Inhibition of heat shock protein (Hsp) 27 potentiates the suppressive effect of Hsp90 inhibitors in targeting breast cancer stem-like cells

Publication year: 2012
Source:Biochimie, Volume 94, Issue 6

Che-Hsin Lee, Hui-Mei Hong, Yu-Ying Chang, Wen-Wei Chang

Heat shock protein (Hsp) 90 is an ATP-dependent chaperone and its expression has been reported to be associated with poor prognosis of breast cancer. Cancer stem cells (CSCs) are particular subtypes of cells in cancer which have been demonstrated to be important to tumor initiation, drug resistance and metastasis. In breast cancer, breast CSCs (BCSCs) are identified as CD24-CD44 + cells or cells with high intracellular aldehyde dehydrogenase activity (ALDH+). Although the clinical trials of Hsp90 inhibitors in breast cancer therapy are ongoing, the BCSC targeting effect of them remains unclear. In the present study, we discovered that the expression of Hsp90? was increased in ALDH + human breast cancer cells. Geldanamycin (GA), a Hsp90 inhibitor, could suppress ALDH + breast cancer cells in a dose dependent manner. We are interesting in the insufficiently inhibitory effect of low dose GA treatment. It was correlated with the upregulation of Hsp27 and Hsp70. By co-treatment with HSP inhibitors, quercetin or KNK437 potentiated BCSCs, which determined with ALDH+ population or mammosphere cells, toward GA inhibition, as well as anti-proliferation and anti-migration effects of GA. With siRNA mediated gene silencing, we found that knockdown of Hsp27 could mimic the effect of HSP inhibitors to potentiate the BCSC targeting effect of GA. In conclusion, combination of HSP inhibitors with Hsp90 inhibitors could serve as a potential solution to prevent the drug resistance and avoid the toxicity of high dose of Hsp90 inhibitors in clinical application. Furthermore, Hsp27 may play a role in chemoresistant character of BCSCs.

Highlights

Posted on 28 May 2012 | 12:49 pm

Mechanisms of DNA methylation and demethylation in mammals

Publication year: 2012
Source:Biochimie

Ghislain Auclair, Michael Weber

Cytosine methylation is an epigenetically propagated DNA modification that can modify how the DNA molecule is recognized and expressed. DNA methylation undergoes extensive reprogramming during mammalian embryogenesis and is directly linked to the regulation of pluripotency and cellular identity. Studying its regulation is also important for a better understanding of the many diseases that show epigenetic deregulations, in particular cancer. In the recent years, a lot of progress has been made to characterize the profiles of DNA methylation at the genome level, which revealed that patterns of DNA methylation are highly dynamic between cell types. Here, we discuss the importance of DNA methylation for genome regulation and the mechanisms that remodel the DNA methylome during mammalian development, in particular the involvement of the rediscovered modified base 5-hydroxymethylcytosine.

Highlights

Posted on 28 May 2012 | 12:49 pm

Editorial board

Publication year: 2012
Source:Biochimie, Volume 94, Issue 5

Posted on 28 May 2012 | 12:49 pm

Differential fumarate binding to Arabidopsis NAD+-malic enzymes 1 and -2 produces an opposite activity modulation

Publication year: 2012
Source:Biochimie, Volume 94, Issue 6

Marcos A. Tronconi, Mariel C. Gerrard Wheeler, María F. Drincovich, Carlos S. Andreo

Arabidopsis mitochondria contain two NAD⁺-malic enzymes, NAD-ME1 and NAD-ME2. These proteins have similar affinity for their substrates but display opposite regulation by fumarate, which strongly stimulates NAD-ME1 but inhibits NAD-ME2 activity. Here, the interaction of NAD-ME1 and -2 with fumarate was investigated by kinetic approaches, urea denaturation assays and intrinsic fluorescence quenching, in the absence and presence of NAD⁺. Fumarate inhibited NAD-ME2 at saturating, but not at low, levels of NAD⁺, and it behaved as competitive inhibitor with respect to L-malate. In contrast, NAD-ME1 fumarate activation was higher at suboptimal NAD⁺ concentrations. In the absence of cofactor, the fluorescence of both NAD-ME1 and -2 is quenched by fumarate. However, for NAD-ME2 the quenching arises from a collisional phenomenon, while in NAD-ME1 the fluorescence decay can be explained by a static process that involves fumarate binding to the protein. Furthermore, the residue Arg84 of NAD-ME1 is essential for fumarate binding, as the mutant protein R84A exhibits a collisional quenching by this metabolite. Together, the results indicate that the differential fumarate regulation of Arabidopsis NAD-MEs, which is further modulated by NAD⁺ availability, is related to the gaining of an allosteric site for fumarate in NAD-ME1 and an active site-associated inhibition by this C4-organic acid in NAD-ME2.

Highlights

Posted on 28 May 2012 | 12:49 pm

The minus 10% syndroma

Publication year: 2012
Source:Biochimie, Volume 94, Issue 6

Jean-Louis Mergny

Posted on 28 May 2012 | 12:49 pm

Antiangiogenic and antivascular effects of a recombinant tumstatin-derived peptide in a corneal neovascularization model

Publication year: 2012
Source:Biochimie, Volume 94, Issue 6

Roman Esipov, Ksenia Beyrakhova, Vera Likhvantseva, Evgenia Stepanova, Vasily Stepanenko, Maria Kostromina, Yulia Abramchik, Anatoly Miroshnikov

Tumstatin, a cleavage fragment of collagen IV, is a potent endogenous inhibitor of angiogenesis. Tumstatin-derived peptide T8 possesses all angiostatic properties of full-length tumstatin and indirectly suppresses tumor growth. The potential of T8 to block pathological angiogenesis in the eye has not been explored yet. Here we assess antiangiogenic effects of a recombinant T8 peptide in rabbit corneal neovascularization models. The fusion protein consisting of T8 and thioredoxin was synthesized in a highly efficient Escherichia coli expression system, isolated using ion-exchange chromatography and cleaved with TEV (tobacco etch virus) protease. The target peptide was purified on an anion-exchange resin and by reversed phase high-performance liquid chromatography. The recombinant peptide suppressed the proliferation of basic fibroblast growth factor-induced SVEC-4-10 endothelial cells (simian virus 40-immortalized murine endothelial cells) and inhibited tube formation in these cells in a dose-dependent manner. In rabbit corneal neovascularization models T8 demonstrated the ability to prevent pathological angiogenesis (when injected simultaneously with the induction of neovascularization) and, moreover, to promote the regression of newly-formed blood vessels (when injected on day 8 after angiogenesis stimulation). Our results suggest that T8 may have a therapeutic potential in the treatment of ocular neovascular diseases.

Highlights

Posted on 28 May 2012 | 12:49 pm

HEXIM1-binding elements on mRNAs identified through transcriptomic SELEX and computational screening

Publication year: 2012
Source:Biochimie

Yuki Fujimoto, Yoshikazu Nakamura, Shoji Ohuchi

The positive transcription elongation factor b (P-TEFb) is one of the main regulatory factors of the transcription mediated by RNA polymerase II (RNAPII). P-TEFb promotes transcriptional elongation by phosphorylating its targets, which include the C-terminal domain of RNAPII. The activity of P-TEFb is negatively regulated by an RNA-binding protein HEXIM1 in association with 7SK snRNA. To search for other cellular RNAs that bind to HEXIM1, we used systematic evolution of ligands by exponential enrichment (SELEX) with the HeLa cDNA library as the initial pool source. We identified cad mRNA as a HEXIM1-binding RNA and confirmed their association in HeLa cells. In vitro mutational analysis showed that cad mRNA binds to HEXIM1 through its bulged stem structure located in exon 11. In addition, a computational search revealed other RNAs with similar stem structures, including brd4 and tcf3 mRNAs, both of which were shown to be coimmunoprecipitable with anti-HEXIM1 antibody in HeLa cells. Our findings suggest a possible role for HEXIM1 in the regulation of specific gene expressions.

Highlights

Posted on 28 May 2012 | 12:49 pm

Molecular dynamics, crystallography and mutagenesis studies on the substrate gating mechanism of prolyl oligopeptidase

Publication year: 2012
Source:Biochimie, Volume 94, Issue 6

Karol Kaszuba, Tomasz Róg, Reinis Danne, Peter Canning, Vilmos Fülöp, Tünde Juhász, Zoltán Szeltner, J.-F. St. Pierre, Arturo García-Horsman, Pekka T. Männistö, Mikko Karttunen, Jyrki Hokkanen, Alex Bunker

Altered prolyl oligopeptidase (PREP) activity is found in many common neurological and other genetic disorders, and in some cases PREP inhibition may be a promising treatment. The active site of PREP resides in an internal cavity; in addition to the direct interaction between active site and substrate or inhibitor, the pathway to reach the active site (the gating mechanism) must be understood for more rational inhibitor design and understanding PREP function. The gating mechanism of PREP has been investigated through molecular dynamics (MD) simulation combined with crystallographic and mutagenesis studies. The MD results indicate the inter-domain loop structure, comprised of 3 loops at residues, 189–209 (loop A), 577–608 (loop B), and 636–646 (loop C) (porcine PREP numbering), are important components of the gating mechanism. The results from enzyme kinetics of PREP variants also support this hypothesis: When loop A is (1) locked to loop B through a disulphide bridge, all enzyme activity is halted, (2) nicked, enzyme activity is increased, and (3) removed, enzyme activity is only reduced. Limited proteolysis study also supports the hypothesis of a loop A driven gating mechanism. The MD results show a stable network of H-bonds that hold the two protein domains together. Crystallographic study indicates that a set of known PREP inhibitors inhabit a common binding conformation, and this H-bond network is not significantly altered. Thus the domain separation, seen to occur in lower taxa, is not involved in the gating mechanism for mammalian PREP. In two of the MD simulations we observed a conformational change that involved the breaking of the H-bond network holding loops A and B together. We also found that this network was more stable when the active site was occupied, thus decreasing the likelihood of this transition.

Graphical abstract

Graphical abstract Highlights

Posted on 28 May 2012 | 12:49 pm

Overexpression of Ipe protein from the coliphage mEp021 induces pleiotropic effects involving haemolysis by HlyE-containing vesicles and cell death

Publication year: 2012
Source:Biochimie, Volume 94, Issue 6

Eva Martínez-Peñafiel, Fernando Fernández-Ramírez, Cecilia Ishida, Ruth Reyes-Cortés, Omar Sepúlveda-Robles, Gabriel Guarneros-Peña, Rosa María Bermúdez-Cruz, Luis Kameyama

Lysogenic Escherichia coli K-12 harbouring the prophage mEp021 displays haemolytic activity. From a genomic library of mEp021, we identified an open reading frame (ORF 4) that was responsible for the haemolytic activity. However, the ORF 4 sequence contains four initiation codons in the same frame: ORF 4.1–ORF 4.4, coding for 83-a.a., 82-a.a., 77-a.a. and 72-a.a. products, respectively. The expression of the cloned ORF 4.3, or inducer of pleiotropic effects (ipe), reproduced the haemolytic phenotype in a native strain carrying the gene hlyE⁺, but not in the mutant hlyE^? strain. The overexpression of Ipe induced several pleiotropic effects, such as the inhibition of cell growth and the deregulation of cell division, which resulted in a mixture of normal and desiccated-like cells: normal-filamentous, desiccated-like-filamentous bacilli, minicells etc. Other effects included abnormalities of the cell membrane, the production of vesicles containing HlyE, and finally, cell death. These events were analysed at the molecular level by microarray assays. The global transcription profile of E. coli K-12 strain MC4100, which expressed Ipe after 4 h, revealed differential expression of various genes, most of which were related either to cell membrane and murein biosynthesis or to cell division. The up-regulation of some of these transcripts was confirmed by qRT-PCR. Additional research is needed to determine whether these effects are directly related to Ipe activity or are consequences of the cellular responses to putative structural damage induced by Ipe.

Highlights

Posted on 28 May 2012 | 12:49 pm

The bactericidal effect of human secreted group IID phospholipase A2 results from both hydrolytic and non-hydrolytic activities

Publication year: 2012
Source:Biochimie, Volume 94, Issue 6

Raquel Fonseca-Maldonado, Tatiana Lopes Ferreira, Richard J. Ward

The Human Secreted Group IID Phospholipase A2 (hsPLA2GIID) may be involved in the human acute immune response. Here we have demonstrated that the hsPLA2GIID presents bactericidal and Ca²⁺-independent liposome membrane-damaging activities and we have compared these effects with the catalytic activity of active-site mutants of the protein. All mutants showed reduced hydrolytic activity against DOPC:DOPG liposome membranes, however bactericidal effects against Escherichia coli and Micrococcus luteus were less affected, with the D49K mutant retaining 30% killing of the Gram-negative bacteria at a concentration of 10?g/mL despite the absence of catalytic activity. The H48Q mutant maintained Ca²⁺-independent membrane-damaging activity whereas the G30S and D49K mutants were approximately 50% of the wild-type protein, demonstrating that phospholipid bilayer permeabilization by the hsPLA2GIID is independent of catalytic activity. We suggest that this Ca²⁺-independent damaging activity may play a role in the bactericidal function of the protein.

Highlights

Posted on 28 May 2012 | 12:49 pm

Molecular characterization of a thermostable l-fucose isomerase from Dictyoglomus turgidum that isomerizes l-fucose and d-arabinose

Publication year: 2012
Source:Biochimie

Seung-Hye Hong, Yu-Ri Lim, Yeong-Su Kim, Deok-Kun Oh

A recombinant thermostable l-fucose isomerase from Dictyoglomus turgidum was purified with a specific activity of 93 U/mg by heat treatment and His-trap affinity chromatography. The native enzyme existed as a 410 kDa hexamer. The maximum activity for l-fucose isomerization was observed at pH 7.0 and 80 °C with a half-life of 5 h in the presence of 1 mM Mn²⁺ that was present one molecular per monomer. The isomerization activity of the enzyme with aldose substrates was highest for l-fucose (with a k cat of 15,500 min^?1 and a K m of 72 mM), followed by d-arabinose, d-altrose, and l-galactose. The 15 putative active-site residues within 5 Å of the substrate l-fucose in the homology model were individually replaced with other amino acids. The analysis of metal-binding capacities of these alanine-substituted variants revealed that Glu349, Asp373, and His539 were metal-binding residues, and His539 was the most influential residue for metal binding. The activities of all variants at 349 and 373 positions except for a dramatically decreased k cat of D373A were completely abolished, suggesting that Glu349 and Asp373 were catalytic residues. Alanine substitutions at Val131, Met197, Ile199, Gln314, Ser405, Tyr451, and Asn538 resulted in substantial increases in K m, suggesting that these amino acids are substrate-binding residues. Alanine substitutions at Arg30, Trp102, Asn404, Phe452, and Trp510 resulted in decreases in k cat, but had little effect on K m.

Highlights

Posted on 28 May 2012 | 12:49 pm

Identification of retinoblastoma related genes with shortest path in a protein-protein interaction network

Publication year: 2012
Source:Biochimie

Bi-Qing Li, Jian Zhang, Tao Huang, Lei Zhang, Yu-Dong Cai

This paper presents a new method for identifying retinoblastoma related genes by integrating gene expression profile and shortest path in a functional linkage graph. With the existing protein-protein interaction data from STRING, a weighted functional linkage graph is constructed. 119 consistently differentially expressed genes between retinoblastoma and normal retina were obtained from the overlap of two gene expression studies of retinoblastoma. Then the shortest paths between each pair of these 119 genes were determined with Dijkstra’s algorithm. Finally, all the genes present on the shortest paths were extracted and ranked according to their betweenness and the 119 shortest genes with a betweenness greater than 100 and with a p-value less than 0.05 were selected for further analysis. We also identified 53 retinoblastoma related miRNAs from published miRNA array data and most of the 238 (119 consistently differentially expressed genes and 119 shortest path genes) retinoblastoma genes were shown to be target genes of these 53 miRNAs. Interestingly, the genes we identified from both the gene expression profiles and the functional protein association network included more cancer genes than did the genes identified from the gene expression profiles alone. In addition, these genes also had greater functional similarity to the reported colorectal cancer genes than did the genes identified from the gene expression profiles alone. This study shows promising results and proves the efficiency of the proposed methods.

Highlights

Posted on 28 May 2012 | 12:49 pm

Ferric haem forms of Mycobacterium tuberculosis catalase-peroxidase probed by EPR spectroscopy: Their stability and interplay with pH

Publication year: 2012
Source:Biochimie, Volume 94, Issue 6

Dimitri A. Svistunenko, Jonathan A.R. Worrall, Snehpriya B. Chugh, Sarah C. Haigh, Reza A. Ghiladi, Peter Nicholls

Low temperature EPR spectroscopy was used to characterise Mycobacterium tuberculosis catalase-peroxidase in its resting ferric haem state. Several high spin ferric haem forms and no low spin forms were found in the enzyme samples frozen in methanol on dry ice. The EPR spectra depended not only on the pH but also on the buffer type. As a general trend, the higher the pH, the greater the ‘rhombic’ fraction of the high spin ferric haem that was observed. The rhombic form was characterised by well separated two lines in the g = 6 region whereas in the ‘axial’ form the two lines overlap. This pH dependence of the equilibrium of axial and rhombic ferric haem forms is also seen in rapidly freeze-quenched samples. Different high spin ferric haem forms were monitored during a 3 week storage of the enzyme at 4 °C. For some forms, extremal dependences, i.e. those progressing via maxima or minima over storage time, were found. This indicates that the mechanism of the time-dependent transition from one high spin ferric haem form to another must be more complex than a simple single site oxidation.

Highlights

Posted on 28 May 2012 | 12:49 pm

A novel Kazal-type trypsin inhibitor from the skin secretion of the Central American red-eyed leaf frog, Agalychnis callidryas

Publication year: 2012
Source:Biochimie, Volume 94, Issue 6

Renjie Li, Hui Wang, Yingchun Jiang, Yang Yu, Lei Wang, Mei Zhou, Yingqi Zhang, Tianbao Chen, Chris Shaw

The chemical complexity of the defensive skin secretion of the red-eyed leaf frog, (Agalychnis callidryas), has not been elucidated in detail. During a systematic study of the skin secretion peptidomes of phyllomedusine frogs, we discovered a novel Kazal-type protein with potent trypsin inhibitory activity (Ki = 1.9 nM) that displays the highest degree of structural similarity with Kazal proteins from bony fishes. The protein was located in reverse-phase HPLC fractions following a screen of such for trypsin inhibition and subsequent partial Edman degradation of the peak active fraction derived the sequen ATKPR-QYIVL-PRILRPV-GT. The molecular mass of the major component in this fraction was established by MALDI-TOF MS as 5893.09 Da. This partial sequence (assuming blank cycles to be Cys residues) was used to design a degenerate primer pool that was employed successfully in RACE-PCR to clone homologous precursor-encoding cDNA that encoded a mature Kazal protein of 52 amino acid residues with a computed molecular mass of 5892.82 Da. The protein was named A. callidryas Kazal trypsin inhibitor (ACKTI). BLAST analysis revealed that ACKTI contained a canonical Kazal motif (C-x(7)-C-x(6)-Y-x(3)-C-x(2,3)-C). This novel amphibian skin Kazal trypsin inhibitor adds to the spectrum of trypsin inhibitors of Kunitz- and Bowman Birk-type reported from this amphibian source.

Highlights

Posted on 28 May 2012 | 12:49 pm

Epigenomics of cancer – emerging new concepts

Publication year: 2012
Source:Biochimie

Melanie R. Hassler, Gerda Egger

The complexity of the mammalian genome is regulated by heritable epigenetic mechanisms, which provide the basis for differentiation, development and cellular homeostasis. These mechanisms act on the level of chromatin, by modifying DNA, histone proteins and nucleosome density/composition. During the last decade it became clear that cancer is defined by a variety of epigenetic changes, which occur in early stages of disease and parallel genetic mutations. With the advent of new technologies we are just starting to unravel the cancer epigenome and latest mechanistic findings provide the first clue as to how altered epigenetic patterns might occur in different cancers. Here we review latest findings on chromatin related mechanisms and hypothesize how their impairment might contribute to the altered epigenome of cancer cells.

Highlights

Posted on 28 May 2012 | 12:49 pm

Transcription factors Sp1 and Sp3 regulate basal transcription of the human IRF-3 gene

Publication year: 2012
Source:Biochimie, Volume 94, Issue 6

Hua-Guo Xu, Rui Jin, Wei Ren, Li Zou, Yi Wang, Guo-Ping Zhou

Interferon regulatory factor 3 (IRF-3) plays a crucial role in initiation and development of the IFN antiviral response. The expression level of human IRF-3 is thought to be closely related to antiviral state of cells. However, the mechanisms of the transcription regulation of IRF-3 have remained largely unknown. We previously reported that transcription factor E2F1 negatively regulates the basal transcriptional activity of IRF-3. Here we demonstrate that transcription factors Sp1 and Sp3 up-regulate the basal transcriptional activity of IRF-3 and increase IRF-3 expression at mRNA level. By transient transfection analysis we revealed that mutation of Sp1/NRF-1 binding site resulted in a profound reduction of IRF-3 promoter activity. Overexpression of Sp1 and Sp3, but not NRF-1, transactivated the IRF-3 promoter activity in reporter gene assays while knocking-down of endogenous Sp1 and Sp3 by a shRNA strategy markedly inhibited IRF-3 promoter activity. Chromatin immunoprecipitation (ChIP) assays showed that Sp1 and Sp3 interact with the IRF-3 promoter in vivo. These results suggest that basal expression level of IRF-3 is regulated by transcription factors Sp1 and Sp3.

Highlights

Posted on 28 May 2012 | 12:49 pm

Purification and identification of two carnosine-cleaving enzymes, carnosine dipeptidase I and Xaa-methyl-His dipeptidase, from Japanese eel (Anguilla japonica)

Publication year: 2012
Source:Biochimie, Volume 94, Issue 6

Takahiro Oku, Seiichi Ando, Hsin-Chun Tsai, Yusuke Yamashita, Hiroshi Ueno, Kazuhiro Shiozaki, Ryuichiro Nishi, Shoji Yamada

Three enzymes, carnosine dipeptidase I (EC 3.4.13.20, CNDP1), carnosine dipeptidase II (EC 3.4.13.18, CNDP2), and Xaa-methyl-His dipeptidase (or anserinase: EC 3.4.13.5, ANSN), are known to be capable of catalyzing the hydrolysis of carnosine (?-alanyl-l-histidine), in vertebrates. Here we report the purification and identification of two unidentified carnosine-cleaving enzymes from Japanese eel (Anguilla japonica). Two different dipeptidases were successfully purified to homogeneity from the skeletal muscle; one exhibited a broad substrate specificity, while the other a narrow specificity. N-terminal amino-acid sequencing, deglycosylation analysis, and genetic analysis clearly revealed that the former is a homodimer of glycosylated subunits, encoded by ANSN, and the latter is another homodimer of glycosylated subunits, encoded by CNDP1; that is, Xaa-methyl-His dipeptidase, and carnosine dipeptidase I respectively. This is the first report on the identification of carnosine dipeptidase I from a non-mammal. Database search revealed presence of a CNDP1 ortholog only from salmonid fishes, including Atlantic salmon and rainbow trout, but not from other ray-finned fish species, such as zebrafish, fugu, and medaka whose genomes have been completely sequenced. The mRNAs of CNDP1 and ANSN are strongly expressed in the liver of Japanese eel, compared with other tissues, while that of CNDP2 is widely distributed in all tissues tested.

Highlights

Posted on 28 May 2012 | 12:49 pm

Structure-based characterization of canine–human chimeric uricases and its evolutionary implications

Publication year: 2012
Source:Biochimie, Volume 94, Issue 6

Chun Zhang, Kai Fan, Weitao Zhang, Ruixin Zhu, Lujia Zhang, Dongzhi Wei

Uricase was lost in hominoids during primate evolution, but the inactivation mechanism remains controversial. To investigate the inactivation process of hominoid uricase, chimeric constructions between canine and human uricase were employed to screen the target regions that may contain labile or inactivated mutations in deduced human uricase. Four chimeric uricases were constructed and showed different enzymatic characteristics. Homology modeling, rational site-directed mutagenesis and DNA alignment were used to analyze the changes. Arg119 is conserved in functional mammalian uricases and its side-chains are crucial in maintaining the stability of the ?-barrel core. A single CGT (Arg) to CAT (His) mutation at codon 119 that is shared by the human and great ape clade greatly reduces this stability and could cause the loss of uricase activity. We speculate that this missense mutation occurred first and inactivated the uricase protein in humans and great apes and that later the known nonsense mutation at codon 33 occurred and silenced the uricase gene. A single GTC (Val) to GCC (Ala) mutation at codon 296 in canine uricase is regarded as deleterious structural mutation, but such kinds of deleterious mutations have been widely accumulated in extant mammalian uricases. We speculate that a reduction in uricase activity has been an evolutionary tendency in mammals. Moreover, from structure–activity analysis of helix 2 in ancestral primate uricase, we suggest that before the inactivation of hominoid uricase, deleterious structural evolutionary changes had occurred in ancestral primates. The loss of hominoid uricase should be caused by progressive multistep mutations rather than a single mutation event.

Highlights

Posted on 28 May 2012 | 12:49 pm

Chemically induced oxidative stress increases polyamine levels by activating the transcription of ornithine decarboxylase and spermidine/spermine-N1-acetyltransferase in human hepatoma HUH7 cells

Publication year: 2012
Source:Biochimie

Olga A. Smirnova, Maria G. Isaguliants, Mervi T. Hyvonen, Tuomo A. Keinanen, Vera L. Tunitskaya, Jouko Vepsalainen, Leena Alhonen, Sergey N. Kochetkov, Alexander V. Ivanov

Biogenic polyamines spermine and spermidine participate in numerous cellular processes including transcription, RNA processing and translation. Specifically, they counteract oxidative stress, an alteration of cell redox balance involved in generation and progression of various pathological states including cancer. Here, we investigated how chemically induced oxidative stress affects polyamine metabolism, specifically the expression and activities of enzymes catalyzing polyamine synthesis (ornithine decarboxylase; ODC) and degradation (spermidine/spermine-N¹-acetyltransferase; SSAT), in human hepatoma cells. Oxidative stress induced the up-regulation of ODC and SSAT gene transcription mediated by Nrf2, and in case of SSAT, also by NF-?B transcription factors. Activation of transcription led to the elevated intracellular activities of both enzymes. The balance in antagonistic activities of ODC and SSAT in the stressed hepatoma cells was shifted towards polyamine biosynthesis, which resulted in increased intracellular levels of putrescine, spermidine, and spermine. Accumulation of putrescine is indicating for accelerated degradation of polyamines by SSAT – acetylpolyamine oxidase (APAO) pathway generating toxic products that promote carcinogenesis, whereas accelerated polyamine synthesis via activation of ODC is favorable for proliferation of cells including those sub-lethally damaged by oxidative stress.

Highlights

Posted on 28 May 2012 | 12:49 pm

MicroRNA-513a-5p mediates TNF-? and LPS induced apoptosis via downregulation of X-linked inhibitor of apoptotic protein in endothelial cells

Publication year: 2012
Source:Biochimie, Volume 94, Issue 6

Sojin Shin, Kyo Chul Moon, Keon Uk Park, Eunyoung Ha

MicroRNAs (miRNAs) are endogenous non-coding small RNAs that have emerged as one of the central players of gene expression regulation. Endothelial cell apoptosis plays a fundamental role in the development of atherosclerosis. This study was designed to determine the effect of miR-513a-5p on apoptosis of human umbilical vein endothelial cells (HUVECs). HUVECs were treated with tumour necrosis factor-? (TNF-?) and lipopolysaccharide (LPS) and miR-513a-5p expression levels were determined. MiR-513a-5p target gene indentification, validation, and signalling pathways were investigated. Treatment of HUVECs with TNF-? and LPS up-regulated miR-513a-5p expressions more than 2-fold compared to control (P < 0.05). Inhibition of miR-513a-5p by antisense (AS) miR-513a-5p reversed TNF-? and LPS induced apoptosis (P < 0.01). Transfection of HUVECs with miR-513a-5p mimics also induced apoptosis (P < 0.01). Treatment of HUVECs with TNF-? and LPS attenuated X-linked inhibitor of apoptosis (XIAP) while increased caspase-3 expression, poly ADP-ribose polymerase (PARP) cleavage, and p53 expression. These effects were reversed by inhibition of miR-513a-5p. Of those miR-513a-5p candidate target genes, we identified and validated XIAP as a miR-513a-5p target gene. Targeting of the XIAP 3?-untranslated region by miR-513a-5p using luciferase reporter assay resulted in attenuated luciferase activity. Transfection of HUVECs with AS miR-513a-5p increased XIAP protein expression while miR-513a-5p mimics attenuated XIAP expression. These results together suggest that miR-513a-5p mediates TNF-? and LPS induced apoptosis via downregulation of XIAP in HUVECs.

Highlights

Posted on 28 May 2012 | 12:49 pm

Interaction of Listeria monocytogenes autolysin amidase with glycosaminoglycans promotes listerial adhesion to mouse hepatocytes

Publication year: 2012
Source:Biochimie, Volume 94, Issue 6

Krisana Asano, Ikuko Kakizaki, Akio Nakane

Adherence to the cell surface is a key event during infection of pathogenic microorganisms. We have previously reported that autolysin amidase (Ami) of Listeria monocytogenes promotes an efficient listerial adherence to mouse hepatocytes and requires for listerial pathogenicity. Cell wall anchoring (CWA) domain of Ami has been shown to bind lipoteichoic acid on listerial cell wall but the binding of Ami to host cell surface molecules remains to be determined. In this study, we present evidence here that Ami promotes efficient adherence of L. monocytogenes to mouse hepatocytes mediated by glycosaminoglycans (GAGs). The adhesion of L. monocytogenes wild type but not Ami-deficient mutant to the hepatocytes was dramatically attenuated by 4-nitrophenyl-?-d-xylopyranoside, a specific inhibitor of GAG association to cell surface. Full-length and truncated Ami were used to investigate the binding of Ami to GAGs and we found that four-repeated CWA of Ami is sufficient to bind GAGs on the host cell surface. Competitive assay and surface plasmon resonance demonstrated that Ami interacts with sulfated GAGs but not non-sulfated GAGs. The results suggest that Ami acts as an adhesin of L. monocytogenes to hepatocytes by interaction via its four-repeated CWA domain and sulfated GAGs.

Highlights

Posted on 28 May 2012 | 12:49 pm

Lactoferrin and lactoferrin chimera inhibit damage caused by enteropathogenic Escherichia coli in HEp-2 cells

Publication year: 2012
Source:Biochimie

Héctor Flores-Villaseñor, Adrian Canizalez-Román, Mireya de la Garza, Kamran Nazmi, Jan G.M. Bolscher, Nidia Leon-Sicairos

Enteropathogenic Escherichia coli (EPEC) is an important cause of infant diarrhea in developing countries. It produces a characteristic intestinal histopathological lesion on enterocytes known as ‘attaching and effacing’ (A/E), and these two steps are mediated by a type-III secretory system. In the present study, we evaluated the effect on the initial host cell attachment step produced by bovine lactoferrin (bLF) and three synthetic peptides: lactoferricin (LFcin), lactoferrampin (LFampin) and LFchimera. A special focus was given to the hemolytic activity and EPEC-induced actin polymerization in HEp-2 cells, as well as to the espA gene expression, which produces the protein responsible for primary contact with the host cells. Results show that EPEC attachment to HEp-2 cells was significantly suppressed by bLF and LFchimera at 125 and 40 ?M, respectively. EPEC-mediated actin polymerization was blocked by bLF and LFchimera at 88 and 99%, respectively. LFchimera inhibited the attachment and A/E lesion caused by EPEC in a dose-dependent manner. In the presence of 125 ?M bLF, the expression level of the espA gene was decreased by 50% compared to the untreated control. LFchimera at concentrations of 20 ?M and 40 ?M diminished the level of espA gene expression 100 and 1,000 fold, respectively (P<0.001). Although bLF, LFchimera, LFcin, and LFampin all significantly blocked the hemolysis produced by EPEC (P<0.001), the two former compounds produced this effect at lower concentrations. These two compounds, bLF and LFchimera, were able to inhibit the first steps of the mechanism of the damage used by EPEC. This data suggests that LFchimera could provide protection against enteropathogens that share this mechanism.

Highlights

Posted on 28 May 2012 | 12:49 pm

Editorial Board

Publication year: 2012
Source:Biochimie, Volume 94, Issue 6

Posted on 28 May 2012 | 12:49 pm

The heart-protective mechanism of nitronyl nitroxide radicals on murine viral myocarditis induced by CVB3

Publication year: 2012
Source:Biochimie

Haibo Wang, Peng Gao, Linlin Jing, Xiangyang Qin, Xiaoli Sun

Our previous researches showed that nitronyl nitroxyl derivatives, NNP and NNVP were good anti-oxidants and provided radioprotective effects in C6 cells. The objective of the present study is to investigate the possible antiviral effects and underlying pharmacological of the two nitronyl nitroxide radicals against CVB3 in vitro and in vivo. The results showed that NNP and NNVP were some of the most potent compounds in terms of their antiviral effects by protecting myocardial cells against oxidative damage of free radicals. Treatment with NNP or NNVP could decrease the intracellular ROS level in vitro. They could lead to a significant decrease in activities of biochemical markers AST, CK and LDH in infected murine serum and regulate SOD, MDA CAT activities to a normal level compared with infected control in vivo. NNP and NNVP could reduce NO production in infected mice by reacting with NO to produce the imino nitroxides which was confirmed by ESR spectrometry. In addition, NNP and NNVP could both decrease the mRNA expression of proinflammatory cytokines, TNF-?, IL-2 and IL-6. In conclusion, nitronyl nitroxide radicals NNP and NNVP were shown to have antiviral activities against CVB3 and they may represent potential therapeutic agents for viral myocarditis.

Graphical abstract

Graphical Abstract Highlights

Posted on 28 May 2012 | 12:49 pm

Bafibrinase: A non-toxic, non-hemorrhagic, direct-acting fibrinolytic serine protease from Bacillus sp. strain AS-S20-I exhibits in vivo anticoagulant activity and thrombolytic potency

Publication year: 2012
Source:Biochimie, Volume 94, Issue 6

Ashis K. Mukherjee, Sudhir K. Rai, Rupamoni Thakur, Pronobesh Chattopadhyay, Santosh K. Kar

A non-toxic, direct-acting fibrinolytic serine protease (Bafibrinase) demonstrating thrombolytic and anticoagulant properties was purified from Bacillus sp. strain AS-S20-I. Bafibrinase was monomeric, with a molecular mass of 32.3 kDa. The peptide mass fingerprinting of Bafibrinase revealed only 8.3% sequence coverage, suggesting it was a novel fibrinolytic enzyme. However, two of the tryptic digested de novo peptide sequences of Bafibrinase demonstrated good similarity with endopeptidases possessing serine in their catalytic triad. Further, catalytic activity of Bafibrinase was inhibited by serine protease inhibitor reinforcing this is a subtilisin-like serine protease. The apparent K m and V max values of Bafibrinase towards fibrin were determined as 0.24 ?M and 2.8 ?mol/min, respectively. It showed a K m value of 0.139 mM towards a chromogenic substrate for plasmin (d-Val-Leu-Lys-p-Nitroanilide dihydrochloride) and optimum activity at physiological conditions (37 °C and pH 7.4). Based on the cleavage pattern of fibrin and fibrinogen, Bafibrinase may be classified as an ?,?-fibrinogenase. Bafibrinase could not degrade collagen and was non-cytotoxic to HT29 cells or mammalian erythrocytes. Further, Bafibrinase at a dose of 2 mg/kg was devoid of toxicity as well as hemorrhagic activity on BALB/c mouse model, supporting its suitability for the development of a better and safer thrombolytic drug. Bafibrinase was also superior to human plasmin in degrading in vitro thrombus. The in vivo anticoagulant nature of Bafibrinase is being explored for the treatment and prevention of thrombosis and other cardiovascular diseases.

Highlights

Posted on 28 May 2012 | 12:49 pm

Temperature adaptations in psychrophilic, mesophilic and thermophilic chloride-dependent alpha-amylases

Publication year: 2012
Source:Biochimie

Alexandre Cipolla, François Delbrassine, Jean-Luc Da Lage, Georges Feller

The functional and structural adaptations to temperature have been addressed in homologous chloride-dependent ?-amylases from a psychrophilic Antarctic bacterium, the ectothermic fruit fly, the homeothermic pig and from a thermophilic actinomycete. This series covers nearly all temperatures encountered by living organisms. We report a striking continuum in the functional properties of these enzymes coupled to their structural stability and related to the thermal regime of the source organism. In particular, thermal stability recorded by intrinsic fluorescence, circular dichroism and differential scanning calorimetry appears to be a compromise between the requirement for a stable native state and the proper structural dynamics to sustain the function at the environmental/physiological temperatures. The thermodependence of activity, the kinetic parameters, the activations parameters and fluorescence quenching support these activity-stability relationships in the investigated ?-amylases.

Highlights

Posted on 28 May 2012 | 12:49 pm

Chromatin-modifying agents in anti-cancer therapy

Publication year: 2012
Source:Biochimie

Carole Seidel, Cristina Florean, Michael Schnekenburger, Mario Dicato, Marc Diederich

Epigenetic alterations are involved in every step of carcinogenesis. The development of chromatin-modifying agents (CMAs) has provided the ability to fight cancer by reversing these alterations. Currently, four CMAs have been approved for cancer treatment; two DNA demethylating agents and two deacetylase inhibitors. A number of promising CMAs are undergoing clinical trials in several cancer types. Moreover, already approved CMAs are still under clinical investigation to improve their efficacy and to extend their use to a broader panel of cancers. Combinatorial treatments with CMAs are already considered a promising strategy to improve clinical benefits and to limit side effects. The real mechanisms by which these CMAs allow the improvement and remission of patients are still obscure. A deeper analysis of the molecular features expressed by responding patients should be performed to reveal this information. In this review, we focus on clinical trials with CMAs, discussing the success and the pitfalls of this new class of anti-cancer drugs.

Highlights

Posted on 28 May 2012 | 12:49 pm

Snap denaturation reveals dimerization by AraC-like protein Rns

Publication year: 2012
Source:Biochimie

Vivienne Mahon, Robert P. Fagan, Stephen G.J. Smith

Here we show that the Rns regulator of Escherichia coli dimerises in vivo and in vitro. Furthermore, we demonstrate that Rns forms aggregates in vitro and describe a methodology to ameliorate aggregation thus permitting the analysis of Rns by cross-linking.

Highlights

Posted on 28 May 2012 | 12:49 pm

Exposure to common food additive carrageenan leads to reduced sulfatase activity and increase in sulfated glycosaminoglycans in human epithelial cells

Publication year: 2012
Source:Biochimie, Volume 94, Issue 6

Bo Yang, Sumit Bhattacharyya, Robert Linhardt, Joanne Tobacman

The commonly used food additive carrageenan, including lambda (?), kappa (?) and iota (?) forms, is composed of galactose disaccharides linked in alpha-1,3 and beta-1,4 glycosidic bonds with up to three sulfate groups per disaccharide residue. Carrageenan closely resembles the endogenous galactose or N-acetylgalactosamine-containing glycosaminoglycans (GAGs), chondroitin sulfate (CS), dermatan sulfate (DS), and keratan sulfate. However, these GAGs have beta-1,3 and beta-1,4 glycosidic bonds, in contrast to the unusual alpha-1,3 glycosidic bond in carrageenan. Since sulfatase activity is inhibited by sulfate, and carrageenan is so highly sulfated, we tested the effect of carrageenan exposure on sulfatase activity in human intestinal and mammary epithelial cell lines and found that carrageenan exposure significantly reduced the activity of sulfatases, including N-acetylgalactosamine-4-sulfatase, galactose-6-sulfatase, iduronate sulfatase, steroid sulfatase, arylsulfatase A, SULF-1,2, and heparan sulfamidase. Consistent with the inhibition of sulfatase activity, following exposure to carrageenan, GAG content increased significantly and showed marked differences in disaccharide composition. Specific changes in CS disaccharides included increases in di-sulfated disaccharide components of CSD (2S6S) and CS-E (4S6S), with declines in CS-A (4S) and CS-C (6S). Specific changes in heparin-heparan sulfate disaccharides included increases in 6S disaccharides, as well as increases in NS and 2S6S disaccharides. Study results suggest that carrageenan inhibition of sulfatase activity leads to re-distribution of the cellular GAG composition with increase in di-sulfated CS and with potential consequences for cell structure and function.

Highlights

Posted on 28 May 2012 | 12:49 pm

Impaired drug-binding capacities of in vitro and in vivo glycated albumin

Publication year: 2012
Source:Biochimie

Jennifer Baraka-Vidot, Alexis Guerin-Dubourg, Emmanuel Bourdon, Philippe Rondeau

Albumin, the major circulating protein in blood, can undergo increased glycation in diabetes. One of the main properties of this plasma protein is its strong affinity to bind many therapeutic drugs, including warfarin and ketoprofen. In this study, we investigated whether or not there were any significant changes related to in vitro or in vivo glycation in the structural properties and the binding of human albumin to both therapeutic drugs. Structural parameters, including redox state and ketoamine contents of in vitro and in vivo glycated purified albumins, were investigated in parallel with their affinity for warfarin and ketoprofen. High-performance liquid chromatography was used to determine the free drug concentrations and dissociation constants according to the Scatchard method. An alternative method based on fluorescence spectroscopy was also used to assess drug-binding properties. Oxidation and glycation levels were found to be enhanced in albumin purified from diabetic patients or glycated with glucose or methylglyoxal, after determination of their ketoamine, free thiol, amino group and carbonyl contents. In parallel, significant impairments in the binding affinity of in vitro and in vivo glycated albumin, as indicated by the higher dissociation constant values and confirmed by higher free drug fractions, were observed. To a lesser extent, this alteration also significantly affected diabetic albumin affinity, indicated by a lower static quenching in fluorescence spectroscopy. This work provides useful information supporting in vivo diabetic albumin could be the best model of glycation for monitoring diabetic physiopathology and should be valuable to know if glycation of albumin could contribute to variability in drugs response during diabetes.

Highlights

Posted on 28 May 2012 | 12:49 pm

The hybrid recombinational repair pathway operates in a ? activity deficient recC1004 mutant of Escherichia coli

Publication year: 2012
Source:Biochimie

Ignacija Vlaši?, Ana Šimatovi?, Krunoslav Br?i?-Kosti?

Homologous recombination is a crucial process for the maintenance of genome integrity. The two main recombination pathways in Escherichia coli (RecBCD and RecF) differ in the initiation of recombination. The RecBCD enzyme is the only component of the RecBCD pathway which acts in the initiation of recombination, and possesses all biochemical activities (helicase, 5?-3? exonuclease, ? cutting and loading of the RecA protein onto single-stranded (ss) DNA) needed for the processing of double stranded (ds) DNA breaks (DSB). When the nuclease and RecA loading activities of the RecBCD enzyme are inactivated, the proteins of the RecF recombination machinery, i.e., RecJ and RecFOR substitute for the missing 5?-3? exonuclease and RecA loading activity respectively. The above mentioned activities of the RecBCD enzyme are regulated by an octameric sequence known as the ? site (5?-GCTGGTGG-3?). One class of recC mutations, designated recC*, leads to reduced ? cutting in vitro. The recC1004 strain (a member of the recC* mutant class) is recombination proficient and resistant to UV radiation. In this paper, we studied the effects of mutations in RecF pathway genes on DNA repair (after UV and ? radiation) and on conjugational recombination in recC1004 and recC1004 recD backgrounds. We found that DNA repair after UV and ? radiation in the recC1004 and recC1004 recD backgrounds depends on recFOR and recJ gene products. We also showed that the recC1004 mutant has reduced survival after ? radiation. This phenotype is suppressed by the recD mutation which abolishes the RecBCD dependent nuclease activity. Finally, the genetic requirements for conjugational recombination differ from those for DNA repair. Conjugational recombination in recC1004 recD mutants is dependent on the recJ gene product. Our results emphasize the importance of the canonical ? recognition activity in DSB repair and the significance of interchange between the components of two recombination machineries in achieving efficient DNA repair.

Highlights

Posted on 28 May 2012 | 12:49 pm

Leptin and leptin receptor-related monogenic obesity

Publication year: 2012
Source:Biochimie

Beatrice Dubern, Karine Clement

The studies based on candidate genes and encoded proteins known to cause severe obesity in rodents, have shown that these genes also contribute to human early-onset obesity especially for those involved in the leptin pathway: the leptin (LEP) and leptin receptor (LEPR) genes. Since 1997, less than 20 individuals carrying a LEP gene mutation have been identified. Patients are mostly characterized by severe early-onset obesity with severe hyperphagia and associated phenotype such hypogonadotrophic hypogonadism, high rate of infection associated with a deficiency in T cell and abnormalities of sympathetic nerve function. Therapeutic option (subcutaneous daily injection of leptin) is available for patients with LEP deficiency. It results in weight loss, mainly of fat mass, with a major effect on reducing food intake and on other dysfunctions including immunity and induction of puberty even in adults. In LEPR deficient subjects, phenotypic similarities with the LEP-deficient subjects were noticed, especially the exhibited rapid weight gain in the first few months of life, with severe hyperphagia and the endocrine abnormalities (hypogonadotrophic hypogonadism, insufficient somatotrophic or thyreotropic secretion). Leptin treatment is useless in the LEPR deficient subjects. Factors that could possibly bypass normal leptin delivery systems are being developed but are not yet currently available for the treatment of these patients. Measurement of circulating leptin may help for the diagnosis of such obesity: it is undetectable in LEP mutation carriers or extremely elevated in LEPR mutation carriers. Thus, LEPR gene screening might be also considered in subjects with the association of severe obesity with endocrine dysfunctions such as hypogonadism and with leptin related to corpulence level.

Highlights

Posted on 28 May 2012 | 12:49 pm

Molecular cloning and characterization of antimicrobial peptides from skin of the broad-folded frog, Hylarana latouchii

Publication year: 2012
Source:Biochimie, Volume 94, Issue 6

Hui Wang, Zhijun Yu, Yuhong Hu, Haining Yu, Ran Ran, Jiangnan Xia, Duo Wang, Shujie Yang, Xiaohong Yang, Jingze Liu

Seven cDNA sequences encoding antimicrobial peptide (AMP) precursors were cloned by screening the skin-derived cDNA library of the broad-folded frog, Hylarana latouchii. Seven of the deduced peptides are highly similar to AMPs in five families of brevinin-2 (brevinin-2LTa, brevinin-2LTb, and brevinin-2LTc), esculentin-1 (esculentin-1LTa), esculentin-2 (esculentin-2LTa), palustrin-2 (palustrin-2LTa), and temporin (temporin-LTe). The actual sequences and characteristics of mature AMPs were analyzed by RP-HPLC and LC–MS/MS-based proteomics approaches in combination of four different protein digestion processes and by LTQ XL in combination of gas-phase fractionation (GPF) analysis. Moreover, most of the peptides found in this study hardly display hemolytic activity in vitro, suggesting they are promising antimicrobial drug candidates.

Highlights

Posted on 28 May 2012 | 12:49 pm

Cholesterol-5,6-epoxides: Chemistry, biochemistry, metabolic fate and cancer

Publication year: 2012
Source:Biochimie

Marc Poirot, Sandrine Silvente-Poirot

In the nineteen sixties it was proposed that cholesterol might be involved in the etiology of cancers and cholesterol oxidation products were suspected of being causative agents. Researchers had focused their attention on cholesterol-5,6-epoxides (5,6-ECs) based on several lines of eviden 1) 5,6-ECs contained an oxirane group that was supposed to confer alkylating properties such as those observed for aliphatic and aromatic epoxides. 2) cholesterol-5,6-epoxide hydrolase (ChEH) was induced in pre-neoplastic lesions of skin from rats exposed to ultraviolet irradiations and ChEH was proposed to be involved in detoxification processes like other epoxide hydrolases. However, 5,6-ECs failed to induce carcinogenicity in rodents which ruled out a potent carcinogenic potential for 5,6-ECs. Meanwhile, clinical studies revealed an anomalous increase in the concentrations of 5,6?-EC in the nipple fluids of patients with pre-neoplastic breast lesions and in the blood of patients with endometrious cancers, suggesting that 5,6-ECs metabolism could be linked with cancer. Paradoxically, ChEH has been recently shown to be totally inhibited by therapeutic concentrations of tamoxifen (Tam), which is one of the main drugs used in the hormonotherapy and the chemoprevention of breast cancers. These data would suggest that the accumulation of 5,6-ECs could represent a risk factor, but we found that 5,6-ECs were involved in the induction of breast cancer cell differentiation and death induced by Tam suggesting a positive role of 5,6-ECs. These observations meant that the biochemistry and the metabolism of 5,6-ECs needed to be extensively studied. We will review the current knowledge and the future direction of 5,6-ECs chemistry, biochemistry, metabolism, and relationship with cancer.

Graphical abstract

Graphical abstract Highlights

Posted on 28 May 2012 | 12:49 pm

Caveolae and non-caveolae lipid raft microdomains of human umbilical vein endothelial cells contain utrophin-associated protein complexes

Publication year: 2012
Source:Biochimie

Israel Ramírez-Sánchez, Patricia Mendoza-Lorenzo, Alejandro Zentella-Dehesa, Enrique Méndez-Bolaina, Eleazar Lara-Padilla, Guillermo Ceballos-Reyes, Patricia Canto, Carlos Palma-Flores, Ramón Mauricio Coral-Vázquez

Several studies have shown the importance of dystrophin-associated protein complex in the development of muscular dystrophies and dilated cardiomyopathy associated to vascular dysfunction. In vascular endothelium, dystrophin is substituted for utrophin (autosomal homolog of dystrophin); however, its role in this tissue is unknown. Therefore, it is important to obtain a more extensive knowledge of utrophin and its associated proteins in endothelial cells. In a previous study, we demonstrated the presence of utrophin-associated protein complex (UAPC) in human umbilical vein endothelial cells HUVEC, which interacts with caveolin-1 (Cav-1) and endothelial nitric oxide synthase (eNOS). Also, some of our observations suggested the presence of this complex in distinct membrane domains. Therefore, the aim of this study was to analyze the presence of the UAPC in caveolae and non-caveolae lipid rafts domains of HUVEC at baseline and with a mechanical stimulus. It was demonstrated, by subcellular fractionation and co-immunoprecipitation assays, the association of UAPC with Cav-1 and eNOS in caveolae domains, as well as its interaction with eNOS in non-caveolae lipid raft domains. Additionally, it was also observed that mechanical stress on endothelial cells induced activation and release of eNOS from both caveolae and non-caveolae lipid raft associated to UAPC. Together these results suggest that UAPC located in caveolae and non-caveolae lipid raft domains of HUVECs may have a mechanosensory function that could participate in the control of eNOS activity.

Highlights

Posted on 28 May 2012 | 12:49 pm

Modification of the lipidome in RAW264.7 macrophage subjected to stable silencing of oxysterol-binding proteins

Publication year: 2012
Source:Biochimie

Terhi Vihervaara, Reijo Käkelä, Gerhard Liebisch, Kirill Tarasov, Gerd Schmitz, Vesa M. Olkkonen

Oxysterol-binding protein homologs (ORPs) are implicated in lipid metabolism, vesicle transport and cell signaling. In this study we generated RAW264.7 cells with ORP1L, ORP3, or ORP8 silenced using shRNA lentiviruses. The lipidome of the cells under basal serum-free culture conditions or as treated with oxidized LDL (oxLDL), enzymatically modified LDL (E-LDL), or lipopolysaccharide (LPS) was analyzed by mass spectrometry. Reduction in each ORP resulted in distinct and complex effects on macrophage lipidome. Under basal conditions, ORP1L silencing had strongest effects on phosphatidylinositols (PI, increase), free cholesterol (FC, increase), and cholesteryl esters (CE, increase). ORP3 silencing affected most the glucosyl ceramides (GluCer, decrease) and PE-plasmalogens (PE-pl, decrease), while ORP8 silencing increased FC and CE, and decreased GluCer and PE-pl. Upon LPS treatment, the ORP effects were modified: under these conditions ORP1L silencing caused increase of Cer, ORP3 silencing decrease of PI, and ORP8 silencing decrease of PI and increase of PE, not detectable under basal conditions. The lipid species data were subjected to multivariate statistical analysis of principal components, revealing numerous specific alterations upon ORP silencing. The cells cultured in basal conditions or treated with LPS showed qualitatively different responses. However, in LPS-stimulated cells silencing of any of the three ORPs decreased the relative amount of arachidonic acid-containing PI species, increased the corresponding PE species, and favored 16-carbon sphingomyelin (SM) species at the expense of the 24-carbon ones. As a conclusion, the present study reveals the distinct and sophisticated roles of different ORP proteins as regulators of macrophage lipid composition, with implications for inflammatory signaling.

Highlights

Posted on 28 May 2012 | 12:49 pm

Glutathione synthetase promotes the reduction of arsenate via arsenolysis of glutathione

Publication year: 2012
Source:Biochimie, Volume 94, Issue 6

Balázs Németi, Mary E. Anderson, Zoltán Gregus

The environmentally prevalent arsenate (As^V) undergoes reduction in the body to the much more toxic arsenite (As^III). Phosphorolytic enzymes and ATP synthase can promote the reduction As^V by converting it into arsenylated products in which the pentavalent arsenic is more reducible by glutathione (GSH) to As^III than in inorganic As^V. Glutathione synthetase (GS) can catalyze the arsenolysis of GSH (?-Glu-Cys-Gly) yielding two arsenylated products, i.e. ?-Glu-Cys-arsenate and ADP-arsenate. Thus, GS may also promote the reduction of As^V by GSH. This hypothesis was tested with human recombinant GS, a Mg²⁺ dependent enzyme. GS markedly increased As^III formation when incubated with As^V, GSH, Mg²⁺ and ADP, but not when GSH, Mg²⁺ or ADP were separately omitted. Phosphate, a substrate competitive with As^V in the arsenolysis of GSH, as well as the products of GSH arsenolysis or their analogs, e.g. glycine and ?-Glu-aminobutyrate, decreased As^V reduction. Replacement of ADP with ATP or an analog that cannot be phosphorylated or arsenylated abolished As^V reduction, indicating that GS-supported As^V reduction requires formation of ADP-arsenate. In the presence of ADP, however, ATP (but not its metabolically inert analog) tripled As^V reduction because ATP permits GS to remove the arsenolysis inhibitory glycine and ?-Glu-Cys by converting them into GSH. GS failed to promote As^V reduction when GSH was replaced with ophthalmic acid, a GSH analog substrate of GS containing no SH group (although ophthalmic acid did undergo GS-catalyzed arsenolysis), indicating that the SH group of GSH is important for As^V reduction. Our findings support the conclusion that GS promotes reduction of As^V by catalyzing the arsenolysis of GSH, thus producing ADP-arsenate, which upon being released from the enzyme is readily reduced by GSH to As^III.

Highlights

Posted on 28 May 2012 | 12:49 pm

Kidins220/ARMS interacts with Pdzrn3, a protein containing multiple binding domains

Publication year: 2012
Source:Biochimie

Massimiliano Andreazzoli, Gaia Gestri, Elena Landi, Beatrice D’Orsi, Manuela Barilari, Angela Iervolino, Marianna Vitiello, Stephen W. Wilson, Luciana Dente

We report the identification of a novel partner of Kidins220/ARMS (Kinase D-interacting substrate of 220 kDa/Ankyrin Repeat-rich Membrane Spanning) an adaptor of neurotrophin receptors playing crucial roles during neurogenesis. Screening a phage display library of brain cDNA products we found that D. rerio Pdzrn3, a protein containing RING-finger and PDZ-domains, interacts with Kidins220/ARMS through its first PDZ-domain. Both zebrafish proteins share high homology with the corresponding mammalian proteins and both genes are developmentally expressed in neural districts where early neurogenesis occurs. The interaction was also confirmed by biochemical assays and by co-localization at the tips of growing neurites of PC12 cells induced with nerve growth factor.

Highlights

Posted on 28 May 2012 | 12:49 pm

Single-cycle kinetic analysis of ternary DNA complexes by surface plasmon resonance on a decaying surface

Publication year: 2012
Source:Biochimie

William Palau, Carmelo Di Primo

Complexes involving three DNA strands were used to demonstrate that the single-cycle kinetics (SCK) method, which consists in injecting sequentially samples at increasing concentrations and until now used exclusively to investigate bimolecular complexes by surface plasmon resonance, can be extended to the kinetic analysis of ternary complexes. DNA targets, B, were designed with sequences of variable lengths on their 3? sides that recognise a surface-immobilized biotinylated DNA anchor, A. These targets displayed on their 5? sides sequences that recognise DNA oligonucleotides of variable lengths, C, namely the analytes. Combinations of B and C DNA oligonucleotides on A generated ternary complexes each composed of two Watson–Crick helices displaying different kinetic properties. The target–analyte B–C duplexes were formed by sequentially injecting three increasing concentrations of the analytes C during the dissociation phase of the target B from the anchor A. The sensorgrams for the target–analyte complexes dissociating from the functionalized surface were successfully fitted by the SCK method while the target dissociated from the anchor, i.e. on a decaying surface. Within the range of applicability of the method which is driven by the rate of dissociation of the target from the anchor, the rate and equilibrium constants characteristic of these target–analyte duplexes of the ternary complexes did not depend on how fast the targets dissociated from the immobilized DNA anchor. In addition the results agreed very well with those obtained when such duplexes were analysed directly as bimolecular complexes, i.e. when the target, modified with a biotin, was directly immobilized onto a streptavidin sensor chip surface rather than captured by an anchor. Therefore the method we named SCKODS (Single-Cycle Kinetics On a Decaying Surface) can also be used to investigate complexes formed during a dissociation phase, in a ternary complex context. The SCKODS method can be combined with the SCK one to fully characterize the two bimolecular complexes of a ternary complex.

Highlights

Posted on 28 May 2012 | 12:49 pm

The SET8 H4K20 protein lysine methyltransferase has a long recognition sequence covering seven amino acid residues

Publication year: 2012
Source:Biochimie

Srikanth Kudithipudi, Arunkumar Dhayalan, Adam Fiseha Kebede, Albert Jeltsch

The SET8 histone lysine methyltransferase, which monomethylates the histone 4 lysine 20 residue plays important roles in cell cycle control and genomic stability. By employing peptide arrays we have shown that it has a long recognition sequence motif covering seven amino acid residues, viz. R¹⁷–H¹⁸–(R¹⁹KY)–K²⁰–(V²¹ILFY)–(L²²FY)–R²³. Celluspots peptide array methylation studies confirmed specific monomethylation of H4K20 and revealed that the symmetric and asymmetric methylation on R¹⁷ of the H4 tail inhibits methylation on H4K20. Similarly, dimethylation of the R located at the ?3 position also reduced methylation of p53 K382 which had been shown previously to be methylated by SET8. Based on the derived specificity profile, we identified 4 potential non-histone substrate proteins. After relaxing the specificity profile, we identified several more candidate substrates and showed efficient methylation of 20 novel non-histone peptides by SET8. However, apart from H4 and p53 none of the identified novel peptide targets was methylated at the protein level. Since H4 and p53 both contain the target lysine in an unstructured part of the protein, we conclude that the long recognition sequence of SET8 makes it difficult to methylate a lysine in a folded region of a protein, because amino acid side chains essential for recognition will be buried.

Graphical abstract

Graphical abstract Highlights

Posted on 28 May 2012 | 12:49 pm

Functional and biochemical characterisation of the Escherichia coli major facilitator superfamily multidrug transporter MdtM

Publication year: 2012
Source:Biochimie, Volume 94, Issue 6

Scarlett R. Holdsworth, Christopher J. Law

Multidrug resistance (MDR) occurs when bacteria simultaneously acquire resistance to a broad spectrum of structurally dissimilar compounds to which they have not previously been exposed. MDR is principally a consequence of the active transport of drugs out of the cell by proteins that are integral membrane transporters. We characterised and purified the putative Escherichia coli MDR transporter, MdtM, a 410 amino acid residue protein that belongs to the large and ubiquitous major facilitator superfamily. Functional characterisation of MdtM using growth inhibition and whole cell transport assays revealed its role in intrinsic resistance of E. coli cells to the antimicrobials ethidium bromide and chloramphenicol. Site-directed mutagenesis studies implied that the MdtM aspartate 22 residue and the highly conserved arginine at position 108 play a role in proton recognition. MdtM was homologously overexpressed and purified to homogeneity in dodecyl-?-d-maltopyranoside detergent solution and the oligomeric state and stability of the protein in a variety of detergent solutions was investigated using size-exclusion HPLC. Purified MdtM is monomeric and stable in dodecyl-?-d-maltopyranoside solution and binds chloramphenicol with nanomolar affinity in the same detergent. This work provides a firm foundation for structural studies on this class of multidrug transporter protein.

Graphical abstract

Highlights

Posted on 28 May 2012 | 12:49 pm

Intracellular accumulation of bilirubin as a defense mechanism against increased oxidative stress

Publication year: 2012
Source:Biochimie

Jaroslav Zelenka, Lucie Muchova, Miroslava Zelenkova, Katerina Vanova, Hendrik J. Vreman, Ronald J. Wong, Libor Vitek

Antioxidant, anti-inflammatory and anti-atherogenic effects have been associated with elevations of unconjugated bilirubin (UCB) in serum and with the induction of heme oxygenase-1 (HO-1), the rate-limiting enzyme in UCB synthesis. The aim of this study was to investigate the intracellular metabolism and antioxidant properties of UCB in human hepatoblastoma HepG2 cells and tissues of Wistar rats exposed to oxidative stressors and lipopolysaccharide (LPS), respectively. Intracellular UCB concentrations in HepG2 cells correlated with its levels in culture media (p < 0.001) and diminished lipid peroxidation in a dose-dependent manner (p < 0.001). Moreover, induction of HO-1 with sodium arsenite led to 2.4-fold (p = 0.01) accumulation of intracellular UCB over basal level while sodium azide-derived oxidative stress resulted in a 60% drop (p < 0.001). This decrease was ameliorated by UCB elevation in media or by simultaneous induction of HO-1. In addition, hyperbilirubinemia and liver HO-1 induction in LPS-treated rats resulted in a 2-fold accumulation of tissue UCB (p = 0.01) associated with enhanced protection against lipid peroxidation (p = 0.02). In conclusion, hyperbilirubinemia and HO-1 induction associated with inflammation and oxidative stress increase intracellular concentrations of UCB, thus enhancing the protection of cellular lipids against peroxidation. Therefore, the previously reported protective effects of hyperbilirubinemia and HO-1 induction are at least in part due to intracellular accumulation of UCB.

Highlights

Posted on 28 May 2012 | 12:49 pm

Early induction of a brown-like phenotype by rosiglitazone in the epicardial adipose tissue of fatty Zucker rats

Publication year: 2012
Source:Biochimie

Emilie Distel, Graziella Penot, Thomas Cadoudal, Irène Balguy, Sylvie Durant, Chantal Benelli

The epicardial adipose tissue (EAT) is “hypertrophied” in the obese. Thiazolidinediones are anti-diabetic, hypolipidemic drugs and are selective agonists for the gamma isoform of peroxisome proliferator-activated receptor (PPAR?). We evaluated the short-term effects of the prototype rosiglitazone (RSG, 5 mg kg^?1 day^?1 for 4 days) on the expression of the genes and proteins (by real-time PCR and Western blot) involved in fatty acid (FA) metabolism in EAT of the obese fatty Zucker rat and compared the levels of expression with those in retroperitoneal adipose tissue (RAT). The glyceroneogenic flux leading to fatty acid re-esterification was assessed by the incorporation of 14C from [1-14C]-pyruvate into neutral lipids. RSG upregulated the mRNA for phosphoenolpyruvate carboxykinase, pyruvate dehydrogenase kinase 4, glycerol kinase, adipocyte lipid binding protein, adipose tissue triglyceride lipase and lipoprotein lipase in both RAT and EAT with a resulting increase in glyceroneogenesis that, however, was more pronounced in EAT than in RAT. Under RSG, fatty acid output was decreased in both tissues but unexpectedly less so in EAT than in RAT. RSG also induced the expression of the key genes for fatty acid oxidation [carnitinepalmitoyl transferase-1, medium chain acyl dehydrogenase and very long chain acyl dehydrogenase (VLCAD)]in EAT and RAT with a resulting significant rise of  the expression of VLCAD protein. In addition, the expression of the genes encoding proteins involved in mitochondrial processing and density PPAR? coactivator 1 alpha (PGC-1?), NADH dehydrogenase 1 and cytochrome oxidase (COX4) were increased by RSG treatment only in EAT, with a resulting significant up-regulation of PGC1-? and COX4 protein. This was accompanied by a rise in the expression of PR domain containing 16 and uncoupling protein 1, two brown adipose tissue-specific proteins. In conclusion, this study reveals that PPAR-? agonist could induce a rapid browning of the EAT that probably contributes to the increase in lipid turnover.

Highlights

Posted on 28 May 2012 | 12:49 pm

Methylenebisphosphonate and triphosphate derivatives of the mevalonate pathway are substrates of yeast UTP:glucose-1-phosphate uridylyltransferase

Publication year: 2012
Source:Biochimie

María Antonia Günther Sillero, Anabel de Diego, Antonio Sillero

UTP:glucose-1-phospate uridylyltransferase (EC 2.7.7.9) from Saccharomyces cerevisiae transfers the uridylyl moiety of UDP-glucose onto methylenebisphosphonate (pCH2p) yielding uridine 5?-(?,?-methylenetriphosphate) (UppCH2p). The following bisphosphonates were not acceptors of UMP: alendronate, pamidronate, clodronate and etidronate. UDP-glucose serves as uridylyl donor to triphosphate derivatives of the mevalonate pathway: farnesyl (far-PPP), geranyl (ger-PPP) and isopentenyl (iso-PPP), with formation of farnesyl-tetraphosphouridine (far-ppppU); geranyl-tetraphosphouridine (ger-ppppU) and isopentenyl-tetraphosphouridine (iso-ppppU). The K m (mM) and V max (mU/mg protein) values determined for these substrates were: 0.32 ± 0.07 and 4.9 ± 0.6; 0.21 ± 0.06 and 5.7 ± 0.8; 0.51 ± 0.14 and 2.0 ± 0.2, respectively. The K m and V max values for methylenebisphosphonate were 1.1 ± 0.2 mM and 4055 ± 96 mU/mg protein, respectively.

Highlights

Posted on 28 May 2012 | 12:49 pm

Erythrocytes express chondroitin sulphate/dermatan sulphate, which undergoes quantitative changes during diabetes and mediate erythrocyte adhesion to extracellular matrix components

Publication year: 2012
Source:Biochimie, Volume 94, Issue 6

C.B. Srikanth, P.V. Salimath, C.D. Nandini

Glycosaminoglycans (GAGs) such as chondroitin sulphate/dermatan sulphate (CS/DS) are complex molecules that are widely expressed on the cell membrane and extracellular matrix (ECM). They play an important role in wide range of biological activities especially during pathological conditions. Diabetes, a metabolic disorder characterized by sustained hyperglycemia, is known to affect GAGs in different tissues and affect erythrocyte adhesion. The present investigation was aimed at exploring the nature of GAGs present in erythrocytes and its role on adhesion of erythrocytes from control and diabetic rats to major extracellular matrix components. GAGs isolated from erythrocytes were demonstrated to be CS/DS and a 2-fold increase was observed in erythrocytes from diabetic rats. Disaccharide composition analysis by HPLC after depolymerization by the enzyme, chondroitinase ABC showed the presence of 4-O sulphated disaccharide units with small amounts of non-sulphated disaccharides, in both control and diabetic erythrocytes. Erythrocytes from diabetic rats, however, showed significantly increased binding to poly-l-ornithine (P-orn), type IV collagen, laminin and fibronectin, which was abrogated on treatment with chondroitinase ABC to various degrees. This study sheds new light on CS/DS in erythrocytes and its likely biological implications in vivo.

Highlights

Posted on 28 May 2012 | 12:49 pm

Microcapillary-like structures prompted by phospholipase A2 activation in endothelial cells and pericytes co-cultures on a polyhydroxymethylsiloxane thin film

Publication year: 2012
Source:Biochimie

Carmelina Daniela Anfuso, Carla Motta, Cristina Satriano, Salvatore Gennaro, Giovanni Marletta, Giovanni Giurdanella, Mario Alberghina, Gabriella Lupo

A thin film of poly(hydroxymethylsiloxane) (PHMS) has been deposited on glass dishes and tested as artificial support material for vascularization from mixed cultures of endothelial cells (EC) and pericytes (PC). The EC/PC co-cultures adhered massively on PHMS, with the formation of net-like microcapillary structures. Such evidence was not found on control glass substrates in the same co-culture conditions neither on PHMS for EC and PC in monocultures. The physicochemical characterization of PHMS and control glass surface by time-of-flight secondary ion mass spectrometry, X-ray photoelectron spectroscopy, water contact angle and atomic force microscopy, pointed to the main role of the polymer hydrophobilicy to explain the observed cellular behavior. Moreover, enhanced intercellular cross-talk was evidenced by the up-regulation and activation of cytoplasmic and Ca²⁺-independent phospholipase A2 (cPLA2 and iPLA2) expression and cPLA2 phosphorylation, leading to the cell proliferation and microcapillary formation on the PHMS surface, as evidenced by confocal microscopy analyses. Co-cultures, established with growth-arrested PCs by treatment with mitomycin C, showed an increase in EC proliferation on PHMS. AACOCF3 or co-transfection with cPLA2 and iPLA2 siRNA reduced cell proliferation. The results highlight the major role played by EC/PC cross-talk as well as the hydrophobic character of the substrate surface, to promote microcapillary formation. Our findings suggest an attractive strategy for vascular tissue engineering and provide new details on the interplay of artificial substrates and capillary formation.

Graphical abstract

Graphical abstract Highlights

Posted on 28 May 2012 | 12:49 pm

Different sensitivity of H69 modification enzymes RluD and RlmH to mutations in Escherichia coli 23S rRNA

Publication year: 2012
Source:Biochimie, Volume 94, Issue 5

Margus Leppik, Rya Ero, Aivar Liiv, Kalle Kipper, Jaanus Remme

Nucleoside modifications are introduced into the ribosomal RNA during the assembly of the ribosome. The number and the localization of the modified nucleosides in rRNAs are known for several organisms. In bacteria, rRNA modified nucleosides are synthesized by a set of specific enzymes, the majority of which have been identified in Escherichia coli. Each rRNA modification enzyme recognizes its substrate nucleoside(s) at a specific stage of ribosome assembly. Not much is known about the specificity determinants involved in the substrate recognition of the modification enzymes. In order to shed light on the substrate specificity of RluD and RlmH, the enzymes responsible for the introduction of modifications into the stem-loop 69 (H69), we monitored the formation of H69 pseudouridines (?) and methylated pseudouridine (m³?) in vitro on ribosomes with alterations in 23S rRNA. While the synthesis of ?s in H69 by RluD is relatively insensitive to the point mutations at neighboring positions, methylation of one of the ?s by RlmH exhibited a much stronger sensitivity. Apparently, in spite of synthesizing modifications in the same region or even at the same position of rRNA, the two enzymes employ different substrate recognition mechanisms.

Highlights

Posted on 28 May 2012 | 12:49 pm

The buccal gland of Lampetra japonica is a source of diverse bioactive proteins

Publication year: 2012
Source:Biochimie, Volume 94, Issue 5

Rong Xiao, Yue Pang, Qing Wei Li

The parasitic phase lampreys (Lampetra japonica) are bloodsuckers in the marine, and their buccal gland secretion (lamphredin) contains various regulators such as anticoagulants, ion channel blockers, and immune suppressors like those from leeches, insects, ticks, vampire bats, and snakes. This review focuses on the functions and characteristics of the active proteins from the buccal gland of L. japonica for the first time, and provides new insights into the parasitic mechanisms of lampreys and the possibilities of developing drugs such as novel anticoagulants, thrombolytic agents, local anesthetics, and immunosuppressants.

Highlights

Posted on 28 May 2012 | 12:49 pm

A 43 kD protein from the leaves of the herb Cajanus indicus L. modulates doxorubicin induced nephrotoxicity via MAPKs and both mitochondria dependent and independent pathways

Publication year: 2012
Source:Biochimie, Volume 94, Issue 6

Sankhadeep Pal, Parames C. Sil

Doxorubicin (Dox), a chemotherapeutic drug, is used for the treatment of different types of cancers. Application of this drug has now been made limited because of its several acute and chronic side effects. The aim of this work is to investigate the nephroprotecive role of a 43 kD protein (CI protein) isolated from the leaves of Cajanus indicus L, against Dox-induced oxidative impairment and kidney tissue damage. Administration of Dox (20 mg/kg body weight, once) significantly enhanced levels of blood urea nitrogen (BUN), creatinine, uric acid, TNF-?, urinary ?-glutamyl transpeptidase (?-GT) activity, total urinary protein and urinary glucose level. Moreover, Dox exposure increased intracellular ROS production, lipid peroxidation, protein carbonylation and ATPases (like Na⁺/K⁺, Mg²⁺.) activities. On the other hand, the same exposure decreased GSH level and the activities of antioxidant enzymes; indicating that Dox-induced renal damage was mediated via oxidative stress. Signal transduction studies showed that Dox markedly decreased mitochondrial membrane potential, disturbed Bcl-2 family protein balance, enhanced cytochrome c release in the cytosol, increased levels of Apaf1, caspase-9/3/8, FAS, cleaved PARP protein and ultimately led to apoptotic cell death. In addition, Dox distinctly increased the phosphorylation of p38, JNK and ERK MAPKs. Post treatment with CI protein (3 mg/kg body weight, once daily for 4 days), however, reduced Dox-induced oxidative stress and suppressed all these apoptotic events. Histological studies also support the beneficial role of the CI protein in this organ pathophysiology. Combining, results suggest that CI protein might act as a beneficial agent in Dox-induced renal dysfunctions.

Graphical abstract

Highlights

Posted on 28 May 2012 | 12:49 pm

Adaptation of aminoacylation identity rules to mammalian mitochondria

Publication year: 2012
Source:Biochimie, Volume 94, Issue 5

Aurélie Fender, Agnès Gaudry, Frank Jühling, Marie Sissler, Catherine Florentz

Many mammalian mitochondrial aminoacyl-tRNA synthetases are of bacterial-type and share structural domains with homologous bacterial enzymes of the same specificity. Despite this high similarity, synthetases from bacteria are known for their inability to aminoacylate mitochondrial tRNAs, while mitochondrial enzymes do aminoacylate bacterial tRNAs. Here, the reasons for non-aminoacylation by a bacterial enzyme of a mitochondrial tRNA have been explored. A mutagenic analysis performed on in vitro transcribed human mitochondrial tRNA^Asp variants tested for their ability to become aspartylated by Escherichia coli aspartyl-tRNA synthetase, reveals that full conversion cannot be achieved on the basis of the currently established tRNA/synthetase recognition rules. Integration of the full set of aspartylation identity elements and stabilization of the structural tRNA scaffold by restoration of D- and T-loop interactions, enable only a partial gain in aspartylation efficiency. The sequence context and high structural instability of the mitochondrial tRNA are additional features hindering optimal adaptation of the tRNA to the bacterial enzyme. Our data support the hypothesis that non-aminoacylation of mitochondrial tRNAs by bacterial synthetases is linked to the large sequence and structural relaxation of the organelle encoded tRNAs, itself a consequence of the high rate of mitochondrial genome divergence.

Highlights

Posted on 28 May 2012 | 12:49 pm