 200 validated interactors in vertebrates. PP1-interacting proteins (PIPs) are ubiquitously expressed but show an exceptional diversity in brain, testis and white blood cells. The binding of PIPs is mainly mediated by short motifs that dock to surface grooves of PP1. Although PIPs often contain variants of the same PP1 binding motifs, they differ in the number and combination of docking sites. This molecular-lego strategy for binding to PP1 creates holoenzymes with unique properties. The PP1 binding code can be described as specific, universal, degenerate, nonexclusive and dynamic. PIPs control associated PP1 by interference with substrate recruitment or access to the active site. In addition, some PIPs have a subcellular targeting domain that promotes dephosphorylation by increasing the local concentration of PP1. The diversity of the PP1 interactome and the properties of the PP1 binding code account for the exquisite specificity of PP1 in vivo.

Posted on 21 March 2012 | 4:43 pm

Insight into the redox regulation of the phosphoglucan phosphatase SEX4 involved in starch degradation

Starch is the major carbohydrate reserve in plants, and is degraded for growth at night. Starch breakdown requires reversible glucan phosphorylation at the granule surface by novel dikinases and phosphatases. The dual-specificity phosphatase starch excess 4 (SEX4) is required for glucan desphosphorylation; however, regulation of the enzymatic activity of SEX4 is not well understood. We show that SEX4 switches between reduced (active) and oxidized (inactive) states, suggesting that SEX4 is redox-regulated. Although only partial reactivation of SEX4 was achieved using artificial reductants (e.g. dithiothreitol), use of numerous chloroplastic thioredoxins recovered activity completely, suggesting that thioredoxins could reduce SEX4 in vivo. Analysis of peptides from oxidized and reduced SEX4 identified a disulfide linkage between the catalytic cysteine at position 198 (Cys198) and the cysteine at position 130 (Cys130) within the phosphatase domain. The position of these cysteines was structurally analogous to that for known redox-regulated dual-specificity phosphatases, suggesting a common mechanism of reversible oxidation amongst these phosphatases. Mutation of Cys130 renders SEX4 more sensitive to oxidative inactivation and less responsive to reductive reactivation. Together, these results provide the first biochemical evidence for a redox-dependent structural switch that regulates SEX4 activity, which represents the first plant phosphatase known to undergo reversible oxidation via disulfide bond formation like its mammalian counterparts.

Posted on 21 March 2012 | 2:27 pm

Sic1 as a timer of Clb cyclin waves in the yeast cell cycle – design principle of not just an inhibitor

Cellular systems biology aims to uncover design principles that describe the properties of biological networks through interaction of their components in space and time. The cell cycle is a complex system regulated by molecules that are integrated into functional modules to ensure genome integrity and faithful cell division. In budding yeast, cyclin-dependent kinases (Cdk1/Clb) drive cell cycle progression, being activated and inactivated in a precise temporal sequence. In this module, which we refer to as the ‘Clb module’, different Cdk1/Clb complexes are regulated to generate waves of Clb activity, a functional property of cell cycle control. The inhibitor Sic1 plays a critical role in the Clb module by binding to and blocking Cdk1/Clb activity, ultimately setting the timing of DNA replication and mitosis. Fifteen years of research subsequent to the identification of Sic1 have lead to the development of an integrative approach that addresses its role in regulating the Clb module. Sic1 is an intrinsically disordered protein and achieves its inhibitory function by cooperative binding, where different structural regions stretch on the Cdk1/Clb surface. Moreover, Sic1 promotes S phase entry, facilitating Cdk1/Clb5 nuclear transport, and therefore revealing a double function of inhibitor/activator that rationalizes a mechanism to prevent precocious DNA replication. Interestingly, the investigation of Clb temporal dynamics by mathematical modelling and experimental validation provides evidence that Sic1 acts as a timer to coordinate oscillations of Clb cyclin waves. Here we review these findings, focusing on the design principle underlying the Clb module, which highlights the role of Sic1 in regulating phase-specific Cdk1/Clb activities.

Posted on 21 March 2012 | 1:37 pm

An extended model for the repression of photosynthesis genes by the AppA/PpsR system in Rhodobacter sphaeroides

Purple bacteria derive energy from aerobic respiration or photosynthesis depending on the availability of oxygen and light. Under aerobic conditions, photosynthesis genes are specifically repressed by the PpsR protein. In Rhodobacter sphaeroides, the repressive action of PpsR is antagonized by the blue-light and redox-sensitive flavoprotein AppA, which sequesters PpsR under anaerobic conditions into transcriptionally inactive complexes. However, under semi-aerobic conditions, blue-light excitation of AppA causes the AppA–PpsR complexes to dissociate, again leading to a repression of photosynthesis genes. We have recently developed a simple mathematical model suggesting that this phenotype arises from the formation of a maximum in the response curve of reduced PpsR at intermediate oxygen concentrations. However, this model focused mainly on the oxygen-dependent interactions whereas light regulation was only implemented in a simplified manner. In the present study, we incorporate a more detailed mechanism for the light-dependent interaction between AppA and PpsR, which now allows for a direct comparison with experiments. Specifically, we take into account that, upon blue–light excitation, AppA undergoes a conformational change, creating a long-lived signalling state causing the dissociation of the AppA–PpsR complexes. The predictions of the extended model are found to be in good agreement with experimental results on the light-dependent repression of photosynthesis genes under semi-aerobic conditions. We also identify the potential kinetic and stoichiometric constraints that the interplay between light and redox regulation imposes on the functionality of the AppA/PpsR system, especially with respect to a possible bistable response.

Posted on 16 March 2012 | 6:52 pm

Laforin, a protein with many faces: glucan phosphatase, adapter protein, et alii

Lafora disease (LD) is a rare, fatal neurodegenerative disorder characterized by the accumulation of glycogen-like inclusions in the cytoplasm of cells from most tissues of affected patients. One hundred years after the first description of these inclusions, the molecular bases underlying the processes involved in LD physiopathology are finally being elucidated. The main cause of the disease is related to the activity of two proteins, the dual-specificity phosphatase laforin and the E3-ubiquitin ligase malin, which form a functional complex. Laforin is unique in humans, as it is composed of a carbohydrate-binding module attached to a cysteine-based catalytic dual-specificity phosphatase domain. Laforin directly dephosphorylates glycogen, but other proteinaceous substrates, if they exist, have remained elusive. Recently, an emerging set of laforin-binding partners apart from malin have been described, suggestive of laforin roles unrelated to its catalytic activity. Further investigations based on different transgenic mouse models have shown that the laforin–malin complex is also involved in other cellular processes, such as response to endoplasmic reticulum stress and misfolded protein clearance by the lysosomal pathway. However, controversial data and some missing links still make it difficult to assess the concrete relationship between glycogen deregulation and neuronal damage leading to the fatal symptoms observed in LD patients, such as myoclonic seizures and epilepsy. Consequently, clinical treatments are far from being achieved. In the present review, we focus on the knowledge of laforin biology, not only as a glucan phosphatase, but also as an adaptor protein involved in several physiological pathways.

Posted on 16 March 2012 | 5:33 pm

Suppression of survival signalling pathways by the phosphatase PHLPP

The recently discovered pleckstrin homology (PH) domain leucine-rich repeat protein phosphatase (PHLPP) family is emerging as a central component in suppressing cell survival pathways. Originally discovered in a rational search for a phosphatase that directly dephosphorylates and inactivates Akt, PHLPP is now known to potently suppress cell survival both by inhibiting proliferative pathways and by promoting apoptotic pathways. In the first instance, PHLPP directly dephosphorylates a conserved regulatory site (termed the hydrophobic motif) on Akt, protein kinase C and S6 kinase, thereby terminating signalling by these pro-survival kinases. In the second instance, PHLPP dephosphorylates and thus activates the pro-apoptotic kinase Mst1, thereby promoting apoptosis. PHLPP is deleted in a large number of cancers and the genetic deletion of one isozyme in a PTEN (phosphatase and tensin homologue located on chromosome 1) +/? (or heterozygous) prostate cancer model results in increased tumourigenesis, underscoring the role of PHLPP as a tumour suppressor. This review summarizes the targets and cellular actions of PHLPP, with emphasis on its role as a tumour suppressor in the oncogenic phosphoinositide 3-kinase (PI3K)/Akt signalling cascade.

Posted on 16 March 2012 | 5:28 pm

Exploiting the selectivity of protein phosphatase 1 for pharmacological intervention

Selective and reversible phosphorylation is one of the most common post-translational modifications of proteins. Although kinase inhibitors are popular in drug development programmes, selective pharmacological manipulation of phosphatase activity has been challenging to achieve. We review recent advances in the development of selective inhibitors of dephosphorylation events and discuss the potential applications of small-molecule phosphatase inhibitors.

Posted on 14 March 2012 | 5:35 pm

Signalling by protein phosphatases and drug development: a systems-centred view

Protein modification cycles catalysed by opposing enzymes, such as kinases and phosphatases, form the backbone of signalling networks. Although, historically, kinases have been at the research forefront, a systems-centred approach reveals predominant roles for phosphatases in controlling the network response times and spatio-temporal profiles of signalling activities. Emerging evidence suggests that phosphatase kinetics are critical for network function and cell-fate decisions. Protein phosphatases operate as both immediate and delayed regulators of signal transduction, capable of attenuating or amplifying signalling. This versatility of phosphatase action emphasizes the need for systems biology approaches to understand cellular signalling networks and predict the cellular outcomes of combinatorial drug interventions.

Posted on 14 March 2012 | 4:30 pm

PP1 and PP2A phosphatases – cooperating partners in modulating retinoblastoma protein activation

The retinoblastoma/pocket protein family is one of the master regulators of the eukaryotic cell cycle. It includes the retinoblastoma protein (Rb) and the related p107 and p130 proteins. The importance of the Rb pathway for homeostasis and tumour suppression is evident from the fact that inactivating mutations in Rb are frequently associated with many cancers. Rbs regulate the cell cycle by controlling the activity of the E2F family of transcription factors. The activity of Rb proteins themselves is modulated by their phosphorylation status at several Ser/Thr residues: phosphorylation by cyclin-dependent kinases inactivates Rb proteins and positively influences the transcription of genes necessary for cell cycle progression. Although the mechanisms of cyclin-dependent kinase-mediated inactivation of Rb proteins are understood in great detail, our knowledge of the process that counteracts Rb phosphorylation is still quite limited. The present review focuses on the Ser/Thr phosphatases that are responsible for the dephosphorylation and thus activation of Rb proteins. Two major scenarios are considered: (a) when pocket proteins are dephosphorylated during regular cell cycle progression and (b) when rapid dephosphorylation is dictated by external stress or growth inhibitory conditions, such as oxidative stress, UV radiation or other DNA-damaging stimuli, and cell differentiation factors. It transpires that protein phosphatase 1 and protein phosphatase 2A can efficiently modulate pocket protein activity in a highly context-dependent manner and both are tightly regulated by the presence of different regulatory subunits or interacting proteins.

Posted on 29 February 2012 | 11:36 am

Structural basis for protein phosphatase 1 regulation and specificity

The ubiquitous serine/threonine protein phosphatase 1 (PP1) regulates diverse, essential cellular processes such as cell cycle progression, protein synthesis, muscle contraction, carbohydrate metabolism, transcription and neuronal signaling. However, the free catalytic subunit of PP1, while an effective enzyme, lacks substrate specificity. Instead, it depends on a diverse set of regulatory proteins (? 200) to confer specificity towards distinct substrates. Here, we discuss recent advances in structural studies of PP1 holoenzyme complexes and summarize the new insights these studies have provided into the molecular basis of PP1 regulation and specificity.

Posted on 24 February 2012 | 6:59 pm

Epigallocatechin-3-gallate and penta-O-galloyl-?-d-glucose inhibit protein phosphatase-1

Protein phosphatase-1 (PP1) and protein phosphatase-2A (PP2A) are responsible for the dephosphorylation of the majority of phosphoserine/threonine residues in cells. In this study, we show that (–)-epigallocatechin-3-gallate (EGCG) and 1,2,3,4,6-penta-O-galloyl-?-d-glucose (PGG), polyphenolic constituents of green tea and tannins, inhibit the activity of the PP1 recombinant ?-isoform of the PP1 catalytic subunit and the native PP1 catalytic subunit (PP1c) with IC50 values of 0.47–1.35 ?m and 0.26–0.4 ?m, respectively. EGCG and PGG inhibit PP2Ac less potently, with IC50 values of 15 and 6.6 ?m, respectively. The structure–inhibitory potency relationships of catechin derivatives suggests that the galloyl group may play a major role in phosphatase inhibition. The interaction of EGCG and PGG with PP1c was characterized by NMR and surface plasmon resonance-based binding techniques. Competitive binding assays and molecular modeling suggest that EGCG docks at the hydrophobic groove close to the catalytic center of PP1c, partially overlapping with the binding surface of microcystin-LR or okadaic acid. This hydrophobic interaction is further stabilized by hydrogen bonding via hydroxyl/oxo groups of EGCG to PP1c residues. Comparative docking shows that EGCG binds to PP2Ac in a similar manner, but in a distinct pose. Long-term treatment (24 h) with these compounds and other catechins suppresses the viability of HeLa cells with a relative effectiveness reminiscent of their in vitro PP1c-inhibitory potencies. The above data imply that the phosphatase-inhibitory features of these polyphenols may be implicated in the wide spectrum of their physiological influence.

Posted on 13 February 2012 | 12:59 pm

Determinants for substrate specificity of the bacterial PP2C protein phosphatase tPphA from Thermosynechococcus elongatus

Members of the Mg2+- or Mn2+-dependent protein phosphatases/PP2C-like serine/threonine phosphatases (PPM/PP2C) are abundant and widely distributed in prokaryotes and eukaryotes, where they regulate diverse signal transduction pathways. Despite low sequence conservation, the structure of their catalytic core is highly conserved except for a flexible loop termed the flap subdomain. Bacterial PPM/PP2C members without C- or N-terminal regulatory domains still recognize their substrates. Based on the crystal structure of tPphA (a PPM/PP2C member from the cyanobacterium Thermosynechococcus elongatus), variants of tPphA were generated by site-directed mutagenesis to identify substrate specificity determinants. Furthermore, a PPM/PP2C chimera containing the tPphA catalytic core and the flap subdomain of human PP2C? was also generated. tPphA variants and the chimera were tested towards different artificial substrates and native phosphorylated PII. A binding assay combining chemical crosslinking and pull-down was designed to analyze the binding of the various phosphatase variants to phosphoprotein PII. Together, these data showed that the metal 1–metal 2 cluster in the catalytic center, but not the catalytically active metal 3, is required for the binding of phosphorylated substrate. Residues outside the catalytic center are pivotal for the recognition and turnover of phosphorylated protein substrate. In particular, a histidine residue (His39) of tPphA was identified to play a specific role in protein substrate dephosphorylation. Furthermore, mutations in the variable flap subdomain can affect enzyme activity as well as substrate specificity.Structured digital abstract•?tPphA binds to P-II by cross-linking study (View Interaction: 1, 2, 3, 4, 5, 6, 7, 8)

Posted on 23 January 2012 | 1:49 pm

Computational study of the covalent bonding of microcystins to cysteine residues – a reaction involved in the inhibition of the PPP family of protein phosphatases

Microcystins (MCs) are cyclic peptides, produced by cyanobacteria, that are hepatotoxic to mammals. The toxicity mechanism involves the potent inhibition of protein phosphatases, as the toxins bind the catalytic subunits of five enzymes of the phosphoprotein phosphatase (PPP) family of serine/threonine-specific phosphatases: Ppp1 (aka PP1), Ppp2 (aka PP2A), Ppp4, Ppp5 and Ppp6. The interaction with the proteins includes the formation of a covalent bond with a cysteine residue. Although this reaction seems to be accessory for the inhibition of PPP enzymes, it has been suggested to play an important part in the biological role of MCs and furthermore is involved in their nonenzymatic conjugation to glutathione. In this study, the molecular interaction of microcystins with their targeted PPP catalytic subunits is reviewed, including the relevance of the covalent bond for overall inhibition. The chemical reaction that leads to the formation of the covalent bond was evaluated in silico, both thermodynamically and kinetically, using quantum mechanical-based methods. As a result, it was confirmed to be a Michael-type addition, with simultaneous abstraction of the thiol hydrogen by a water molecule, transfer of hydrogen from the water to the ?,?-unsaturated carbonyl group of the microcystin and addition of the sulfur to the ?-carbon of the microcystin moiety. The calculated kinetics are in agreement with previous experimental results that had indicated the reaction to occur in a second step after a fast noncovalent interaction that inhibited the enzymes per se.

Posted on 9 January 2012 | 6:33 pm

Crystal structures of the Chromobacterium violaceum?-transaminase reveal major structural rearrangements upon binding of coenzyme PLP

SummaryThe bacterial ?-transaminase from Chromobacterium violaceum (Cv-?TA, EC 2.6.1.18) catalyzes industrially important transamination reactions by use of the coenzyme pyridoxal 5’-phosphate (PLP). Here, we present four crystal structures of Cv-?TA: two in the apo form, one in the holo form and one in an intermediate state, at resolutions between 1.35 and 2.4 Å. The enzyme is a homodimer with a molecular weight of approximately 100 kDa. Each monomer has an active site at the dimeric interface that involves amino acid residues from both subunits. The apo-Cv-?TA structure reveals unique “relaxed” conformations of three critical loops involved in structuring the active site, that have not previously been seen in a transaminase. Analysis of the four crystal structures reveals major structural rearrangements involving elements of the large and small domains of both monomers that reorganize the active site in the presence of PLP. The conformational change appears to be triggered by binding of the phosphate group of PLP. Furthermore, one of the apo structures shows a disordered “roof” over the PLP binding site, while in the other apo form and the holo form the “roof” is ordered. Comparison with other known transaminase crystal structures suggests that ordering of the “roof” structure may be associated with substrate binding in Cv-?TA and some other transaminases.Structured digital abstract-transaminases and -transaminases bind by dynamic light scattering (View interaction) -transaminase and -transaminase bind by x-ray crystallography (View interaction) -transaminase and -transaminase bind by x-ray crystallography (View interaction)

Posted on 6 January 2012 | 12:11 pm

Corrigendum

Posted on 23 January 2009 | 6:00 am

Corrigendum

Posted on 9 September 2008 | 7:00 am

Corrigendum

Posted on 30 July 2008 | 7:00 am