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International audience; While GASP-1 and GASP-2 proteins are known to regulate myogenesis by inhibiting myostatin, their structural organization suggests a putative role as multivalent protease inhibitors controlling different protease activities. In this study, we show the noncompetitive and competitive antitrypsin activities of the full-length GASP-1 and GASP-2 proteins, respectively, by using a bacterial system production and in vitro enzymatic experiments. The role of the second Kunitz domain in this functional duality is described by assessing the antitrypsin activity of GASP-1/2 chimeric proteins. Molecular dynamics simulations support the experimental data to rationalize differences in binding modes between trypsin and the GASP-1 and GASP-2 second Kunitz domains. A new inhibition mechanism was evidenced for the second Kunitz domain of GASP-2, in which the conventional cationic residue of trypsin inhibitors was substituted by the strongly interacting glutamine residue.

The family of serpins is known to fold into a metastable state that is required for the proteinase inhibition mechanism. One of the consequences of this conformational flexibility is the tendency of some mutated serpins to form polymers, which occur through the insertion of the reactive center loop of one serpin molecule into the A-sheet of another. This “A-sheet polymerization” has remained an attractive explanation for the molecular mechanism of serpinopathies. Polymerization of serpins can also take place in vitro under certain conditions (e.g., pH or temperature). Surprisingly, on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, bovSERPINA3-3 extracted from skeletal muscle or expressed in Escherichia coli was mainly observed as a homodimer. Here, in this report, by site-directed mutagenesis of recombinant bovSERPINA3-3, with substitution D371A, we demonstrate the importance of D371 for the intermolecular linkage observed in denaturing and reducing conditions. This residue influences the electrophoretic and conformational properties of bovSERPINA3-3. By structural modeling of mature bovSERPINA3-3, we propose a new “non-A-sheet swap” model of serpin homodimer in which D371 is involved at the molecular interface.

α 1 -Antichymotrypsin is encoded by the unique SERPINA3 gene in humans, while it is encoded by a cluster of eight closely related genes in cattle. BovSERPINA3 proteins present a high degree of similarity and significant divergences in the reactive centre loop (RCL) domains which are responsible for the antiprotease activity. In this study, we analysed their expression patterns in a range of cattle tissues. Even if their expression is ubiquitous, we showed that the expression levels of each serpin vary in different tissues of 15-month-old Charolais bulls. Our results led us to focus on bovSERPINA3-7, one of the two most divergent members of the bovSERPINA3 family. Expression analyses showed that bovSERPINA3-7 protein presents different tissue-specific patterns with diverse degrees of N -glycosylation. Using a specific antibody raised against bovSERPINA3-7, Western blot analysis revealed a specific 96 kDa band in skeletal muscle. BovSERPINA3-7 immunoprecipitation and mass spectrometry revealed that this 96 kDa band corresponds to a complex of bovSERPINA3-7 and creatine kinase M-type. Finally, we reported that the bovSERPINA3-7 protein is present in slow-twitch skeletal myofibres. Precisely, bovSERPINA3-7 specifically colocalized with myomesin at the M-band region of sarcomeres where it could interact with other components such as creatine kinase M-type. This study opens new prospects on the bovSERPINA3-7 function in skeletal muscle and promotes opportunities for further understanding of the physiological role(s) of serpins.

The structural gene for translation initiation factor IF2 (infB) was isolated from the myxobacterium Stigmatella aurantiaca on a 5.18-kb BamHI genomic restriction fragment. The infB gene (ca. 3.16 kb) encodes a 1,054-residue polypeptide with extensive homology within its G domain and C terminus with the equivalent regions of IF2s from Escherichia coli, Bacillus subtilis, Bacillus stearothermophilus, and Streptococcus faecium. The N-terminal region does not display any significant homology to other known proteins. The S. aurantiaca infB gene encodes a single protein which cross-reacted with antiserum to E. coli IF2 and was able to complement an E. coli infB mutant. The S. aurantiaca IF2 is distinguished from all other IF2s by a sequence of 160 residues near the N terminus that has an unusual composition, made up essentially of alanine, proline, valine, and glutamic acid. Within this sequence, the pattern PXXXAP is repeated nine times. Complete deletion of this sequence did not affect the factor's function in initiation of translation and even increased its capacity to complement the E. coli infB mutant.

During the decoding of messenger RNA, each step of the translational cycle requires the intervention of protein factors and the hydrolysis of one or more GTP molecule(s). Of the prokaryotic translational factors, IF2, EF-Tu, SELB, EF-G and RF3 are GTP-binding proteins. In this review we summarize the latest findings on the structures and the roles of these GTPases in the translational process.

An acidic endoprotease (MAEP) secreted during vegetative growth by Myxococcus xanthus DK101 was purified to homogeneity by a series of chromatographic procedures. The endoprotease cleaved the Phe-Met bond of kappa-casein under acidic conditions (pH 5.9). Its apparent molecular mass and its isoelectric point have been estimated to be 12 kDa and 4.5, respectively. From the N-terminal amino acid sequence, a set of two primers for polymerase chain reaction have been designed. Amplification of the corresponding DNA fragment (84 bp) generated a probe, then used to screen an expression DNA library of M. xanthus and to isolate a recombinant plasmid which contained a 2127-bp insert. The nucleotide sequence included an open reading frame (ORF) of 585 nucleotides, encoding 195 amino acids, that exhibited a high degree of similarity with the N-terminal amino acid sequence of the purified MAEP. The polypeptide sequence inferred from this ORF revealed that the mature enzyme should contain 131 amino acids arising from a 195-amino-acid precursor protein.

International audience; Several reports suggested the activation of caspases in postmortem muscle implicating the onset of a caspase-dependent cell death process after animal bleeding. It has been further well established that apoptosis and necrosis are the two major cell death pathways. The questions addressed in the present work were as follows: (a) in postmortem muscle, do cells die as in vivo? and (b) if so, by which dying process this goal is achieved? Selected hallmarks of apoptosis (phosphatidylserine externalization (PS), cell shrinkage, actin degradation) were analyzed in postmortem rat muscles and compared to usual cell behavior in apoptotic and necrotic processes. Results presented clearly demonstrate a rapid PS externalization and cell shrinkage extending during the first 24 h postexsanguination together with a progressive degradation of cytoskeletal and thin filaments of actin. It was therefore concluded that, in postmortem muscle, cells commit suicide soon after animal bleeding through apoptosis.

Serpins are a superfamily of structurally conserved proteins. Inhibitory serpins use a suicide substrate-like mechanism. Some are able to inhibit cysteine proteases in cross-class inhibition. Here, we demonstrate for the first time the strong inhibition of initiator and effector caspases 3 and 8 by two purified bovine SERPINA3s. SERPINA 3-1 (uniprotkb: Q9TTE1 ) binds tighly to human CASP3 (uniprotkb: P42574 ) and CASP8 (uniprotkb: Q14790 ) with kass of 4.2 × 105 and 1.4 × 106 M−1 s−1, respectively. A wholly similar inhibition of human CASP3 and CASP8 by SERPINA3-3 (uniprotkb: Q3ZEJ6 ) was also observed with kass of 1.5 × 105 and 2.7 × 106 M−1 s−1, respectively and form SDS-stable complexes with both caspases. By site-directed mutagenesis of bovSERPINA3-3, we identified Asp371 as the potential P1 residue for caspases. The ability of other members of this family to inhibit trypsin and caspases was analysed and discussed. Structured summary MINT- 7234656 : CASP8 (uniprotkb: Q14790 ) and SERPINA3-1 (uniprotkb: Q9TTE1 ) bind (MI: 0407 ) by biochemical (MI: 0401 ) MINT- 7234634 : SERPINA3-3 (uniprotkb: Q3ZEJ6 ) and CASP3 (uniprotkb: P42574 ) bind (MI: 0407 ) by biochemical (MI: 0401 ) MINT- 7234663 : CASP8 (uniprotkb: Q14790 ) and SERPINA3-3 (uniprotkb: Q3ZEJ6 ) bind (MI: 0407 ) by biochemical (MI: 0401 ) MINT- 7234625 : SERPINA3-1 (uniprotkb: Q9TTE1 ) and CASP3 (uniprotkb: P42574 ) bind (MI: 0407 ) by biochemical (MI: 0401 )

Background The superfamily of ser ine p roteinase in hibitors (serpins) is involved in numerous fundamental biological processes as inflammation, blood coagulation and apoptosis. Our interest is focused on the SERPINA3 sub-family. The major human plasma protease inhibitor, α1-antichymotrypsin, encoded by the SERPINA3 gene, is homologous to genes organized in clusters in several mammalian species. However, although there is a similar genic organization with a high degree of sequence conservation, the reactive-centre-loop domains, which are responsible for the protease specificity, show significant divergences. Results We provide additional information by analyzing the situation of SERPINA3 in the bovine genome. A cluster of eight genes and one pseudogene sharing a high degree of identity and the same structural organization was characterized. Bovine SERPINA3 genes were localized by radiation hybrid mapping on 21q24 and only spanned over 235 Kilobases. For all these genes, we propose a new nomenclature from SERPINA3-1 to SERPINA3-8. They share approximately 70% of identity with the human SERPINA3 homologue. In the cluster, we described an original sub-group of six members with an unexpected high degree of conservation for the reactive-centre-loop domain, suggesting a similar peptidase inhibitory pattern. Preliminary expression analyses of these bovSERPINA3s showed different tissue-specific patterns and diverse states of glycosylation and phosphorylation. Finally, in the context of phylogenetic analyses, we improved our knowledge on mammalian SERPINAs evolution. Conclusion Our experimental results update data of the bovine genome sequencing, substantially increase the bovSERPINA3 sub-family and enrich the phylogenetic tree of serpins. We provide new opportunities for future investigations to approach the biological functions of this unusual subset of serine proteinase inhibitors.

In the present work, a new endopin-like serpin designed mEndopin 1B was purified from bovine muscle. Biochemical characterizations (amino acid sequencing and Maldi-Tof mass spectrometry peptide mapping) demonstrated that the purified protein is different from the previously described Endopin 1, renamed mEndopin 1A. The genes and cDNA of both endopins were characterized. The cDNA sequence of mEndopin 1B encodes a predicted protein of 411 amino-acids with a molecular mass of 43808Da. The mEndopin 1B gene comprised four coding exons and an additional 5' untranslated exon. The reactive site sequence of mEndopin 1B is somewhat different from that of mEndopin 1A. Nevertheless, both serpins have a similar peptidase inhibitory pattern against examined proteases (elastase, trypsin, plasmin and chymotrypsin). The high expression of both mEndopin 1A and 1B in bovine serum and tissues and their high efficiency to inhibit elastase (k(ass) approximately 10(6)-10(7) M(-1) s(-1)) suggested that these serpins might play a major role in inflammatory processes.

In the present work, an endopin-like elastase inhibitor was purified for the first time from bovine muscle. A three-step chromatography procedure was developed including successively SP-Sepharose, Q-Sepharose and EMD-DEAE 650. This procedure provides about 300 mu g of highly pure inhibitor from 500 g of bovine diaphragm muscle. The N-terminal sequence of the muscle elastase inhibitor, together with the sequence of a trypsin-generated peptide, showed 100% similarity with the cDNA deduced sequence of chromaffin cell endopin 1. Hence, the muscle inhibitor was designated muscle endopin 1 (mEndopin 1). mEndopin 1 had a molecular mass of 70 kDa, as assessed by both gel filtration and SDS/PAGE. According to the association rates determined, mEndopin 1 is a potent inhibitor of elastase (k(ass) = 2.41 x 10(7) M-1 . s(-1)) and trypsin (k(ass) = 3.92 x 10(6) M-1 . s(-1)), whereas plasmin (k(ass) 1.78 x 10(3) M-1 . s(-1)) and chymotrypsin (k(ass) = 1.0 x 10(2) M-1 . s(-1)) were only moderately inhibited. By contrast, no inhibition was detected against several other selected serine proteinases, as well as against cysteine proteinases of the papain family. The cellular location of mEndopin in muscle tissue and its tissue distribution were investigated using a highly specific rabbit antiserum. The results obtained demonstrate an intracellular location and a wide distribution in bovine tissues.

Calpain 1 behaviour toward cytoskeletal targets was investigated using two alpha-actinin isoforms from smooth and skeletal muscles. These two isoforms which are, respectively, sensitive and resistant to calpain cleavage, interact with the protease when using in vitro binding assays. The stability of the complexes in EGTA [Kd(-Ca2+) = 0.5 +/- 0.1 microM] was improved in the presence of 1 mm calcium ions [Kd(+Ca2+) = 0.05 +/- 0.01 microM]. Location of the binding structures shows that the C-terminal domain of alpha-actinin and each calpain subunit, 28 and 80 kDa, participates in the interaction. In particular, the autolysed calpain form (76/18) affords a similar binding compared to the 80/28 intact enzyme, with an identified binding site in the catalytic subunit, located in the C-terminal region of the chain (domain III-IV). The in vivo colocalization of calpain 1 and alpha-actinin was shown to be likely in the presence of calcium, when permeabilized muscle fibres were supplemented by exogenous calpain 1 and the presence of calpain 1 in Z-line cores was shown by gold-labelled antibodies. The demonstration of such a colocalization was brought by coimmunoprecipitation experiments of calpain 1 and alpha-actinin from C2.7 myogenic cells. We propose that calpain 1 interacts in a resting state with cytoskeletal targets, and that this binding is strengthened in pathological conditions, such as ischaemia and dystrophies, associated with high calcium concentrations.

The tufB gene, encoding elongation factor Tu (EF-Tu), from the myxobacterium Stigmatella aurantiaca was cloned and sequenced. It is preceded by four tRNA genes, the first ever described in myxobacteria. The tRNA synthesized from these genes and the general organization of the locus seem identical to that of Escherichia coli, but differences of potential importance were found in the tRNA sequences and in the intergenic regions. The primary structure of EF-Tu was deduced from the tufB DNA sequence. The factor is composed of 396 amino acids, with a predicted molecular mass of 43.4 kDa, which was confirmed by expression of tufB in maxicells. Sequence comparisons between S.aurantiaca EF-Tu and other bacterial homologues from E.coli, Salmonella typhimurium and Thermus thermophilus displayed extensive homologies (75.9%). Among the variable positions, two Cys residues probably involved in the temperature sensitivity of E.coli and S.typhimurium EF-Tu are replaced in T.thermophilus and S.aurantiaca EF-Tu. Since two or even three tuf genes have been described in other bacterial species, the presence of multiple tuf genes was sought for. Southern and Northern analysis are consistent with two tuf genes in the genome of S.aurantiaca. Primer extension experiments indicate that the four tRNA genes and tufB are organized in a single operon.

Skeletal muscles, responsible for voluntary movements such as locomotion or posture maintenance, represent about 40% of body mass. This muscle mass is maintened by several signaling pathways that regulate , among other things, the balance between synthesis and degradation of myofibrillar proteins. By targeting the Akt/mTOR pathway, myostatin is a negative regulator of myogenesis. It inhibits myogenic differentiation and cell turnover. Among the various endogenous molecular factors that regulate myostatin, proteins GASP (Growth and differentiation factor Associated Serum Protein) have been described as antagonists of its activity. The Animal Genetics Unit has developed several strategies to understand the molecular mechanisms that govern the role (s) of GASP proteins during muscle development. Thus, the creation of the transgenic mouse line named surGasp-1-20 has shown that overexpression of Gasp-1 results in a hypermuscular phenotype associated with myofibril hypertrophy. An analysis of gene expression in myoblasts derived from satellite cells showed overexpression of myostatin correlating with an absence of hyperplasia in Gasp-1-20 mice. Similar studies currently underway for the protein GASP-2 should clarify its role in the muscular context. Proteins GASP are also defined as compound heterotypic inhibitors characterized by several inhibitory domains that can modulate the activity of different proteases. Among these different modules, the second Kunitz domain of GASP-2 was previously been described as able to inhibit trypsin. In this work, we have shown that the two whole proteins conserve this capacity of inhibition. However, our results indicate that GASP-1 and GASP-2 exhibit a difference in specificity due to the composition of the second Kunitz domain and not to the molecular environment present in each of the proteins. Finally, by modeling, we propose a structural model of the second Kunitz domain of GASP-1 and GASP-2 implicated in the antitrypsin inhibition specificity.; Les muscles squelettiques, responsables des mouvements volontaires tels que la locomotion ou le maintien de la posture, représentent environ 40% de la masse corporelle.Cette masse musculaire est maintenue par plusieurs voies de signalisation qui régulent entre autres l’équilibre entre la synthèse et la dégradation des protéines myofibrillaires. En ciblant la voie de signalisation Akt/mTOR, la myostatine est un régulateur négatif de la myogenèse. Elle inhibe la différentiation myogénique et le renouvellement cellulaire. Parmi les différents facteurs moléculaires extracellulaires qui régulent la myostatine, les protéines GASP (Growth and differentiation factor Associated Serum Protein) ont été décrites comme des antagonistes de son activité. L’Unité de Génétique Animale a développé plusieurs stratégies qui permettent d’appréhender les mécanismes moléculaires qui régissent le(s) rôle(s) des protéines GASP au cours du développement musculaire. Ainsi, la création de la lignée murine appelée surGasp-1-20 a permis de montrer que la surexpression de Gasp-1 entraîne un phénotype hypermusclé associé à une hypertrophie des myofibrilles. Une analyse de l’expression génique dans des myoblastes dérivés des cellules satellites montre une surexpression de la myostatine corrélant avec une absence d’hyperplasie chez les souris surGasp-1-20. Des études similaires actuellement en cours pour la protéine GASP-2 devraient permettre de préciser son rôle dans le contexte musculaire. Les protéines GASP sont également définies comme des inhibiteurs composés hétérotypiques caractérisés par plusieurs domaines inhibiteurs pouvant moduler l’activité de différentes protéases. Parmi ces différents domaines,le second domaine Kunitz de GASP-2 a été précédemment décrit comme pouvant inhiber la trypsine. Dans ce travail, nous avons pu montrer que les deux protéines entières conservent cette capacité d’inhibition. Nos résultats indiquent cependant que GASP-1 et GASP-2 présentent une différence de spécificité due à la composition du second domaine Kunitz et non à l’environnement moléculaire présent dans chacune des protéines. Enfin, nous proposons un modèle structural du second domaine Kunitz impliqué dans la dualité fonctionnelle dans l'inhibition anti-trypsine de GASP-1 et GASP-2.

Skeletal muscles, responsible for voluntary movements such as locomotion or posture maintenance, represent about 40% of body mass. This muscle mass is maintened by several signaling pathways that regulate , among other things, the balance between synthesis and degradation of myofibrillar proteins. By targeting the Akt/mTOR pathway, myostatin is a negative regulator of myogenesis. It inhibits myogenic differentiation and cell turnover. Among the various endogenous molecular factors that regulate myostatin, proteins GASP (Growth and differentiation factor Associated Serum Protein) have been described as antagonists of its activity. The Animal Genetics Unit has developed several strategies to understand the molecular mechanisms that govern the role (s) of GASP proteins during muscle development. Thus, the creation of the transgenic mouse line named surGasp-1-20 has shown that overexpression of Gasp-1 results in a hypermuscular phenotype associated with myofibril hypertrophy. An analysis of gene expression in myoblasts derived from satellite cells showed overexpression of myostatin correlating with an absence of hyperplasia in Gasp-1-20 mice. Similar studies currently underway for the protein GASP-2 should clarify its role in the muscular context. Proteins GASP are also defined as compound heterotypic inhibitors characterized by several inhibitory domains that can modulate the activity of different proteases. Among these different modules, the second Kunitz domain of GASP-2 was previously been described as able to inhibit trypsin. In this work, we have shown that the two whole proteins conserve this capacity of inhibition. However, our results indicate that GASP-1 and GASP-2 exhibit a difference in specificity due to the composition of the second Kunitz domain and not to the molecular environment present in each of the proteins. Finally, by modeling, we propose a structural model of the second Kunitz domain of GASP-1 and GASP-2 implicated in the antitrypsin inhibition specificity.; Les muscles squelettiques, responsables des mouvements volontaires tels que la locomotion ou le maintien de la posture, représentent environ 40% de la masse corporelle.Cette masse musculaire est maintenue par plusieurs voies de signalisation qui régulent entre autres l’équilibre entre la synthèse et la dégradation des protéines myofibrillaires. En ciblant la voie de signalisation Akt/mTOR, la myostatine est un régulateur négatif de la myogenèse. Elle inhibe la différentiation myogénique et le renouvellement cellulaire. Parmi les différents facteurs moléculaires extracellulaires qui régulent la myostatine, les protéines GASP (Growth and differentiation factor Associated Serum Protein) ont été décrites comme des antagonistes de son activité. L’Unité de Génétique Animale a développé plusieurs stratégies qui permettent d’appréhender les mécanismes moléculaires qui régissent le(s) rôle(s) des protéines GASP au cours du développement musculaire. Ainsi, la création de la lignée murine appelée surGasp-1-20 a permis de montrer que la surexpression de Gasp-1 entraîne un phénotype hypermusclé associé à une hypertrophie des myofibrilles. Une analyse de l’expression génique dans des myoblastes dérivés des cellules satellites montre une surexpression de la myostatine corrélant avec une absence d’hyperplasie chez les souris surGasp-1-20. Des études similaires actuellement en cours pour la protéine GASP-2 devraient permettre de préciser son rôle dans le contexte musculaire. Les protéines GASP sont également définies comme des inhibiteurs composés hétérotypiques caractérisés par plusieurs domaines inhibiteurs pouvant moduler l’activité de différentes protéases. Parmi ces différents domaines,le second domaine Kunitz de GASP-2 a été précédemment décrit comme pouvant inhiber la trypsine. Dans ce travail, nous avons pu montrer que les deux protéines entières conservent cette capacité d’inhibition. Nos résultats indiquent cependant que GASP-1 et GASP-2 présentent une différence de spécificité due à la composition du second domaine Kunitz et non à l’environnement moléculaire présent dans chacune des protéines. Enfin, nous proposons un modèle structural du second domaine Kunitz impliqué dans la dualité fonctionnelle dans l'inhibition anti-trypsine de GASP-1 et GASP-2.