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Mitogen-Activated Protein Kinases (MAPKs) are signaling components highly conserved among eukaryotes. Their diverse biological functions include cellular differentiation and responses to different extracellular stress stimuli. Although some substrates of MAPKs have been identified in plants, no information is available about whether other amino acids in the primary sequence other than proline-directed phosphorylation (pS-P) contribute to kinase specificity towards substrates. Here, we used a random positional peptide library to search for consensus phosphorylation sequences for Arabidopsis MAPKs, MPK3 and MPK6. These experiments indicated a preference towards the sequence L/P-P/X-S-P-R/K for both kinases. After bioinformatic processing, a number of novel candidate MAPK substrates were predicted and subsequently confirmed by in vitro kinase assays using bacterially expressed native Arabidopsis proteins as substrates. MPK3 and MPK6 phosphorylated all tested proteins more efficiently than did another MAPK, MPK4. These results indicate that the amino acid residues in the primary sequence surrounding the phosphorylation site of Arabidopsis MAPK substrates can contribute to MAPK specificity. Further characterization of one of these new substrates confirmed that At1g80180.1 was phosphorylated in planta in a MAPK-dependent manner. Phenotypic analyses of Arabidopsis expressing phosphorylation site mutant forms of At1g80180.1 showed clustered stomata and higher stomatal index in cotyledons expressing the phosphomimetic form of At1g80180.1, providing a link between this new MAPK substrate and the defined role for MPK3 and MPK6 in stomatal patterning.
Silencing of GATA5 gene expression as a result of promoter hypermethylation has been observed in lung, gastrointestinal, and ovarian cancers. However, the regulation of GATA5 gene expression has been poorly understood. Here, we demonstrate that an enhancer (E)-box in the GATA5 promoter (bp -118 to -113 in mouse; bp -164 to -159 in the human) positively regulates GATA5 transcription by binding upstream stimulatory factor 1 (USF1). Using site-directed mutagenesis, EMSA, and affinity chromatography, we found that USF1 specifically binds to the E-box sequence (5’-CACGTG-3’), but not to a mutated E-box. CpG methylation of this E-box significantly diminished its binding of transcription factors. Mutation of the E-box within a GATA5 promoter fragment significantly decreased promoter activity in a luciferase reporter assay. Chromatin immunoprecipitation identified that USF1 physiologically interacts with the GATA5 promoter E-box in mouse intestinal mucosa, which has the highest GATA5 gene expression in mouse. Co-transfection with USF1 expression plasmid significantly increased GATA5 promoter-driven luciferase transcription. Furthermore, real-time and RT-PCR analyses confirmed that overexpression of USF1 activates endogenous GATA5 gene expression in human bronchial epithelial cells. This study presents the first evidence that USF1 activates GATA5 gene expression through E-box motif, and suggests a potential mechanism (disruption of the E-box) by which GATA5 promoter methylation reduces GATA5 expression in cancer.
Gene expression during oocyte maturation and early embryogenesis until zygotic genome activation requires translational activation of maternally-derived mRNAs. Embryonic poly(A) binding protein (EPAB) is the predominant poly(A) binding protein during this period in Xenopus, mouse, and human. In Xenopus oocytes, EPAB stabilizes maternal mRNAs and promotes their translation. To assess the role of EPAB in mammalian reproduction, we generated Epab knockout mice. While Epab-/- males and Epab+/- of both sexes were fertile, Epab-/- female mice were infertile, and could not generate embryos or mature oocytes in vivo or in vitro. Epab-/- oocytes failed to achieve translational activation of maternally-stored mRNAs upon stimulation of oocyte maturation, including Cyclin B1 and Dazl mRNAs. Microinjection of Epab mRNA into Epab-/- germinal vesicle stage oocytes did not rescue maturation, suggesting that EPAB is also required for earlier stages of oogenesis. In addition, late antral follicles in the ovaries of Epab-/- mice exhibited impaired cumulus expansion, and a 8-fold decrease in ovulation, associated with a significant down-regulation of mRNAs encoding EGF-like growth factors Areg, Ereg, and Btc, and their downstream regulators, Ptgs2, Has2, and Tnfaip6. Our findings indicate that EPAB is necessary for oogenesis, folliculogenesis, and female fertility in mice.
Humoral and tumoral factors collectively promote cancer-induced skeletal muscle wasting by increasing protein degradation. While several humoral proteins, namely TNF-α and IL-6, have been shown to induce skeletal muscle wasting, there is dearth of information regarding the tumoral factors that contribute to the atrophy of muscle during cancer cachexia. Therefore, we have characterized the secretome of C26 colon cancer cells to identify the tumoral factors involved in cancer-induced skeletal muscle wasting. In this report, we show that Myostatin, a procachectic TGF-β superfamily member, is abundantly secreted by C26 cells. Consistent with Myostatin signaling during cachexia, treating differentiated C2C12 myotubes with C26 conditioned medium (CM) resulted in myotubular atrophy due to the up-regulation of muscle-specific E3 ligases, Atrogin-1 and MuRF1, and enhanced activity of the ubiquitin-proteasome pathway. Furthermore, the C26 CM also activated ActRIIB/Smad and NF-κB signaling, and reduced activity of the IGF-1/PI3K/Akt pathway, three salient molecular features of Myostatin action in skeletal muscles. Antagonists to Myostatin prevented C26 CM-induced wasting in muscle cell cultures, further confirming that tumoral Myostatin may be a key contributor in the pathogenesis of cancer cachexia. Finally, we show that treatment with C26 CM induced the autophagy-lysosome pathway and reduced mitochondria number in myotubes. These two previously unreported observations were recapitulated in skeletal muscles collected from C26 tumor-bearing mice.
A new peptide trypsin inhibitor named BWI-2c was obtained from buckwheat (Fagopyrum esculentum) seeds by sequential affinity, ion exchange and reversed phase chromatography. The peptide was sequenced and found to contain 41 amino acid residues, with four cysteines involved in two intramolecular disulfide bonds. Recombinant BWI-2c identical to the natural peptide was produced in Escherichia coli in a form of a cleavable fusion with thioredoxin. The 3D structure of the peptide in solution was determined by NMR spectroscopy revealing two antiparallel α-helices stapled by disulfide bonds. Together with trypsin inhibitor from veronica (Veronica hederifolia; VhTI), BWI-2c represents a new family of protease inhibitors with the unusual α-helical hairpin fold. The linker sequence between the helices represents the so-called trypsin inhibitory loop responsible for direct binding to the active site of the enzyme that cleaves BWI-2c at the functionally important residue arginine-19. The inhibition constant was determined for BWI-2c against trypsin (1.7×10-10 M), and the peptide was tested on other enzymes including those from various insect digestive systems revealing high selectivity to trypsin-like proteases. Structural similarity shared by BWI-2c, VhTI and several other plant defense peptides leads to the acknowledgement of a new widespread family of plant peptides termed α-hairpinins.
CYP2B proteins in rat hepatocytes undergo NO-dependent proteolytic degradation, but the mechanisms and the reasons for the specificity towards only certain P450 enzymes are yet unknown. Here, we found that down-regulation of CYP2B proteins by the NO donor NOC-18 is accelerated by pretreatment of the hepatocytes with interleukin-1b (IL-1) in the presence of a nitric oxide synthase inhibitor, suggesting that an NO-independent action of IL-1 contributes to the lability of CYP2B proteins. The immunoproteasome subunit LMP2 was significantly expressed in hepatocytes under basal conditions, and IL-1 induced LMP2 within 6-12 h of treatment. CYP2B protein degradation in response to IL-1 was attenuated by the selective LMP2 inhibitor UK-101, but not by the LMP7 inhibitor IPSI. The results show that LMP2 contributes to the NO-dependent degradation of CYP2B proteins, and suggest that induction of LMP2 may be involved in the potentiation of this degradation by IL-1.
Autosomal-dominant missense mutations in leucine-rich repeat kinase 2 (LRRK2) are a common genetic cause of Parkinson’s disease (PD). LRRK2 is a multidomain protein with kinase and GTPase activities. Dominant mutations are found in the domains that have these two enzyme activities including the common G2019S mutation that increases kinase activity by 2-3 fold. However, there is also a genetic variant in some populations, G2385R, that lies in a C-terminal WD40 domain of LRRK2 and acts as a risk factor for PD. In this study we show that the G2385R mutation causes a partial loss of the kinase function of LRRK2 and deletion of the C-terminus completely abolishes kinase activity. This effect is strong enough to overcome the kinase activating effects of the G2019S mutation in the kinase domain. Hsp90 has an increased affinity to G2385R variant compare to wild type LRRK2 and inhibition of the chaperone binding combined with proteasome inhibition leads to association of mutant LRRK2 with high molecular weight native fractions that likely represent proteasome degradation pathways. The loss of function of G2385R correlates with several cellular phenotypes that have been proposed to be kinase dependent. These results suggest that the C-terminus of LRRK2 plays an important role in maintaining enzymatic function of the protein and that G2385R may be associated with PD in a way that is different from kinase activating mutations. These results may be important in understanding the differing mechanism(s) by which mutations in LRRK2 act and may also have implications for therapeutic strategies for PD.
Plants contain both cytosolic and chloroplastic glyceraldehyde-3-phosphate dehydrogenases (GAPDHs). In Arabidopsis thaliana, cytosolic GAPDH is involved in the glycolytic pathway and is represented by two differentially expressed isoforms (GapC1, GapC2) that are 98% identical in amino acid sequence. Here we show that GapC1 is a phosphorylating, NAD-specific GAPDH with enzymatic activity strictly dependent on Cys-149. Catalytic Cys-149 is the only solvent-exposed cysteine of the protein and its thiol is relatively acidic (pKa 5.7). This property makes GapC1 sensitive to oxidation by H2O2, which appears to inhibit enzyme activity by converting the thiolate of Cys-149 (-Sˉ) to irreversible oxidized forms (-SO2ˉ, -SO3ˉ) via a labile sulphenate intermediate (-SOˉ). Reduced glutathione (GSH) prevents this irreversible process by reacting with Cys-149 sulphenates to give rise to a mixed disulphide (Cys-149-SSG), as demonstrated by both mass spectrometry and biotinylated GSH. Glutathionylated GapC1 can be fully reactivated either by cytosolic glutaredoxin, via a GSH-dependent monothiol mechanism or, less efficiently, by cytosolic thioredoxins physiologically reduced by NADPH:thioredoxin reductase. Potential relevance of these findings is discussed in the light of the multiple functions of GAPDH in eukaryotic cells (e.g. glycolysis, control of gene expression, apoptosis) that appear to be influenced by the redox state of the catalytic Cys-149.
In humans, assembly of spliceosomal snRNPs begins in the cytoplasm where the multi-protein SMN complex mediates formation of a seven-membered ring of Sm proteins onto a conserved site of the snRNA. The SMN complex contains the survival of motor neuron (SMN) protein, Gemin2, and several additional 'Gemins' that participate in snRNP biosynthesis. SMN was first identified as the product of a gene found to be deleted or mutated in patients with the neurodegenerative disease spinal muscular atrophy (SMA), the leading genetic cause of infant mortality. Here, we report the solution structure of Gemin2 bound to the Gemin2-binding domain of SMN determined by NMR spectroscopy. This complex reveals the structure of Gemin2, how Gemin2 binds to SMN, and the roles of conserved SMN residues near the binding interface. Surprisingly, several conserved SMN residues, including the sites of two SMA patient mutations, are not required for binding to Gemin2. Instead, they form a conserved SMN/Gemin2 surface that may be functionally important for snRNP assembly. The SMN-Gemin2 structure explains how Gemin2 is stabilized by SMN and establishes a framework for structure-function studies to investigate snRNP biogenesis as well as biological processes involving Gemin2 that do not involve snRNP assembly.
Selenium is an essential trace element in mammals, but is toxic at high levels. It is best known for its cancer prevention activity, but cancer cells are more sensitive to selenite toxicity than normal cells. Since selenite treatment leads to oxidative stress, and the thioredoxin system is a major antioxidative system, we examined the interplay between thioredoxin reductase 1 (TR1) and thioredoxin 1 (Trx1) deficiencies and selenite toxicity in DT cells, a malignant mouse cell line, and the corresponding parental NIH3T3 cells. TR1 deficient cells were far more sensitive to selenite toxicity than Trx1-deficient or control cells. In contrast, this effect was not seen in cells treated with hydrogen peroxide, suggesting that the increased sensitivity of TR1 deficiency to selenite was not due to oxidative stress caused by this compound. Further analyses revealed that only TR1-deficient cells manifested strongly enhanced production and secretion of glutathione, which was associated with increased sensitivity of the cells to selenite. The data uncover a new role of TR1 in cancer that is independent of Trx reduction and compensated for by the glutathione system. The data also suggest that the enhanced selenite toxicity of cancer cells and simultaneous inhibition of TR1 can provide a new avenue for cancer therapy.
Mimitin, a novel mitochondrial protein, has been shown to act as a molecular chaperone for the mitochondrial complex I and to regulate ATP synthesis. During type 1 diabetes development proinflammatory cytokines induce mitochondrial damage in pancreatic beta cells, inhibit ATP synthesis, and reduce glucose-induced insulin secretion. Mimitin was expressed in rat pancreatic islets including beta cells and decreased by cytokines. In the ob/ob mouse, a model of insulin resistance and obesity, mimitin expression was downregulated in liver and brain, upregulated in heart and kidney, but not affected in islets. To further analyze the impact of mimitin on beta cell function two beta cell lines, one with a low (INS1E) and another with a higher mimitin expression (MIN6) were studied. Mimitin overexpression protected INS1E cells against cytokine-induced caspase-3 activation, mitochondrial membrane potential reduction and ATP production inhibition, independently from the NFkB-iNOS pathway. Mimitin overexpression increased basal and glucose-induced insulin secretion and prevented cytokine-mediated suppression of insulin secretion. Mimitin knock-down in MIN6 cells had opposite effects to those observed after overexpression. Thus mimitin has the capacity to modulate pancreatic islet function and to reduce cytokine toxicity.
A major multidrug transporter, MDR1, belonging to the Major Facilitator Superfamily (MFS) invariably contributes to an increased efflux of commonly used azoles and thus corroborates their direct involvement in multidrug resistance (MDR) in Candida albicans. The MDR1 protein with two transmembrane domains, each comprising six transmembrane helices, is interconnected with extracellular loops and intracellular loops (ICLs). The introduction of deletions and insertions through mutagenesis was used to address the role of the largest inter-domain ICL3 of the Mdr1 protein. Most of the progressive deletants, when overexpressed, eliminated the drug resistance. Notably, restoration of the length of the ICL3 by insertional mutagenesis did not restore the functionality of the protein. Interestingly, most of the insertion and deletant variants of ICL3 became amenable to trypsinization, yielding peptide fragments. The homology model of the Mdr1 protein showed that the molecular surface charge distribution was perturbed in most of the ICL3 mutant variants. Taken together, these results provide the first evidence that the central cytoplasmic loop (CCL) of the fungal MFS transporter of the DHA1 family is critical for the function of MDR. Unlike other homologous proteins, ICL3 has no apparent role in imparting substrate specificity or in the recruitment of the transporter protein.
Enteropathogenic (EPEC) and enterohemorrhagic Escherichia coli (EHEC) are attaching and effacing pathogens frequently associated with infectious diarrhoea. EPEC and EHEC use a type III secretion system (T3SS) to translocate effectors that subvert different cellular processes to sustain colonization and multiplication. The eukaryotic proteins Na+/H+ exchanger regulatory factor 2 (NHERF2) and Annexin A2 (AnxA2), which are involved in regulation of intestinal ion channels are recruited to the bacterial attachment sites. Using a stable HeLa-HA-NHERF2 cell line we found partial colocalization of AnxA2 and NHERF2; in EPEC infected cells AnxA2 and NHERF2 were extensively recruited to the site of bacterial attachment. We confirmed that NHERF2 dimerises and found that NHERF2 interacts with AnxA2. Moreover, we found that AnxA2 also binds both the N- and C-terminal domains of the bacterial efector Tir through its C-terminal domain. Immunofluorescence of HeLa cells infected with EPEC showed that AnxA2 is recruited to the site of bacterial attachment in a Tir-dependent manner, but independently of Tir-induced actin polymerization. Our results suggest that AnxA2 and NHERF2 form a scaffold complex that links adjacent Tir molecules at the plasma membrane forming a lattice that could be involved in retention and dissemination of other effectors at the bacterial attachment site.
Actin dynamics are implicated in various cellular processes, not only through the regulation of cytoskeletal organization, but also via the control of gene expression. Here we show that the Src family kinase substrate p130Cas influences actin remodeling and concomitant muscle-specific gene expression, thereby regulating myogenic differentiation. In C2C12 myoblasts, silencing of p130Cas expression by RNA interference impaired F-actin formation and nuclear localization of the SRF co-activator MAL following the induction of myogenic differentiation. Consequently, formation of multinucleated myotubes was abolished. Re-introduction of wild-type p130Cas, but not its phosphorylation-defective mutant, into p130Cas-knockdown myoblasts restored F-actin assembly, MAL nuclear localization and myotube formation. Depletion of the adhesion molecule integrin b3, a key regulator of myogenic differentiation as well as actin cytoskeletal organization, attenuated p130Cas phosphorylation and MAL nuclear localization during C2C12 differentiation. Moreover, knockdown of p130Cas led to the activation of the F-actin severing protein cofilin. The introduction of a dominant-negative mutant of cofilin into p130Cas-knockdown myoblasts restored muscle-specific gene expression and myotube formation. These results suggest that p130Cas phosphorylation, mediated by integrin b3, facilitates cofilin inactivation and promotes myogenic differentiation through modulating actin cytoskeleton remodeling.
In Saccharomyces cerevisiae the Pho84 phosphate transporter acts as the main provider of phosphate to the cell using a proton symport mechanism, but also mediates rapid activation of the protein kinase A (PKA) pathway. These two features led to recognition of Pho84 as a transceptor. Although the physiological role of Pho84 has been studied in depth, the mechanisms underlying the transport- and sensor function are unclear. To obtain more insight into the structure-function relationships of Pho84, we have rationally designed and analyzed site-directed mutants. Using a three-dimensional model of Pho84 created on the basis of the GlpT permease, complemented with multiple-sequence alignments, we selected Arg-168 and Lys-492, and Asp-178, Asp-358 and Glu-473 as residues potentially involved in phosphate or proton binding, respectively, during transport. We found that Asp-358 (Helix 7) and Lys-492 (Helix 11) are critical for the transport function, and might be part of the putative substrate binding pocket of Pho84. Moreover, we show that alleles mutated in the putative proton-binding site Asp-358 are still capable of strongly activating PKA pathway targets despite their severely reduced transport activity. This indicates that signaling does not require transport and suggests that mutagenesis of amino acid residues involved in binding of the cotransported ion may constitute a promising general approach to separate the transport and signaling functions in transceptors.
The TRPM7 channel has been shown to play a pivotal role in cell survival during brain ischemia as well as in the survival of other cell types challenged with apoptotic stimuli. Ca2+ is thought to be central to the channel’s ability to regulate reactive oxygen species (ROS) production. However, channel-mediated entry of Mg2+ and Zn2+ have also been implicated in cell death. Here we show that depletion of TRPM7 by RNA interference in fibroblasts increases cell resistance to apoptotic stimuli by decreasing ROS levels in a Mg2+-dependent manner. Depletion of TRPM7 lowered cellular Mg2+, decreased the concentration of ROS and lessened p38 MAP kinase and JNK activation as well as decreased caspase-3 activation and PARP cleavage in response to apoptotic stimuli. Re-expression of TRPM7 or of a kinase-inactive mutant of TRPM7 in TRPM7-knockdown cells increased cellular Mg2+ and ROS levels, as did expression of the Mg2+ transporter SLC41A2. In addition, expression of SLC41A2 increased TRPM7-knockdown cells’ sensitivity to apoptotic stimuli as well as boosted ROS generation in response to cell stress. Together these data uncover an essential role for Mg2+ in TRPM7’s control of cell survival and in the regulation of cellular ROS levels.
MEKK1 is a mitogen activated protein kinase kinase kinase (MAP3K) that regulates MAPK activation, and is the only known mammalian kinase that is also a ubiquitin ligase. MEKK1 contains a RING domain within its amino-terminal regulatory region, and MEKK1 has been shown to ubiquitylate the AP-1 transcription factor protein c-Jun, but the mechanism by which MEKK1 interacts with c-Jun to induce ubuiquitylation has not been defined. Proximal to RING domain is a SWI2/SNF2 and MuDR (SWIM) domain of undetermined function. In this report, we demonstrate that the MEKK1 SWIM domain, but not the RING domain directly associates with the c-Jun DNA binding domain, and that the SWIM domain is required for MEKK1-dependent c-Jun ubiquitylation. We further show that this MEKK1 SWIM/Jun interaction is specific, as SWIM domains from other proteins failed to bind c-Jun. We reveal that although the Jun and Fos DNA-binding domains are highly conserved, the MEKK1 SWIM domain does not bind Fos. Finally, we identify the sequence unique to Jun proteins required for specific interaction with the MEKK1 SWIM domain. Therefore we propose that the MEKK1 SWIM domain represents a novel substrate-binding domain necessary for direct interaction between c-Jun and MEKK1 that promotes MEKK1-dependent c-Jun ubiquitylation.
DNA damage detection and repair take place in the context of chromatin, and histone proteins play important roles in these events. Post-translational modifications of histone proteins are involved in repair and DNA damage signalling processes in response to genotoxic stresses. In particular, histone H3 and H4 acetylation plays important roles in the mammalian and yeast DNA damage response and survival under genotoxic stress. However, the role of post-translational modifications to histones during the plant DNA damage response is currently poorly understood. Several different acetylated H3 and H4 N-terminal peptides following X-ray treatment were identified using MS analysis of purified histones, revealing previously unseen patterns of histone acetylation in Arabidopsis. Immunoblot analysis revealed an increase in relative abundance of the H3 acetylated N-terminus, and a global decrease in hyperacetylation of H4 in response to DNA damage induced by X-rays. Conversely, mutants in the key DNA damage signalling factor ATAXIA TELANGIECTASIA MUTATED (ATM) display increased histone acetylation upon irradiation, linking the DNA damage response with dynamic changes in histone modification in plants.
N-linked glycosylation is a critical determinant of protein structure and function, regulating processes such as protein folding, stability, localization, ligand-receptor binding and intracellular signaling. The type II TGF-b receptor (TbRII) plays a crucial role in the TGF-b signaling pathway. Although N-linked glycosylation of TbRII was first demonstrated over a decade ago, it was unclear how this modification influenced TbRII biology. Here, we show that inhibiting N-linked glycosylation process successfully hinders binding of TGF-b1 to TbRII and subsequently renders cells resistant to TGF-b signaling. Lung cancer cell line A549, gastric carcinoma cell line MKN1 and immortal cell line HEK293 exhibit reduced TGF-b signaling when either treated with two inhibitors, including tunicamycin (a potent N-linked glycosylation inhibitor) and kifunensine (an inhibitor of ER and Golgi mannosidase I family members), or introduced with the non-glycosylated mutant version of TbRII. We demonstrate that defective N-linked glycosylation prevents TbRII proteins from being transported to the cell surface. Moreover, we clearly show that not only complex type but also high-mannose type of TbRII can be localized on the cell surface. Collectively, these findings demonstrate that N-linked glycosylation is essentially required for the successful cell surface transportation of TbRII, suggesting a novel mechanism, which the TGF-b sensitivity can be regulated by N-linked glycosylation levels of TbRII.
E2 conjugating enzymes are the central enzymes in the ubiquitination pathway and are responsible for the transfer of ubiquitin and ubiquitin-like proteins onto target substrates. The secondary structural elements of the catalytic domain of these enzymes is highly conserved, including the sequence conservation of a three-residue HPN motif located upstream of the active site cysteine residue used for ubiquitin conjugation. Despite the vast structural knowledge of E2 enzymes, the catalytic mechanism of these enzymes remains poorly understood, in large part due to variation in the arrangements of the residues in the HPN motif in existing E2 structures. In this work, we used the E2 enzyme HIP2 to probe the structures of the HPN motif in several other E2 enzymes. A combination of chemical shift analysis, determination of the histidine protonation states and amide temperature coefficients were used to determine the orientation of the histidine ring and hydrogen bonding arrangements within the HPN motif. Unlike many three-dimensional structures, we find that a conserved hydrogen bond between the histidine imidazole ring and the asparagine backbone amide proton, a common histidine protonation state, and a common histidine orientation exists for all E2 enzymes examined. These results indicate that the histidine within the HPN motif is oriented to structurally stabilize a tight turn motif in all E2 enzymes and is not oriented to interact with the asparagine side chain as proposed in some mechanisms. These results suggest that a common catalysis mechanism likely exists for all E2 conjugating enzymes to facilitate ubiquitin transfer.
Activated Cdc42-associated tyrosine kinase (ACK or TNK2) is activated in response to multiple cellular signals, including cell adhesion, growth factor receptors and heterotrimeric G-protein coupled receptor signaling. However, the molecular mechanism underlying activation of ACK remains largely unclear. In this report, we demonstrated that interaction of the SH3 domain with the EGFR-binding domain (EBD) in ACK1 forms an auto-inhibition of the kinase activity. Release of this auto-inhibition is a key step for activation of ACK1. Mutation of the SH3 domain caused activation of ACK1 independent of cell adhesion, suggesting that cell adhesion-mediated activation of ACK1 is through releasing the auto-inhibition. A region at the N-terminus of ACK1 (L10-L14) is essential for cell adhesion-mediated activation. In activation of ACK1 by EGFR signaling, Grb2 mediates the interaction of ACK1 with EGFR through binding to the EBD and activates ACK1 by releasing the auto-inhibition. Furthermore, we found that mutation of serine 445 to proline caused constitutively activation of ACK1. Taken together, our studies have revealed a novel molecular mechanism underlying activation of ACK1.
Urea is exploited as a nitrogen source by bacteria, and its breakdown products, ammonia and bicarbonate, are employed to counteract stomach acidity in pathogens such as Helicobacter pylori. Uptake in the latter is mediated by UreI, a Urea Amide Channel family member. Here we describe the structure and function of UACBc, a homologue from Bacillus cereus. The purified channel was found to be permeable not only to urea but also to other small amides. Circular dichroism and infrared spectroscopy revealed a structure comprising mainly α-helices, oriented approximately perpendicular to the membrane. Consistent with this finding, site-directed fluorescent labelling indicated the presence of 7 transmembrane (TM) helices, with a cytoplasmic C-terminus. In detergent UACBc exists largely as a hexamer, as demonstrated both by cross-linking and size exclusion chromatography. A 9 Å resolution projection map obtained by electron cryomicroscopy of 2-D crystals shows that the six protomers are arranged in a planar hexameric ring. Each exhibits six density features attributable to TM helices, surrounding a putative central channel, while an additional helix is peripherally located. Bioinformatic analyses allowed individual TM regions to be tentatively assigned to the density features, the resultant model enabling identification of residues likely to contribute to channel function.
The permeabilized cells and muscle fibers technique allows to study the functional properties of mitochondria without their isolation, thus preserving intact all contacts with cellular structures, mostly cytoskeleton, to study the whole population of mitochondria in the cell in their natural surrounding and is increasingly used both in experimental and clinical studies. The functional parameters (affinity for ADP in regulation of respiration) of mitochondria in permeabilized myocytes or myocardial fibers are very different from those in isolated mitochondria in vitro. In this article we analyze the data showing the dependence of this parameter upon muscle contractile state. Most remarkable is the effect of recently described calcium- independent contraction of permeabilized muscle fibers induced by elevated temperatures (30-37°C). We show that very similar strong spontaneous calcium-independent contraction can be produced by proteolytic treatment of permeabilized muscle fibers that resulting in a disorganization of mitochondrial arrangement leading to significant increase of affinity for ADP. These data show that calcium-insensitive contraction may be related to the destruction of cytoskeleton structures by intracellular proteases. Therefore, use of their inhibitors is strongly advised at the step of permeabilization with careful washing of fibers or cells afterwards. Possible physiologically relevant relationship between calcium-regulated, ATP-dependent contraction and mitochondrial functional parameters is also discussed.
In higher plants biosynthesis of cysteine is catalyzed by O-acetylserine(thiol)lyase (OAS-TL), which replaces the activated acetyl group of O-acetylserine with sulfide. The enzyme is present in cytosol, plastids and mitochondria of plant cells. Solely the knockout of mitochondrial OAS-TL activity (oastlC) leads to significant reduction of growth in Arabidopsis thaliana. The reason for this phenotype is still enigmatic, since mitochondrial OAS-TL accounts only for approximately 5 % of total OAS-TL activity. In this study we demonstrate that sulfide specifically intoxicates complex IV activity but not electron transport through complex II + III in isolated mitochondria of oastlC plants. Loss of mitochondrial OAS-TL activity resulted in significant inhibition of dark respiration under certain developmental conditions. The abundance of mitochondrial encoded proteins and iron-sulfur cluster containing proteins was not affected in oastlC. Furthermore, oastlC seedlings were insensitive to cyanide, which is detoxified by b-cyano-alanine synthase in mitochondria at the expense of cysteine. These results indicate that in-situ biosynthesis of cysteine in mitochondria is not mandatory for translation, Fe-S cluster assembly and cyanide detoxification. Finally, we uncover an OAS-TL-independent detoxification system for sulfide in mitochondria of Arabidopsis that allows oastlC plants to cope with high sulfide levels caused by abiotic stresses.
The citrullination of enolase by peptidylarginine deiminase (PAD) has emerged as an important post-translational modification in human disorders; however, the physiological function of citrullination remains unknown. Here, we report that citrullination diversely regulates the biological functions of α-enolase (ENO1) and neuron-specific enolase (NSE). We developed three mouse IgG1 monoclonal antibodies with specificity to the following: (1) citrullination of arginine at position 9 of ENO1 (ENO1Cit9; anti-CE1), (2) citrullination of arginine at position 9 in ENO1 and NSE (ENO1Cit9/NSECit9; anti-CE1/2), and (3) citrullination of arginine at position 429 of NSE (NSECit429; anti-CE2). Regardless of the total protein expression level, the levels of ENO1Cit9 and NSECit429 were elevated, and their immunoreactivities were also increased in cortical neuronal cells or around blood vessels in the frontal cortex of patients with sporadic Creutzfeldt-Jakob disease and Alzheimer’s disease compared to controls. In a time- and dose-dependent manner, PAD negatively regulated enolase activity via citrullination, and enolase in diseased patients was more inactive than in controls. Interestingly, the citrullination of enolase effectively promoted its proteolytic degradation by calcium-dependent calpain-1, and leupeptin (calpain inhibitor I) abrogated this degradation. Surprisingly, using an affinity assay, the citrullination of enolase enhanced its plasminogen binding affinity, which was blocked by the lysine analog ε-aminocaproic acid. These findings suggest that PAD-mediated citrullination regulates the diverse physiological activities of enolase and that citrullinated enolase may be a candidate diagnostic/prognostic factor for degenerative diseases.
Glucose transporter GLUT4 is responsible for the insulin-induced uptake of glucose by muscle and fat cells. In non-stimulated (basal) cells, GLUT4 is retained intracellularly, while insulin stimulation leads to its translocation from storage compartments towards the cell surface. How GLUT4 is retained intracellularly is largely unknown. Previously, aberrant GLUT4 N-glycosylation has been linked to increased basal cell surface levels while N-glycosylation-deficient GLUT4 was found to be quickly degraded. As recycling and degradation of GLUT4 are positively correlated, we hypothesized that incorrect N-glycosylation of GLUT4 might reduce its intracellular retention resulting in an increased cell surface recycling, in increased basal cell surface levels, and in enhanced GLUT4 degradation. Here, we have studied N-glycosylation-deficient GLUT4 in detail in 3T3-L1 preadipocytes, 3T3-L1 adipocytes, and L6 myoblasts. We have found no alterations in retention, insulin response, internalization, or glucose transport activity. Degradation of the mutant molecule was increased, though once present at the cell surface, its degradation was identical to that of wild-type GLUT4. Our findings indicate that N-glycosylation is important for efficient trafficking of GLUT4 to its proper compartments, but once the transporter has arrived there, N-glycosylation plays no further major role in its intracellular traffic, nor in its functional activity.
Legionella pneumophila is an opportunistic pathogen and the causative agent of Legionnaires’ disease. Despite being exposed to many chemical compounds in its natural and man-made habitats (natural aquatic biotopes and man-made water systems), L. pneumophila is able to adapt and survive in these environments. The molecular mechanisms by which this bacterium detoxifies these chemicals remain poorly understood. In particular, the expression and functions of xenobiotic-metabolizing enzymes (XME) that could contribute to chemical detoxification in L. pneumophila have been poorly documented at the molecular and functional levels. We report here the identification and biochemical and functional characterization of a unique acetyltransferase that metabolizes aromatic amine chemicals in three characterized clinical strains of L. pneumophila (Paris, Lens, and Philadelphia). Strain-specific sequence variations in this enzyme, an atypical member of the arylamine N-acetyltransferase family (EC 2.3.1.5), produce enzymatic variants with different structural and catalytic properties. Functional inactivation and complementation experiments showed that this acetyltransferase allows L. pneumophila to detoxify aromatic amine chemicals and grow in their presence. Our study provides a new enzymatic mechanism by which the opportunistic pathogen L. pneumophila biotransforms and detoxifies toxic aromatic chemicals. These data also emphasize the role of xenobiotic-metabolizing enzymes in the environmental adaptation of certain prokaryotes.
The conventional analysis of enzyme evolution is to regard one single salient feature as a measure of fitness, expressed in a milieu exposing the possible selective advantage at a given time and location. Given that a single protein may serve more than one function, fitness should be assessed in several dimensions. Here we have explored individual mutational steps leading to a triple-point-mutated human GST A2-2 displaying enhanced activity with azathioprine. Eight alternative substrates were used to monitor the diverse evolutionary trajectories. The epistatic effects of mutations on catalytic activities were variable in sign and magnitude and depended on the substrate used, showing that epistasis is a multidimensional quality. Evidently, the multidimensional fitness landscape can lead to alternative trajectories resulting in enzymes optimized for features other than the selectable markers relevant at the origin of the evolutionary process. In this manner the evolutionary response is robust and can adapt to changing environmental conditions.
Eukaryotic phosphofructokinase, a key regulatory enzyme in glycolysis, has homologous N- and C-terminal domains thought to result from duplication, fusion, and divergence of an ancestral prokaryotic gene. It has been suggested that both the active site and the fructose 2,6-P2 allosteric site are formed by opposing N- and C-termini of subunits oriented antiparallel in a dimer. On the contrary, we show here that in fact the N-terminal halves form the active site, since expression of the N-terminal half of the enzymes from Dictyostelium discoideum and human muscle in phosphofructokinase-deficient yeast restored growth on glucose. However, the N-terminus alone was not stable in vitro. The C-terminus is not catalytic but is needed for stability of the enzyme, as is the connecting peptide that normally joins the two domains (here included in the N-terminus). Co-expression of homologous, but not heterologous, N- and C-termini yielded stable, fully active enzymes in vitro with sizes and kinetic properties similar to those of the wild type tetrameric enzymes. This indicates that the separately translated domains can fold sufficiently well to bind to each other, that such binding of complementary domains is stable and that the alignment is sufficiently accurate and tight as to preserve metabolite binding sites and allosteric interactions.
Candida albicans RCH1 encodes a protein of 10-transmembrane domains, homologous to human SLC10A7, and localizes in the plasma membrane. Deletion of RCH1 confers hypersensitivity to high concentrations of extracellular calcium and tolerance to azoles and lithium, which phenocopies the deletion of CaPMC1 encoding the vacuolar calcium pump. Additive to CaPMC1 mutation, lack of RCH1 alone shows an increase in calcium sensitivity, calcium uptake and cytosolic calcium level. The calcium hypersensitivity is abolished by cyclosporin A and magnesium. In addition, deletion of RCH1 elevates the expression of CaUTR2, a downstream target of the calcium/calcineurin signaling. Mutational and functional analysis indicates that the Rch1p TM8 domain, but not the TM9 and TM10 domains, are required for its protein stability, cellular functions and subcellular localization. Therefore, Rch1p is a novel regulator of cytosolic calcium homeostasis, which expands the functional spectrum of the vertebrate SLC10 family.
S-(2-succino)-cysteine (2SC) is a chemical modification formed by a Michael addition reaction of fumarate with cysteine residues in proteins. Formation of 2SC, termed succination of proteins, increases in adipocytes grown in high glucose medium and in adipose tissues of type 2 diabetic mice. However, the metabolic mechanisms leading to increased fumarate and succination of protein in the adipocyte are unknown. Treatment of 3T3 cells with high glucose (30 mM vs. 5 mM) caused a significant increase in cellular ATP/ADP, NADH/NAD+, and the mitochondrial membrane potential. There was also a significant increase in cellular fumarate concentration and succination of proteins, which may be attributed to the increase in NADH/NAD+ and subsequent inhibition of Krebs cycle NAD+-dependent dehydrogenases. Chemical uncouplers, which dissipated the mitochondrial membrane potential and reduced the NADH/NAD+ ratio, also decreased fumarate concentration and protein succination. High glucose plus metformin, an inhibitor of Complex I in the electron transport chain, caused an increase in fumarate and succination of protein. Thus, excess fuel supply (glucotoxicity) appears to create a pseudohypoxic environment (high NADH/NAD+ without hypoxia), which drives the increase in succination of protein. We propose that increased succination of proteins is an early marker of glucotoxicity and mitochondrial stress in adipose tissue in diabetes.
Caenopores are antimicrobial and pore-forming polypeptides of C. elegans belonging to the saposin-like protein superfamily and considered important elements of the nematode´s intestinal immune system. Here, we demonstrate that unlike the other members of the multifarious gene family (spps) coding for caenopores, spp-12 is expressed exclusively in two pharyngeal neurons. Recombinantly expressed SPP-12 binds to phospholipid membranes and forms pores in a pH-dependent manner characteristic for caenopores. Moreover, SPP-12 kills viable gram-positive bacteria, yeast cells and amoebae by permeabilizing their membranes suggesting a wide target cell spectrum. A spp-12 knock-out mutant is more susceptible to pathogenic Bacillus thuringiensis than wild-type worms and is tolerant to non-pathogenic bacteria. By contrast, SPP-1, a caenopore which gene is expressed in the intestine only and reported to be regulated by the same pathway as spp-12, is apparently non-protective against pathogenic B. thuringiensis, although displaying antimicrobial activity as well. The transcription of spp-1 is down-regulated in wild-type worms in the presence of pathogenic B. thuringiensis and a spp-1 knock-out mutant is hyposusceptible to this bacterium. This implies that SPP-12 but not SPP-1 contributes to resistance against B. thuringiensis, a natural pathogen of the nematode.
Rac1, a small GTPase, regulates macrophage matrix metalloproteinase-9 (MMP-9) in an ERK- and SP-1-dependent manner. SP-1 contains a PEST domain that may modulate protein stability. We hypothesize that T578, S586, and/or S587 in the PEST domain are required for SP-1 stability and MMP-9 expression secondary to activation of ERK, a serine/threonine kinase. We determined the effects of Rac1 and ERK on MMP-9 expression driven by SP-1WT and SP-1 mutants, T578A, S586A and S587A. Expression of WT and mutant SP-1 increased MMP-9 promoter activity in alveolar macrophages. However, constitutively active Rac1 suppressed MMP-9 promoter activity in cells expressing SP-1WT, SP-1T578A, and SP-1S587A, but not SP-1S586A. Furthermore, constitutive ERK activation, which was inhibited by Rac1, significantly increased MMP-9 transcription in cells expressing SP-1WT but not SP-1S586A. Because Rac1 activation and ERK inactivation increased degradation of SP-1WT and not SP-1S586A, our results suggest that SP-1 stability mediated at S586 regulates MMP-9 transcription. Ex vivo, alveolar macrophages obtained from asbestosis patients had less MMP-9 expression that was associated with decreased SP-1 expression and ERK activation. These observations demonstrate that S586 in the PEST domain of SP-1 is important for MMP-9 gene expression in alveolar macrophages and highlight the importance of these proteins in pulmonary fibrosis.
The uncharacterized α/β-hydrolase protein OLEI01171 from the psychrophilic marine bacterium Oleispira antarctica belongs to the PF00756 family of putative esterases, which also includes human esterase D. Here we show that purified recombinant OLEI01171 exhibites high esterase activity against the model esterase substrate α-naphthyl acetate at 5 °C – 30 °C with maximal activity at 15 °C – 20 °C. The esterase activity of OLEI01171 was stimulated 3-8 times by the addition of chloride or several other anions (0.1 M – 1.0 M). Compared to mesophilic PF00756 esterases, OLEI01171 exhibited lower overall protein thermostability. Two crystal structures of OLEI01171 were solved at 1.75 and 2.1 Å resolution and revealed a classical Ser hydrolase catalytic triad and the presence of a chloride or bromide ion bound in the active site close to the catalytic Ser148. Both anions were found to coordinate a potential catalytic water molecule located in the vicinity of the catalytic triad His257. Our data suggest that the bound anion perhaps contributes to the polarization of the catalytic water molecule and increases the rate of the hydrolysis of an acyl-enzyme intermediate. Alanine replacement mutagenesis of OLEI01171 identified ten amino acid residues important for esterase activity. The replacement of Asn225 by Lys had no significant effect on the OLEI01171 activity or thermostability, but resulted in a detectable increase of activity at 35 °C to 45 °C. Our work has provided insight into the molecular mechanisms of activity of a cold-active and anion-activated carboxyl esterase.
Caspase-2 was initially identified as a neuronally expressed, developmentally down-regulated gene and has been shown to be required for neuronal death induced by several stimuli, including NGF deprivation and β-amyloid. In non-neuronal cells the PIDDosome, composed of caspase-2 and two death adaptor proteins, PIDD and RAIDD, has been proposed as the caspase-2 activation complex, although the absolute requirement for the PIDDosome is not clear. To investigate the requirement for the PIDDosome in caspase-2 dependent neuronal death we examined the necessity for each component in induction of active caspase-2 and in execution of caspase-2 dependent neuronal death. We find that both NGF deprivation and Aβ treatment of neurons induce active caspase-2 and that induction of this activity depends on expression of RAIDD but is independent of PIDD expression. We show that treatment of wild-type or PIDD null neurons with Aβ or NGF deprivation induces formation of a complex of caspase-2 and RAIDD. We also show that caspase-2 dependent execution of neurons requires RAIDD, not PIDD. Caspase-2 activity can be induced in neurons from PIDD null mice and NGF deprivation or Aβ utilize caspase-2 and RAIDD to execute death of these neurons.
We describe the 2.3 Å X-ray structure of alpha1-microglobulin (a1m), an abundant protein in human blood plasma, which reveals the beta-barrel fold typical for lipocalins with a deep pocket lined by four loops at its open rim. Loop #1 harbours the residue Cys34 that is responsible for covalent cross-linking with plasma IgA. A single disulfide bond between Cys72 and Cys169 connects the C-terminal segment to the beta-barrel as in many other lipocalins. The exposed imidazole side chains of His122 and His123 in loop #4 give rise to a double Ni2+-binding site together with a crystallographic neighbour. The closest structural relatives of a1m are the complement protein component C8gamma, the L-prostaglandin D synthase, and lipocalin 15, three other structurally characterized members of the lipocalin family in humans that have only distant sequence similarity. In contrast with these, a1m is initially expressed as a bifunctional fusion protein with the protease inhibitor bikunin. Neither the electron density nor ESI-MS provide evidence for a chromophore bound to the recombinant a1m, also known as "yellow-brown lipocalin". However, the three side chains of Lys92, Lys118, and Lys130 that were reported to be involved in covalent chromophore binding appear to be freely accessible to ligands accommodated in the hydrophobic pocket. A structural feature similar to the well known CP haem-binding motif indicates the presence of a haem-binding site within the loop region of a1m, which explains previous biochemical findings and supports a physiological role in haem scavenging as well as redox-mediated detoxification
Symplocarpus renifolius and Arum maculatum are known to produce significant heat during the course of their floral development but using different regulatory mechanisms i.e., homoeothermic versus transient thermogenesis. To further clarify the molecular basis of species-specific thermogenesis in plants, we have here analyzed the native structures and expression patterns of the mitochondrial respiratory components in S. renifolius and A. maculatum. Our comparative analysis using blue native polyacrylamide gel electrophoresis combined with nano LC-MS/MS has revealed that the constituents of the respiratory complexes in both plants were basically similar, but that several mitochondrial components appeared to be differently expressed in their thermogenic organs. Namely, complex II in S. renifolius was detected as a 340 kDa product, suggesting an oligomeric or supramolecular structure in vivo. Moreover, the expression of an external NAD(P)H dehydrogenase was found to be higher in A. maculatum than in S. renifolius whereas an internal NAD(P)H dehydrogenase was expressed to a similar level in both species. Alternative oxidase was detected as smear-like signals that were elongated on the first dimension with a peak at around 200 kDa in both species. The significance and implication of these data are discussed in terms of thermoregulation in plants.
The closely-related pathogenic Neisseria species N. meningitidis and N. gonorrhoeae are able to respire in the absence of oxygen, using nitrite as an alternative electron acceptor. Nitrite reductase (encoded by aniA) is tightly regulated by four transcriptional regulators: FNR, NarP, FUR and NsrR. The four regulators control expression of aniA in N. meningitidis by binding to specific and distinct regions of the promoter. We show here that FUR and NarP are both required for induction of expression of aniA in N. meningitidis, and that they bind adjacent to one another in a non-cooperative manner. Activation via FUR/NarP is dependent on their topological arrangement relative to the RNA polymerase binding site. Analysis of the sequence of the aniA promoters from multiple N. meningitidis and N. gonorrhoeae strains indicates that there are species-specific single nucleotide polymorphisms, in regions predicted to be important for regulator binding. These sequence differences alter both the in vitro DNA binding and the promoter activation in intact cells by key activators FNR (oxygen sensor) and NarP (which is activated by nitrite in N. meningitidis). The weak relative binding of FNR to the N. gonorrhoeae aniA promoter (compared to N. meningitidis) is compensated for by a higher affinity of the gonococcal aniA promoter for NarP. Despite containing nearly identical genes for catalysing and regulating denitrification, variations in the promoter for the aniA gene appear to have been selected to enable the two pathogens to tune differentially their responses to environmental variables during the aerobic-anaerobic switch.
A disintegrin and metalloproteinase 17 (ADAM17) is a membrane-bound protease that
cleaves various cell surface proteins including cytokines and cytokine receptors.
Recently it was shown that ADAM17 is highly expressed on the surface of many cancer
cells whereas normal cells express low levels of ADAM17, implying ADAM17 as a
potential immunotherapeutic target. We have generated a monoclonal antibody against
human ADAM17, which recognized the membrane proximal cysteine-rich extension of
the ADAM17 protein. Unlike normal cells, tumor cell lines such as a prostate cancer
cell line, pancreatic cancer cell lines, a breast cancer cell line and a non-small lung
cancer cell line expressed ADAM17 on the cell surface. Using the sequence of the
antibody we generated an ADAM17-specific single-chain antibody (scFv) and fused
this to a CD3-specific scFv to generate a bispecific T-cell engager antibody
(A300E-BiTE). Specificity was demonstrated on cells in which ADAM17 was knocked
down with a specific shRNA. A300E-BiTE recognized ADAM17 and CD3 on the cell
surface of tumor cells and T-cells, respectively. In the presence of primary human
peripheral blood mononuclear cells or human T-cells addition of A300E-BiTE led to
ADAM17 specific killing of prostate tumor cells indicating a novel strategy for the
treatment of cancer.
Aquaporins (AQPs) are conserved in all kingdoms of life and facilitate the rapid diffusion of water and/or other small solutes across cell membranes. Amongst the different plant AQPs, Plasma Membrane Intrinsic proteins (PIPs), which fall into two phylogenetic groups, PIP1 and PIP2, play key roles in plant water transport processes. PIPs form tetramers in which each monomer acts as a functional channel. The intermolecular interactions that stabilize PIP oligomer complexes and are responsible for the resistance of PIP dimers to denaturating conditions are not well characterized. Here, we identified a highly conserved cysteine residue in loop A of PIP1 and PIP2 proteins and demonstrated by mutagenesis that it is involved in the formation of a disulphide bond between two monomers. While this cysteine seems not to be involved in regulation of trafficking to the plasma membrane, activity, substrate selectivity or oxidative gating of Zea mays ZmPIP1s, ZmPIP2s and hetero-oligomers, it increases oligomer stability under denaturating conditions. In addition, when PIP1 and PIP2 are co-expressed, the loop A cysteine of ZmPIP1;2, but not that of ZmPIP2;5, is involved in the mercury sensitivity of the channels.
Homer is a family of cytoplasmic adaptor proteins that plays different roles in cell function, including the regulation of G protein-coupled receptors. These proteins contain an Ena/VASP homology 1 domain that binds to the PPXXF sequence motif, which is present in different Ca2+-handling proteins such as IP3 receptors and TRPC channels. Here we present evidence for a role of Homer proteins in the STIM1/Orai1 association, as well as in the TRPC1/IP3RII interaction, which might be of relevance in platelet function. Treatment of human platelets with thapsigargin or thrombin, results in Ca2+-independent association of Homer1 with TRPC1 and IP3RII. In addition, thapsigargin and thrombin enhanced association of Homer1 with STIM1 and Orai1 in a Ca2+-dependent manner. Interference with Homer function by introduction into cells of the synthetic PPKKFR peptide, which emulates the proline-rich sequences of the PPXXF motif, reduced both STIM1/Orai1 and TRPC1/ IP3RII associations, as compared to the introduction of the inactive PPKKRR peptide. The PPKKFR peptide attenuates thrombin-evoked Ca2+ entry and the maintenance of thapsigargin-induced store-operated Ca2+ entry. Finally, the PPKKFR peptide attenuated thrombin-induced platelet aggregation. These findings support an important role for Homer proteins in thrombin-stimulated platelet function, which is likely mediated by the support of agonist-induced Ca2+ entry.
The binding mechanism of a new class of lipid competitive, ATP non-competitive, p110a isoform selective phosphoinositide-3-kinase (PI3K) inhibitors has been elucidated. Using the novel technique of isoform reciprocal mutagenesis of non-conserved amino acids in the p110alpha and p110beta isoforms we have identified three unique binding mechanisms for the p110alpha-selective inhibitors, PIK-75, A-66S and J-32. Each inhibitor’s p110alpha-isoform selective binding was found to be due to interactions with different amino acids within p110. PIK-75 interaction bound the non-conserved region 2 amino acid, p110alpha S773, A-66S bound the region 1 non-conserved amino acid, p110alpha Q859, and J-32 binding had indirect interaction with K776 and I771. The isoform reciprocal mutagenesis technique is shown to be an important analytical tool for the rational design of isoform-selective inhibitors.
Polyubiquitin chains serve a variety of physiological roles. Typically, the chains are bound covalently to a protein substrate, and in many cases target it for degradation by the 26S proteasome. However, several studies have demonstrated the existence of free polyubiquitin chains which are not linked to a specific substrate. Several physiological functions have been attributed to these chains, among them playing a role in signal transduction and serving as storage of ubiquitin for utilization under stress. In this study, we have established a system for detection of free ubiquitin chains and monitoring their level under changing conditions. Using this system, we show that UFD4, a HECT domain ubiquitin ligase, is involved in free chains generation. We also show that generation of these chains is stimulated in response to a variety of stresses, particularly that caused by DNA damage. However, it appears that the stress-induced synthesis of free chains is catalyzed by a different ligase, HUL5, which is also a HECT domain E3.
Oocyte maturation and early embryonic development require the cytoplasmic polyadenylation and concomitant translational activation of stored maternal mRNAs. Embryonic poly(A)-binding protein (ePAB, also known as ePABP and PABPc1-like) is a multi-functional post-transcriptional regulator that binds to poly(A) tails. Here we find that ePAB is a dynamically modified phosphoprotein in Xenopus laevis oocytes and show by mutation that phosphorylation at a four residue cluster is required for oocyte maturation. We further demonstrate that these phosphorylations are critical for cytoplasmic polyadenylation but not for ePAB’s inherent ability to promote translation. Our data provide the first insight into the role of post-translational modifications in regulating PABP protein activity in vivo.
This study demonstrates the important structural features of ceramide required for proper regulation, binding and identification by both pro-apoptotic and anti-apoptotic Bcl-2 family proteins. The C4:C5 trans double bond has little influence on the ability of Bax and Bcl-xL to identify and bind to these channels. The stereochemistry of the head group and access to the amide N-H group of ceramide is indispensible for Bax binding indicating that Bax may be interacting with the polar portion of the ceramide channel facing the bulk phase. On the contrary, Bcl-xL binding to ceramide channels is tolerant of stereochemical changes in the head group. This study also revealed that Bcl-xL has an optimal interaction with long-chain ceramides that are elevated early in apoptosis while short-chain ceramides are not well regulated. Inhibitors specific for the hydrophobic groove of Bcl-xL including 2-methoxyantimycin A3, ABT-737, and ABT-263 provide insights into the region of Bcl-xL involved in binding to ceramide channels. Molecular docking simulations of the lowest energy binding poses of ceramides and Bcl-xL inhibitors to Bcl-xL were consistent with the results of our functional studies and propose potential binding modes.
The EphA4 receptor tyrosine kinase interacts with ephrin ligands to regulate many processes,
ranging from axon guidance and nerve regeneration to cancer malignancy. Thus, antagonists that
inhibit ephrin binding to EphA4 could be useful for a variety of research and therapeutic
applications. Here we characterize the binding features of three antagonistic peptides (KYL,
APY and VTM) that selectively target EphA4 among the Eph receptors. Isothermal titration
calorimetry analysis demonstrates that all three peptides bind to the ephrin-binding domain of
EphA4 with low micromolar affinity. Furthermore, the effects of a series of EphA4 mutations
suggest that the peptides interact in different ways with the ephrin-binding pocket of EphA4.
Chemical shifts observed by NMR spectroscopy upon binding of the KYL peptide involve many
EphA4 residues, consistent with extensive interactions and possibly receptor conformational
changes. Additionally, systematic replacement of each of the 12 amino acids of KYL and VTM
identify the residues critical for EphA4 binding. The peptides exhibit a long half-life in cell
culture medium, which with their substantial binding affinity and selectivity for EphA4 makes
them excellent research tools to modulate EphA4 function.
Cells in mechanically challenged environments cope with high amplitude exogenous forces that can lead to cell death but the mechanisms that mediate force-induced apoptosis and the identity of mechanoprotective cellular factors are not defined. We assessed apoptosis in 3T3 and HEK cells exposed to tensile forces applied through beta1 integrins. Apoptosis was mediated by Rac-dependent activation of p38alpha. Depletion of Pak1, a downstream effector of Rac, prevented force-induced p38 activation and apoptosis. Rac was recruited to sites of force transfer by filamin A, which inhibited force-induced apoptosis mediated by Rac and p38alpha. We conclude that in response to tensile force, filamin A regulates Rac-dependent signals, which induce apoptosis through Pak1 and p38.
β 2-GPI (β 2-glycoprotein I) is a plasma glycoprotein ascribed with an anti-angiogenic function; however, the biological role and molecular basis of its action in cell migration remain unknown. The aim of this study was to assess the contribution of β 2-GPI to HAEC (human aortic endothelial cell) migration and the details of its underlying mechanism. Using wound healing and Boyden chamber assays, we found that β 2-GPI inhibited endothelial cell migration, which was restored by the neutralizing antibody. NF-кB (nuclear factor-кB) inhibitors and lentiviral siRNA (small interfering RNA) silencing of NF-кB significantly attenuated the inhibitory effect of β 2-GPI on cell migration. Moreover, β 2-GPI was found to induce IκBa (inhibitor of NF-кB) phosphorylation, and translocation of p65 and p50. We further demonstrated that mRNA and protein levels of eNOS (endothelial nitric oxide synthase) and NO (nitric oxide) production were all increased by β2-GPI and these effects were remarkably inhibited by NF-кB inhibitors and siRNAs of p65 and p50. Furthermore, β2-GPI-mediated inhibition of cell migration was reversed by eNOS inhibitors and eNOS siRNAs. Our findings provide novel insight into the ability of β2-GPI to inhibit endothelial cell migration predominantly through NF-кB/eNOS/NO signaling pathway, which indicates a potential direction for clinical therapy in vascular diseases.
We demonstrate that the cytostatic and antiviral activity of pyrimidine nucleoside analogues is markedly decreased by a Mycoplasmahyorhinis infection and show that the phosphorolytic activity of the mycoplasmas is responsible for this. Since mycoplasmas are (i) an important cause of secondary infections in immunocompromised (e.g. HIV-infected) patients, and (ii) known to preferentially colonize tumor tissue in cancer patients, catabolic mycoplasma enzymes may compromise efficient chemotherapy of virus infections and cancer. In the genome of M. hyorhinis, a thymidine phosphorylase (TP) gene has been annotated. This gene was cloned, expressed in Escherichia coli and kinetically characterized. Whereas the mycoplasma TP efficiently catalyzes the phosphorolysis of thymidine (Km = 473 µM) and deoxyuridine (Km = 578 µM), it prefers uridine (Km = 92 µM) as a substrate. Our kinetic data and sequence analysis revealed that the annotated M. hyorhinis TP belongs to the nucleoside phosphorylase (NP)-II class pyrimidine nucleoside phosphorylases (PyNP), and is distinct from the NP-II class TP and NP-I class uridine phosphorylases (UP).M. hyorhinis PyNP also markedly differs from TP and UP in its substrate specificity towards therapeutic nucleoside analogues and susceptibility to clinically relevant drugs. Several kinetic properties of mycoplasma PyNP were explained by in silico analyses.
IL-6, an established growth factor for multiple myeloma cells, induces myeloma therapy resistance but the resistance mechanisms remain unclear. This study determines the role of IL-6 in re-establishing intracellular redox homeostasis in the context of myeloma therapy. IL-6 treatment increased myeloma cell resistance to agents that induce oxidative stress including ionizing radiation (IR) and dexamethasone (Dex). Relative to IR alone, myeloma cells treated with IL-6 plus IR demonstrated reduced annexin/propidium iodide staining, caspase-3 activation, PARP cleavage, and mitochondrial membrane depolarization with increased clonogenic survival. IL-6 combined with IR or Dex increased early, intracellular pro-oxidants levels that were causally related to activation of NF-kB as determined by the ability of N-acetyl-cysteine to suppress both pro-oxidant levels and NF-kB activation. In myeloma cells, upon combination with hydrogen peroxide treatment, relative to TNF-a, IL-6 induced an early perturbation in reduced glutathione level and increased NF-kB-dependent manganese superoxide dismutase (MnSOD) expression. Furthermore, knockdown of MnSOD suppressed the IL-6-induced myeloma cell resistance to radiation. MitoSOX Red staining showed that IL-6 treatment attenuated late mitochondrial oxidant production in irradiated myeloma cells. This study provides evidence that increases in MnSOD expression mediate IL-6-induced resistance to Dex and radiation in myeloma cells. Our results indicate that inhibition of antioxidant pathways could enhance myeloma cell responses to radiotherapy and/or chemotherapy.
Factor H (FH) with 20 short complement regulator (SCR) domains is a major serum regulator of complement, and genetic defects in this are associated with inflammatory diseases. Heparan sulphate is a cell surface glycosaminoglycan composed of sulphated S-domains and unsulphated NA-domains. To elucidate the molecular mechanism of binding of FH to glycosaminoglycans, we performed ultracentrifugation, X-ray scattering and surface plasmon resonance with FH and glycosaminoglycan fragments. Ultracentrifugation showed that FH formed up to 63% of well-defined oligomers with purified heparin fragments (equivalent to S-domains), and indicated a dissociation constant KD of about 0.5 µM. FH structures that are bivalently cross-linked at SCR-7 and SCR-20 with heparin explained the sedimentation coefficients of the FH-heparin oligomers. The X-ray radius of gyration RG of FH in the presence of heparin fragments 18 to 36 monosaccharide units long increased significantly from 10.4 to 11.7 nm, and the maximum lengths of FH increased from 35 nm to 40 nm, confirming that large compact oligomers had formed. Surface plasmon resonance of immobilised heparin with full-length FH gave KD values of 1-3 µM, and similar but weaker KDvalues of 4-20 µM for the SCR-6/8 and SCR-16/20 fragments, confirming co-operativity between the two binding sites. The use of minimally-sulphated heparan sulphate fragments that correspond largely to NA-domains showed much weaker binding, proving the importance of S-domains for this interaction. This bivalent and co-operative model of FH binding to heparan sulphate provided novel insights on the immune function of FH at host cell surfaces.
Salt-inducible kinase (SIK) 2 is a member of the AMP-activated protein kinase (AMPK) family of kinases and is highly expressed in adipocytes. We investigated the regulation of SIK2 in adipocytes in response to cellular stimuli with relevance for adipocyte function and/or AMPK signalling. None of the treatments, including insulin, cAMP-inducers or AICAR, affected SIK2 activity towards peptide or protein substrates in vitro. However, stimulation with the cAMP-elevating agent forskolin and the beta-adrenergic receptor agonist CL 316,243 resulted in a PKA-dependent phosphorylation and 14-3-3 binding of SIK2. Phosphopeptide mapping of SIK2 revealed several sites phosphorylated in response to cAMP-induction, including Ser358. Site-directed mutagenesis demonstrated that phosphorylation of Ser358, but not the previously reported PKA site Ser587, was required for 14-3-3 binding. Immunocytochemistry illustrated that the localisation of exogenously expressed SIK2 in HEK293 cells was exclusively cytosolic and remained unchanged after cAMP elevation. Fractionation of adipocytes however, revealed a significant increase of wild type, but not Ser358Ala, HA-SIK2 in the cytosol and a concomitant decrease in a particulate fraction after CL 316,243 treatment. This supports a phosphorylation-dependent re-localisation in adipocytes. We hypothesize that regulation of SIK2 by cAMP could play a role for the critical effects of this second messenger on lipid metabolism in adipocytes.
Bacterioferritin (BFR) is an iron storage and detoxification protein that differs from other ferritins by its ability to bind heme cofactors. Heme bound to BFR is believed to be involved in iron release, and was previously thought not to play a role in iron core formation. Investigation of the effect of bound heme on formation of the iron core has been enabled in the present work by development of a method for reconstitution of BFR from Escherichia coli with exogenously added heme at elevated temperature in the presence of a relatively high concentration of sodium chloride. Kinetic analysis of iron oxidation by E. coli BFR preparations containing varying amounts of heme revealed that heme bound to BFR decreases the rate of iron oxidation at the dinuclear iron ferroxidase sites but increases the rate of iron core formation. Similar kinetic analysis of BFR reconstituted with cobalt-heme revealed that this heme derivative has no influence on the rate of iron core formation. These observations argue that heme bound to E. coli BFR accelerates iron core formation by an electron transfer-based mechanism.
Recent studies have highlighted the fact that cancer cells have an altered metabolic phenotype, and this metabolic reprogramming is required to drive biosynthesis pathways necessary for rapid replication and proliferation. Specifically, the importance of citric acid cycle-generated intermediates in the regulation of cancer cells proliferation has been recently appreciated. One function of monocarboxylate transporters (MCTs) is to transport the citric acid cycle substrate pyruvate across the plasma membrane and into mitochondria, and inhibition of MCTs has been proposed as a therapeutic strategy to target metabolic pathways in cancer. Here, we examined the effect of different metabolic substrates (glucose and pyruvate) on mitochondrial function and proliferation in breast cancer cells. We demonstrated that cancer cells proliferate more rapidly in the presence of exogenous pyruvate when compared to lactate. Pyruvate supplementation fueled mitochondrial oxygen consumption and the reserve respiratory capacity, and this increase in mitochondrial function correlated with proliferative potential. In addition, inhibition of cellular pyruvate uptake using the MCT inhibitor α-cyano-4-hydroxycinnamic acid impaired mitochondrial respiration and decreased cell growth. These data demonstrate the importance of mitochondrial metabolism in proliferative responses and highlight a novel mechanism of action for MCT inhibitors through suppression of pyruvate-fueled mitochondrial respiration.
CymA is a member of the NapC/NirT-family of quinol dehydrogenases. Essential for the anaerobic respiratory flexibility of shewanellae, CymA transfers electrons from menaquinol to various dedicated systems for the reduction of terminal electron acceptors including fumarate and insoluble minerals of Fe(III). Spectroscopic characterisation of CymA from Shewanella oneidensis MR-1 identifies three low-spin His/His coordinated c-hemes and a single high-spin c-heme with His/H2O coordination lying adjacent to the quinol binding site. At pH 7, binding of the menaquinol analogue, 2-heptyl-4-hydroxyquinoline-N-oxide, does not alter the mid-point potentials of the high-spin (ca. -240 mV) and low-spin (ca. -110, -190 and -265 mV) hemes that appear biased to transfer electrons from the high- to low-spin centres following quinol oxidation. CymA is reduced with menadiol (Em -80 mV) in the presence of NADH (Em -320 mV) and an NADH:menadione oxidoreductase, but not by menadiol alone. In cytoplasmic membranes reduction of CymA may then require the thermodynamic driving force from NADH, formate or H2 oxidation as the redox poise of the menaquinol pool in isolation is insufficient. Spectroscopic studies suggest that CymA requires a non-heme cofactor for quinol oxidation and that the reduced enzyme forms a 1:1 complex with its redox partner Fcc3. The implications for CymA supporting the respiratory flexibility of shewanellae are discussed.
NAC (NAM/ATAF/CUC) plant transcription factors regulate essential processes in development, stress responses and nutrient distribution in important crop and model plants (rice, Populus, Arabidopsis), which makes them highly relevant in the context of crop optimization and bioenergy production. The structure of the DNA-binding NAC domain of ANAC019 has previously been determined by X-ray crystallography, revealing a dimeric and predominantly β-fold structure, but the mode of binding to cognate DNA has remained elusive. In this study, information from low resolution X-ray structures and small angle X-ray scattering on complexes with oligonucleotides, mutagenesis and (DNaseI and uranyl photo-) footprinting, is combined to form a structural view of DNA-binding, and for the first time provide experimental evidence for the speculated relationship between plant specific NAC proteins, WRKY transcription factors and the mammalian GCM transcription factors, which all use a b-strand motif for DNA-binding. The structure shows how the NAC domain inserts the edge of its core β-sheet in the major groove, while leaving the DNA largely un-distorted. The structure of the NAC-DNA complex and a new crystal form of the unbound NAC also indicate limited flexibility of the NAC dimer arrangement which could be important in recognizing suboptimal binding sites.
Dickkopf 1 (DKK1) is a secreted inhibitor of the Wnt signaling pathway and a critical modulator of tumor promotion and the tumor microenvironment. However, mechanisms regulating DKK1 expression are understudied. DNAJB6 is an HSP40 family member whose expression is compromised during progression of breast cancer and melanoma. Inhibition of Wnt/β-catenin signaling pathway, by up-regulation of DKK1, is one of the key mechanisms by which DNAJB6 suppresses tumor, metastasis and epithelial-mesenchymal transition (EMT). Analysis of the DKK1 promoter to define the cis-site responsible for its up regulation by DNAJB6 revealed the presence of two binding sites for a transcriptional repressor, MSX1 (muscle segment homeobox gene). Our investigations showed that MSX1 binds the DKK1 promoter and inhibits DKK1 transcription. Interestingly, silencing DNAJB6 resulted in up-regulation of MSX1 concomitant with increased stabilization of β-catenin. ChIP studies revealed that β-catenin binds MSX1 promoter and stabilization of β-catenin elevates MSX1 transcription, indicating that β-catenin works as a transcription co-activator for MSX1. Functionally, exogenous expression of MSX1 in DNAJB6 expressing cells promotes the mesenchymal phenotype by suppression of DKK1. Thus we have identified a novel regulatory mechanism of DNAJB6 mediated DKK1 transcription up-regulation that can influence epithelial-mesenchymal transition. DKK1 is a feedback regulator of β-catenin levels. Thus our studies also define an additional negative control of this β-catenin-DKK1 feedback loop by MSX1, which may potentially contribute to excessive stabilization of β-catenin.
We previously revealed that tumor suppressor ATBF1 formed an autoregulatory feedback loop with estrogen-ERα signaling to regulate estrogen-dependent cell proliferation in breast cancer cells. In this loop, ATBF1 inhibits the function of estrogen-ERα signaling while ATBF1 protein levels are fine-tuned by estrogen-induced transcriptional upregulation as well as ubiquitin proteasome pathway (UPP)-mediated protein degradation. Here we show that the estrogen-responsive finger protein (EFP) is an E3 ubiquitin ligase mediating estrogen-induced ATBF1 protein degradation. Knockdown increases but overexpression of EFP decreases ATBF1 protein levels. EFP interacts with and ubiquitinates ATBF1 protein. Furthermore, we show that EFP is an important factor in estrogen-induced ATBF1 protein degradation in which some other factors are also involved. In human primary breast tumors, due to both as directly-upregulated ERα target gene products, the levels of ATBF1 protein are positively correlated with the levels of EFP protein. However, the ratio of ATBF1 protein to EFP protein is negatively correlated with EFP protein levels. Functionally, ATBF1 antagonizes EFP-mediated cell proliferation. These findings not only establish EFP as the E3 ubiquitin ligase for estrogen-induced ATBF1 protein degradation, but further support the autoregulatory feedback loop between ATBF1 and estrogen-ERα signaling and thus implicate ATBF1 in estrogen-dependent breast development and carcinogenesis.
EHD proteins participate in several endocytic events, such as the internalization and the recycling processes. There are four Eps15 homology (EH) domain containing proteins (EHDs) in mammalian cells (EHD1-EHD4), each with diverse roles in the recycling pathway of endocytosis. EHD2 is a plasma membrane associated member of the EHD family, which regulates internalization. Since several endocytic proteins have been shown to undergo nucleocytoplasmic shuttling and have been assigned roles in regulation of gene expression, we tested the possibility that EHDs also shuttle to the nucleus. Our results showed that among three EHDs (EHD1-EHD3), which were tested, only EHD2 accumulates in the nucleus under nuclear export inhibition treatment. Moreover, presence of nuclear localization signal (NLS) was essential for its entry to the nucleus. Nuclear exit of EHD2 depended partially on its nuclear export signal (NES). Elimination of a potential SUMOylation site in EHD2 resulted in a major accumulation of the protein in the nucleus, indicating the involvement of SUMOylation in nuclear exit of EHD2. We confirmed SUMOylation of EHD2 employing the yeast two-hybrid system. Using GAL4-based transactivation assay as well as Krüppel-like factor 7 (KLF7)-dependent transcription assay of p21WAF1/Cip1 gene we could show that EHD2 represses transcription. Quantitative RT-PCR of RNA from cells overexpressing EHD2 or of RNA from cells knocked down for EHD2 confirmed that EHD2 represses transcription of the p21WAF1/Cip1 gene.
Glucokinase activators (GKAs) are promising agents for the therapy of Type-2 diabetes, but little is known about their effects on hepatic intermediary metabolism. We monitored the fate of 13C-labeled glucose in both liver perfusion system and isolated hepatocytes. Mass spectrometry and nuclear magnetic resonance spectroscopy were deployed to measure isotopic enrichment. The results demonstrate that the stimulation of glycolysis by GKA led to numerous changes in hepatic metabolism: (i) Augmented flux through the TCA cycle, as evidenced by larger incorporation of 13C into the cycle (anaplerosis) and increased generation of 13C isotopomers of citrate, glutamate and aspartate (cataplerosis); (ii) Lowering of hepatic [Pi] and elevated [ATP], denoting greater phosphorylation potential and energy state; (iii) Stimulation of glycogen synthesis from glucose but inhibition of glycogen synthesis from 3-carbon precursors; (iv) Increased synthesis of N-acetlylglutamate and consequently augmented ureagenesis; (v) Increased synthesis of glutamine, alanine, serine and glycine; and (vi) Increased production and outflow of lactate. The current study provides a deeper insight into the hepatic actions of GKAs and uncovers the potential benefits and risks of GKA for treatment of diabetes. GKA improved hepatic bioenergetics, ureagenesis and glycogenesis but decreased gluconeogenesis with a potential risk of lactic acidosis and fatty liver.
The hotdog fold is one of the basic protein folds widely present in bacteria, archaea, and eukaryotes. Many of these proteins exhibit thioesterase activity against fatty acyl-CoAs and play important roles in lipid metabolism, cellular signaling, and degradation of xenobiotics. The genome of the opportunistic pathogen Pseudomonas aeruginosa contains over 20 genes encoding predicted hotdog-fold proteins, none of which have been experimentally characterized. We have found that two P. aeruginosa hotdog proteins display high thioesterase activity against 3-hydroxy-3-methylglutaryl-CoA and glutaryl-CoA (PA5202), and octanoyl-CoA (PA2801). Crystal structures of these proteins were solved (1.70 and 1.75 Å) and revealed a hotdog fold with a potential catalytic carboxylate residue located on the long alpha helix (Asp57 in PA5202 and Glu35 in PA2801). Alanine replacement mutagenesis of PA5202 identified four residues (Asn42, Arg43, Asp57, and Thr76), which are critical for activity and are located in the active site. A P. aeruginosa PA5202 deletion strain showed an increased secretion of the antimicrobial pigment pyocyanine and an increased expression of genes involved in pyocyanin biosynthesis suggesting a functional link between the PA5202 activity and pyocyanin production. Thus, the P. aeruginosa hotdog thioesterases PA5202 and PA2801 have similar structures, but exhibit different substrate preferences and functions.
Drosophila have emerged as a model system to study mammalian neurodegenerative diseases. Here we have generated Drosophila transgenic for ovine PrP to begin to establish an invertebrate model of ovine prion disease. We generated Drosophila transgenic for polymorphic variants of ovine PrP by PhiC31 site-specific germ line transformation under expression control by the bi-partite GAL4/UAS system. Site-specific transgene insertion in the fly genome allowed us to test the hypothesis that single amino acid codon changes in ovine PrP modulate prion protein levels and phenotype of the fly when expressed in the Drosophila nervous system. The Arg154 ovine PrP variants showed higher levels of prion protein expression in neuronal cell bodies and insoluble PrP conformer than did His154 variants. High levels of ovine PrP protein expression in Drosophila were associated with phenotypic effects including reduced locomotor activity and decreased survival. Significantly, our studies highlight a critical role for helix-1 in the formation of distinct conformers of ovine PrP since expression of His154 variants were associated with decreased survival in the absence of high levels of prion protein accumulation. Collectively, our studies show that variants of the ovine prion protein are associated with different spontaneous detrimental effects in ovine PrP transgenic Drosophila.
BcChi-A, a GH19 chitinase from the moss Bryum coronatum, is an endo-acting enzyme that hydrolyzes glycosidic bonds of chitin, a β-1,4-linked polysacharide of N-acetylglucosamine, (GlcNAc)n, through an inverting mechanism. When the wild-type enzyme was incubated with alpha-(GlcNAc)2 fluoride [alpha-(GlcNAc)2-F] in the absence or presence of (GlcNAc)2, (GlcNAc)2 and hydrogen fluoride were found to be produced through the Hehre resynthesis-hydrolysis mechanism. To convert BcChi-A into a glycosynthase, we employed the strategy reported by Honda et al. (J. Biol. Chem. 281, 1426-1431 (2006); Glycobiology, 18, 325-330 (2008)); that is, Ser102 holding a nucleophilic water molecule and Glu70 acting as a catalytic base were mutated, producing S102A, S102C, S102D, S102G, S102H, S102T, E70G, and E70Q. In all mutated enzymes except S102T, hydrolytic activity toward (GlcNAc)6 was not detected under the conditions we employed. Among the inactive BcChi-A mutants, S102A, S102C, S102G, and E70G were found to successfully synthesize (GlcNAc)4 as a major product from alpha-(GlcNAc)2-F in the presence of (GlcNAc)2. The S102A mutant showed the greatest glycosynthase activity due to its enhanced F- releasing activity and its suppressed hydrolytic activity. This is the first report on a glycosynthase that employs amino sugar fluoride as a donor substrate.
Reduction of phytate is a major goal of plant breeding programs to improve the nutritional quality of crops. Remarkably, except for storage organs of crops such as barley, maize and soybean, we know little of the stereoisomeric composition of inositol phosphates in plant tissues. To investigate the metabolic origins of higher inositol phosphates in photosynthetic tissues, we have radiolabelled leaf tissue of Solanum tuberosum with myo-[2-3H]inositol, undertaken a detailed analysis of inositol phosphate stereoisomerism and permeabilized mesophyll protoplasts in media containing inositol phosphates. We describe the inositol phosphate composition of leaf tissue and identify pathways of inositol phosphate metabolism that we reveal to be common to other kingdoms. Our results identify the metabolic origins of a number of higher inositol phosphates including ones that are precursors of cofactors, or cofactors of plant hormone/receptor complexes. Our work affords alternative explanations of the effects of disruption of inositol phosphate metabolism reported in other species, and identifies different inositol phosphates from that described in photosynthetic tissue of the monocot Spirodela polyrhiza. We define pathways of inositol hexakisphosphate turnover and shed light on the occurrence of a number of inositol phosphates identified in animals, for which metabolic origins have not been defined.
Dengue is the major arthropod-borne human viral disease, for which no vaccine or specific treatment are available. We used nuclear magnetic resonance, zeta potential measurements and atomic force microscopy to study the structural features of the interaction between dengue virus capsid (C) protein and lipid droplets (LDs), organelles crucial for infectious particles formation. C protein binding sites to LD were mapped, revealing a new function for a conserved segment in the N-terminal disordered region, and indicating that conformational selection is involved in recognition. The results suggest that C protein positively-charged N-terminal region prompts the interaction with negatively-charged LDs, after which a conformational rearrangement enables the access of the central hydrophobic patch to LD surface. Altogether, the results allowed the design of a peptide with inhibitory activity of C protein-LD binding, paving the way for new drug development approaches against dengue.
In vitro, the tumor suppressor PTEN displays intrinsic phosphatase activity towards both protein and lipid substrates. In vivo, the lipid phosphatase activity of PTEN, through which it dephosphorylates the 3 position in the inositol sugar of phosphatidylinositol derivatives, is important for its tumor suppressor function; however, the significance of its protein phosphatase activity remains unclear. Utilizing two-photon laser scanning microscopy and biolistic gene delivery of GFP-tagged constructs into organotypic hippocampal slice cultures, we have developed an assay of PTEN function in living tissue. Using this bioassay, we have demonstrated that overexpression of wild-type PTEN led to a decrease in spine density in neurons. Furthermore, it was the protein phosphatase activity, but not the lipid phosphatase activity, of PTEN that was essential for this effect. The ability of PTEN to decrease neuronal spine density depended upon the phosphorylation status of Ser and Thr residues in its C-terminal segment and the integrity of the C-terminal PDZ-binding motif. Our studies reveal a new aspect of the function of this important tumor suppressor and suggest that in addition to dephosphorylating the 3 position in phosphatidylinositol phospholipids, the critical protein substrate of PTEN may be PTEN itself.
Structural studies place the VDAC N-terminal region within the channel pore. Biochemical and functional studies, however, reveal that the N-terminal domain is cytoplasmically exposed. Here, the location and translocation of the VDAC1 N-terminal domain, and its role in voltage-gating and as a target for anti-apoptotic proteins, were addressed. Site-directed mutagenesis and cysteine substitution, together with a thiol-specific cross-linker, served to show that the VDAC1 N-terminal region exists in a dynamic equilibrium, located within the pore or exposed outside the β-barrel. Using single cysteine-bearing VDAC1, we demonstrate that the N-terminal region lies inside the pore. However, the same region can be exposed outside the pore, where it dimerizes with the N-terminal domain of a second VDAC molecule. When the N-terminal region a-helix structure was perturbed, intra-molecular cross-linking was abolished and dimerization was enhanced. This mutant also displays reduced voltage-gating and less binding of hexokinase but not of the anti-apoptotic proteins, Bcl2 and Bcl-xL. Replacing glycines in the N-terminal domain glycine-rich sequence (GRS) yielded less intra-molecular cross-linked product but more dimerization, suggesting that GRS provides the flexibility needed for N-terminal translocation from the internal pore to the channel face. N-terminal mobility may thus contribute to channel gating and interaction with anti-apoptotic proteins.
Posted on 8 March 2012 | 1:29 pm
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