The Universal Protein Resource (UniProt) in 2010.

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Methicillin-resistant Staphylococcus aureus resensitization by protein synthesis inhibitors : proposal for a mechanism

RESUME Staphylococcus aureus est un important pathogène à gram-positif, à la fois responsable d'infections nosocomiales et communautaires. Le S. aureus résistant à la méthicilline est intrinsèquement résistant aux bêta-lactamines, inhibiteurs de la synthèse de la paroi bactérienne, grâce à une enzyme nouvellement acquise, la protéine liant la pénicilline 2A, caractérisée par une faible affinité pour ces agents et pouvant poursuivre la synthèse de la paroi, alors que les autres enzymes sont bloquées. Ce micro-organisme a également développé des résistances contre quasiment tous les antibiotiques couramment utilisés en clinique. Parallèlement au développement de molécules entièrement nouvelles, il peut être utile d'explorer d'éventuelles caractéristiques inattendues de médicaments déjà existants, par exemple en les combinant, dans l'espoir d'un potentiel effet synergique. Comprendre les mécanismes de tels effets synergiques pourrait contribuer à la justification de leur utilisation clinique potentielle. Récemment, un effet synergique contre le S. aureus résistant à la méthicilline a été décrit entre la streptogramine quinupristine-datfopristine et les bêta-lactamines, aussi bien in vitro qu'in vivo. Le présent travail a pour but de proposer un modèle pour le mécanisme de cette interaction positive et de l'étendre à d'autres classes d'antibiotiques. Premièrement, un certain nombre de méthodes microbiologiques ont permis de mieux cerner la nature de cette interaction, en montrant qu'elle agissait spécifiquement sur le S. aureus résistant à la méthicilline et qu'elle était restreinte à l'association entre inhibiteurs de la synthèse des protéines et bêta-lactamines. Deuxièmement, L'observation de l'influence des inhibiteurs de la synthèse des protéines sur la machinerie de la paroi bactérienne, c'est-à-dire sur l'expression des protéines liant la pénicilline, responsables de la synthèse du peptidoglycan, a montré une diminution de la quantité de ta protéine liant la pénicilline 2, connue pour posséder une activité de transglycosylation, indispensable au bon fonctionnement de la protéine liant la pénicilline 2A, responsable de la résistance à la méthicilline. Troisièmement, l'analyse fine de la composition du peptidoglycan extrait de bactéries, avant ou après traitement par des inhibiteurs de la synthèse des protéines, a montré des altérations corrélant avec leur capacité à agir en synergie avec les bêta-lactamines contre S. aureus résistant à ta méthicilline. Ces altérations dans les muropeptides pourraient représenter une signature de la diminution de la quantité de la protéine liant la pénicilline 2. Le modèle mécanistique retenu considère que les inhibiteurs de la synthèse des protéines pourraient diminuer l'expression de la protéine Liant la pénicilline 2, indispensable à la résistance à la méthiciltine, et que ce déséquilibre dans les enzymes synthétisant la paroi bactérienne pourrait générer une signature dans les muropeptides. SUMMARY Staphylococcus aureus is a major gram-positive pathogen causing both hospital-acquired and community-acquired infections. Methicillin- resistant Staphylococcus aureus is intrinsically resistant to the cell wall inhibitors beta-lactams by virtue of a newly acquired cell-wall-building enzyme, tow-affinity penicillin-binding protein 2A, which can build the wall when other penicillin-binding proteins are blocked. Moreover, the microorganism has developed resistance to virtually all non-experimental antibiotics. In addition of producing entirely new molecules, it is useful to explore unexpected features of existing drugs, for example by using them in combination, expecting drug synergisms. Understanding the mechanisms of such synergisms would help justify their putative clinical utilization. Recently, a synergism between the streptogramin quinupristin-dalfopristin and beta-lactams was reported against methicillin-resistant S. aureus, both in vitro and in vivo. The present work intends to propose a model for the mechanism of this positive interaction and to extend it to other drug classes. First, microbiological experimentation helped better defining the nature of this interaction, restricting it to methicillin-resistant S. aureus, and to the association of protein synthesis inhibitors with beta-lactams. Second, the observation of inhibitors of protein synthesis influence on the cell-wall-building machinery, i.e. on the expression of penicillin-binding proteins responsible for peptidoglycan synthesis, showed a decrease in the amount of penicillin-binding protein 2, known to provide a transglycosylase activity for glycan chain elongation, indispensable for the functionality of the low-affinity penicillin-binding protein 2A responsible for methicillin resistance. Third, the fine analysis of the peptidoglycan composition purified from bacteria before or after treatment with inhibitors of protein synthesis showed alterations that correlated with their ability to synergize with beta-lactams against methicillin-resistant S. aureus. These muropeptide alterations could be the signature of decrease in the amount of penicillin-binding protein 2. The retained mechanistic model is that inhibitors of protein synthesis could decrease the expression of penicillin-binding protein 2, wich is indispensable for methicillin-resistance, and that this imbalance in cell-wall-building enzymes could generate a muropeptide signature.

Cross-presenting dendritic cells are required for control of Leishmania major infection.

Leishmania major infection induces self-healing cutaneous lesions in C57BL/6 mice. Both IL-12 and IFN-γ are essential for the control of infection. We infected Jun dimerization protein p21SNFT (Batf3(-/-) ) mice (C57BL/6 background) that lack the major IL-12 producing and cross-presenting CD8α(+) and CD103(+) DC subsets. Batf3(-/-) mice displayed enhanced susceptibility with larger lesions and higher parasite burden. Additionally, cells from draining lymph nodes of infected Batf3(-/-) mice secreted less IFN-γ, but more Th2- and Th17-type cytokines, mirrored by increased serum IgE and Leishmania-specific immunoglobulin 1 (Th2 indicating). Importantly, CD8α(+) DCs isolated from lymph nodes of L. major-infected mice induced significantly more IFN-γ secretion by L. major-stimulated immune T cells than CD103(+) DCs. We next developed CD11c-diptheria toxin receptor: Batf3(-/-) mixed bone marrow chimeras to determine when the DCs are important for the control of infection. Mice depleted of Batf-3-dependent DCs from day 17 or wild-type mice depleted of cross-presenting DCs from 17-19 days after infection maintained significantly larger lesions similar to mice whose Batf-3-dependent DCs were depleted from the onset of infection. Thus, we have identified a crucial role for Batf-3-dependent DCs in protection against L. major.

The Ca(V)3.3 calcium channel is the major sleep spindle pacemaker in thalamus.

Low-threshold (T-type) Ca(2+) channels encoded by the Ca(V)3 genes endow neurons with oscillatory properties that underlie slow waves characteristic of the non-rapid eye movement (NREM) sleep EEG. Three Ca(V)3 channel subtypes are expressed in the thalamocortical (TC) system, but their respective roles for the sleep EEG are unclear. Ca(V)3.3 protein is expressed abundantly in the nucleus reticularis thalami (nRt), an essential oscillatory burst generator. We report the characterization of a transgenic Ca(V)3.3(-/-) mouse line and demonstrate that Ca(V)3.3 channels are indispensable for nRt function and for sleep spindles, a hallmark of natural sleep. The absence of Ca(V)3.3 channels prevented oscillatory bursting in the low-frequency (4-10 Hz) range in nRt cells but spared tonic discharge. In contrast, adjacent TC neurons expressing Ca(V)3.1 channels retained low-threshold bursts. Nevertheless, the generation of synchronized thalamic network oscillations underlying sleep-spindle waves was weakened markedly because of the reduced inhibition of TC neurons via nRt cells. T currents in Ca(V)3.3(-/-) mice were <30% compared with those in WT mice, and the remaining current, carried by Ca(V)3.2 channels, generated dendritic [Ca(2+)](i) signals insufficient to provoke oscillatory bursting that arises from interplay with Ca(2+)-dependent small conductance-type 2 K(+) channels. Finally, naturally sleeping Ca(V)3.3(-/-) mice showed a selective reduction in the power density of the σ frequency band (10-12 Hz) at transitions from NREM to REM sleep, with other EEG waves remaining unaltered. Together, these data identify a central role for Ca(V)3.3 channels in the rhythmogenic properties of the sleep-spindle generator and provide a molecular target to elucidate the roles of sleep spindles for brain function and development.

Leishmania major metacaspase can replace yeast metacaspase in programmed cell death and has arginine-specific cysteine peptidase activity.

The human protozoan parasite Leishmania major has been shown to exhibit several morphological and biochemical features characteristic of a cell death program when differentiating into infectious stages and under a variety of stress conditions. Although some caspase-like peptidase activity has been reported in dying parasites, no caspase gene is present in the genome. However, a single metacaspase gene is present in L. major whose encoded protein harbors the predicted secondary structure and the catalytic dyad histidine/cysteine described for caspases and other metacaspases identified in plants and yeast. The Saccharomyces cerevisiae metacaspase YCA1 has been implicated in the death of aging cells, cells defective in some biological functions, and cells exposed to different environmental stresses. In this study, we describe the functional heterologous complementation of a S. cerevisiae yca1 null mutant with the L. major metacaspase (LmjMCA) in cell death induced by oxidative stress. We show that LmjMCA is involved in yeast cell death, similar to YCA1, and that this function depends on its catalytic activity. LmjMCA was found to be auto-processed as occurs for caspases, however LmjMCA did not exhibit any activity with caspase substrates. In contrast and similarly to Arabidopsis thaliana metacaspases, LmjMCA was active towards substrates with arginine in the P1 position, with the activity being abolished following H147A and C202A catalytic site mutations. These results suggest that metacaspases are members of a family of peptidases with a role in cell death conserved in evolution notwithstanding possible differences in their catalytic activity.

Hypertension differentially affects the expression of the gap junction protein connexin43 in cardiac myocytes and aortic smooth muscle cells.

Electrical and mechanical coupling of myocytes in heart and of smooth muscle cells in the aortic wall is thought to be mediated by intercellular channels aggregated at gap junctions. Connexin43 (Cx43) is one of the predominant membrane proteins forming junctional channels in the cardiovascular system. This study was undertaken to assess its expression during experimental hypertension. Rats were made hypertensive by clipping one renal artery (two-kidney, one-clip renal hypertension) or by administering deoxycorticosterone and salt (DOCA-salt hypertension). After four weeks, rats from both models showed a similar increase in intra-arterial mean blood pressure, as well as in the thickness of both aorta and heart walls. Northern blot analysis showed that, compared to controls, hypertensive rats expressed twice more Cx43 in aorta, but not in heart. These results suggest that localized mechanical forces induced by hypertension are major tissue-specific regulators of Cx43 expression.

Microtubule-associated protein-2 immunoreactivity: a useful tool in the differential diagnosis of low-grade neuroepithelial tumors.

Complex and variable morphological phenotypes pose a major challenge to the histopathological classification of neuroepithelial tumors. This applies in particular for low-grade gliomas and glio-neuronal tumors. Recently, we and others have identified microtubule-associated protein-2 (MAP2) as an immunohistochemical marker expressed in the majority of glial tumors. Characteristic cell morphologies can be recognized by MAP2 immunoreactivity in different glioma entities, i.e., process sparse oligodendroglial versus densely ramified astrocytic elements. Here, we describe MAP2-immunoreactivity patterns in a large series of various neuroepithelial tumors and related neoplasms (n = 960). Immunohistochemical analysis led to the following conclusions: (1) specific pattern of MAP2-positive tumor cells can be identified in 95% of glial neoplasms; (2) ependymal tumors do not express MAP2 in their rosette-forming cell component; (3) tumors of the pineal gland as well as malignant embryonic tumors are also characterized by abundant MAP2 immunoreactivity; (4) virtually no MAP2 expression can be observed in the neoplastic glial component of glio-neuronal tumors, i.e. gangliogliomas; (5) malignant glial tumor variants (WHO grade III or IV) exhibit different and less specific MAP2 staining patterns compared to their benign counterparts (WHO grade I or II); (6) with the exception of melanomas and small cell lung cancers, MAP2 expression is very rare in metastatic and non-neuroepithelial tumors; (7) glial MAP2 expression was not detected in 56 non-neoplastic lesions. These data point towards MAP2 as valuable diagnostic tool for pattern recognition and differential diagnosis of low-grade neuroepithelial tumors.

MAR elements as tools to increase protein production by CHO cells

One of the major hurdles of isolating stable, inducible or constitutive high-level producer cell lines is the time-consuming selection, analysis and characterization of the numerous clones required to identify one with the desired characteristics. Various boundary elements, matrix attachment regions, and locus control regions were screened for for their ability to augment the expression of heterologous genes in CHO and other cells. The 5'-matrix-attachment region (MAR) of the chicken lysozyme gene was found to significantly increase stable gene expression, in culture dishes and in bioreactors. These MAR elements can be easily combined with various existing expression systems, as they can be added in trans (i.e. on a separate plasmid) in co-transfections with previously constructed expression vectors. Using cell population analysis, we found that the use of the MAR increases the proportion of high-producing CHO cell clones, thus reducing the number of cell lines that need to be screened while increasing maximal productivity. Random cDNA cloning and sequencing indicated that over 12% of the ESTs correspond to the transgene. Thus, productivity is no longer limited by transcriptional events in such MAR-containing cell lines. The identification of small and more convenient active MAR portions will also be summarized. Finally, we will show examples of how MAR elements can be combined with short term expression to increase the simultaneous synthesis of many proteins in parallel by CHO cells. Overall, we conclude that the MAR sequence is a versatile tool to increase protein expression in short and long term production processes.

Antigenicity and immunogenicity of a novel chimeric peptide antigen based on the P. vivax circumsporozoite protein.

BACKGROUND: Plasmodium vivax circumsporozoite (PvCS) protein is a major sporozoite surface antigen involved in parasite invasion of hepatocytes and is currently being considered as vaccine candidate. PvCS contains a dimorphic central repetitive fragment flanked by conserved regions that contain functional domains. METHODS: We have developed a chimeric 137-mer synthetic polypeptide (PvCS-NRC) that includes the conserved region I and region II-plus and the two natural repeat variants known as VK210 and VK247. The antigenicity of PvCS-NRC was tested using human sera from PNG and Colombia endemic areas and its immunogenicity was confirmed in mice with different genetic backgrounds, the polypeptide formulated either in Alum or GLA-SE adjuvants was assessed in inbred C3H, CB6F1 and outbred ICR mice, whereas a formulation in Montanide ISA51 was tested in C3H mice. RESULTS: Antigenicity studies indicated that the chimeric peptide is recognized by a high proportion (60-70%) of residents of malaria-endemic areas. Peptides formulated with either GLA-SE or Montanide ISA51 adjuvants induced stronger antibody responses as compared with the Alum formulation. Sera from immunized mice as well as antigen-specific affinity purified human IgG antibodies reacted with sporozoite preparations in immunofluorescence and Western blot assays, and displayed strong in vitro inhibition of sporozoite invasion (ISI) into hepatoma cells. CONCLUSIONS: The polypeptide was recognized at high prevalence when tested against naturally induced human antibodies and was able to induce significant immunogenicity in mice. Additionally, specific antibodies were able to recognize sporozoites and were able to block sporozoite invasion in vitro. Further evaluation of this chimeric protein construct in preclinical phase e.g. in Aotus monkeys in order to assess the humoral and cellular immune responses as well as protective efficacy against parasite challenge of the vaccine candidate must be conducted.

Exome sequencing identifies DYNC2H1 mutations as a common cause of asphyxiating thoracic dystrophy (Jeune syndrome) without major polydactyly, renal or retinal involvement.

BACKGROUND: Jeune asphyxiating thoracic dystrophy (JATD) is a rare, often lethal, recessively inherited chondrodysplasia characterised by shortened ribs and long bones, sometimes accompanied by polydactyly, and renal, liver and retinal disease. Mutations in intraflagellar transport (IFT) genes cause JATD, including the IFT dynein-2 motor subunit gene DYNC2H1. Genetic heterogeneity and the large DYNC2H1 gene size have hindered JATD genetic diagnosis. AIMS AND METHODS: To determine the contribution to JATD we screened DYNC2H1 in 71 JATD patients JATD patients combining SNP mapping, Sanger sequencing and exome sequencing. RESULTS AND CONCLUSIONS: We detected 34 DYNC2H1 mutations in 29/71 (41%) patients from 19/57 families (33%), showing it as a major cause of JATD especially in Northern European patients. This included 13 early protein termination mutations (nonsense/frameshift, deletion, splice site) but no patients carried these in combination, suggesting the human phenotype is at least partly hypomorphic. In addition, 21 missense mutations were distributed across DYNC2H1 and these showed some clustering to functional domains, especially the ATP motor domain. DYNC2H1 patients largely lacked significant extra-skeletal involvement, demonstrating an important genotype-phenotype correlation in JATD. Significant variability exists in the course and severity of the thoracic phenotype, both between affected siblings with identical DYNC2H1 alleles and among individuals with different alleles, which suggests the DYNC2H1 phenotype might be subject to modifier alleles, non-genetic or epigenetic factors. Assessment of fibroblasts from patients showed accumulation of anterograde IFT proteins in the ciliary tips, confirming defects similar to patients with other retrograde IFT machinery mutations, which may be of undervalued potential for diagnostic purposes.

Protein energy malnutrition increases arginase activity in monocytes and macrophages.

BACKGROUND: Protein energy malnutrition is commonly associated with immune dysfunctions and is a major factor in susceptibility to infectious diseases. METHODS: In this study, we evaluated the impact of protein energy malnutrition on the capacity of monocytes and macrophages to upregulate arginase, an enzyme associated with immunosuppression and increased pathogen replication. RESULTS: Our results show that monocytes and macrophages are significantly increased in the bone marrow and blood of mice fed on a protein low diet. No alteration in the capacity of bone marrow derived macrophages isolated from malnourished mice to phagocytose particles, to produce the microbicidal molecule nitric oxide and to kill intracellular Leishmania parasites was detected. However, macrophages and monocytes from malnourished mice express significantly more arginase both in vitro and in vivo. Using an experimental model of visceral leishmaniasis, we show that following protein energy malnutrition, the increased parasite burden measured in the spleen of these mice coincided with increased arginase activity and that macrophages provide a more permissive environment for parasite growth. CONCLUSIONS: Taken together, these results identify a novel mechanism in protein energy malnutrition that might contributes to increased susceptibility to infectious diseases by upregulating arginase activity in myeloid cells.

G-protein coupled receptor-evoked glutamate exocytosis from astrocytes: role of prostaglandins.

Astrocytes are highly secretory cells, participating in rapid brain communication by releasing glutamate. Recent evidences have suggested that this process is largely mediated by Ca(2+)-dependent regulated exocytosis of VGLUT-positive vesicles. Here by taking advantage of VGLUT1-pHluorin and TIRF illumination, we characterized mechanisms of glutamate exocytosis evoked by endogenous transmitters (glutamate and ATP), which are known to stimulate Ca(2+) elevations in astrocytes. At first we characterized the VGLUT1-pHluorin expressing vesicles and found that VGLUT1-positive vesicles were a specific population of small synaptic-like microvesicles containing glutamate but which do not express VGLUT2. Endogenous mediators evoked a burst of exocytosis through activation of G-protein coupled receptors. Subsequent glutamate exocytosis was reduced by about 80% upon pharmacological blockade of the prostaglandin-forming enzyme, cyclooxygenase. On the other hand, receptor stimulation was accompanied by extracellular release of prostaglandin E2 (PGE2). Interestingly, administration of exogenous PGE2 produced per se rapid, store-dependent burst exocytosis of glutamatergic vesicles in astrocytes. Finally, when PGE2-neutralizing antibody was added to cell medium, transmitter-evoked exocytosis was again significantly reduced (by about 50%). Overall these data indicate that cyclooxygenase products are responsible for a major component of glutamate exocytosis in astrocytes and that large part of such component is sustained by autocrine/paracrine action of PGE2.

Mapping of a gene coding for a major late structural polypeptide on the vaccinia virus genome.

Cell-free translation of total RNA isolated from vaccinia virus-infected cells late in infection results in a complex mixture of polypeptides. A monospecific antibody directed against one of the major structural proteins of the virus particle immunoprecipitated a single polypeptide with a molecular weight of 11,000 (11K) from this mixture. Immunoprecipitation was therefore used to identify the structural polypeptide among the in vitro translation products of RNA purified by hybridization selection to restriction fragments of the vaccinia virus genome. This allowed us to map the mRNA coding for the 11K polypeptide to the extreme left-hand end of the HindIII E fragment. Detailed transcriptional mapping of this region of the genome by nuclease S1 analysis revealed the presence of a late RNA transcribed from the rightward-reading strand. Its 5' end mapped at ca. 130 base pairs to the left of the HindIII site at the junction between the HindIII F and E fragments. The map position of this RNA coincided precisely with the map position of the late message coding for the 11K polypeptide.

" Leishmania major " metacaspase in programmed cell death

Abstract: The human protozoan parasite Leishmania major has been shown to exhibit several morphological and biochemical features characteristic of a programmed cell death (PCD) when differentiating into infectious stages and under a variety of stress conditions. In mammalian cells, the principal effector molecules of PCD or apoptosis are caspases. Although some caspase-like peptidase activity has been reported in dying parasites, no caspase gene is present in the L. major genome. However, a single metacaspase gene is present in L. major whose encoded protein harbors the predicted secondary structure and the catalytic dyad histidine/cysteine described for caspases and other metacaspases identified in plants and yeast. Metacaspases are also present in other protozoan parasites such as Trypanosoma and Plasmodium species and are not present in mammalian cells, which make them a possible drug target for the treatment of the parasitic diseases they cause. The Saccharomyces cerevisiae metacaspase YCA1 has been implicated in the death of aging cells, cells defective in some biological functions, and cells exposed to different environmental stresses. In this study, we evaluated the functional heterologous complementation of a S. cerevisiae ycal null mutant with the L. major metacaspase (LmjMCA} in cell death induced by oxidative stress. We show that LmjMCA is involved in yeast cell death, similar to YCA1, and that this function depends on its catalytic activity. LmjMCA was found to be auto-processed as occurs for caspases, however, LmjMCA did not exhibit any activity with caspase substrates. In contrast, LmjMCA was active towards substrates with arginine in the P1 position, with the activity being abolished following H147A and C202A catalytic site mutations and addition of the arginal inhibitor leupeptin. In order to identify the L. major proteins that may function as substrates, inhibitors, or may bind and recruit LmjMCA, a yeast two-hybrid screening with cDNA libraries from different life cycle stages of the parasite was conducted. Proteins putatively involved in PCD were identified as interacting with LmjMCA, however, the interaction of LmjMCA with proteins involved in other physiological processes such as vesicle transport, suggests that LmjMCA could have additional roles in the different life cycle stages of the parasite. Résumé: Plusieurs caractéristiques morphologiques et biochimiques rappelant la mort cellulaire programmée ont été identifiées dans les stades infectieux et sous des conditions de stress, chez le parasite protozoaire humain, Leishmania major. Dans les cellules de mammifères, les caspases sont les molécules effectrices principales impliquées dans la mort cellulaire programmée et l'apoptose. Bien qu'une activité caspase ait été retrouvée dans des parasites en mon` cellulaire, le génome de Leishmania ne contient aucun gène qui pourrait coder pour une caspase. À la place, on retrouve un gène unique codant pour une métacaspase. Une prédiction de la structure secondaire de la métacaspase montre que cette métacaspase a un domaine catalytique contenant la dyade histidine/cystéine présente dans les caspases et les autres métacaspases décrites chez les plantes et la levure. Les métacaspases sont aussi présentes dans d'autres parasites protozoaires tels que Trypanosome et Plasmodium, mais ne sont pas présentes dans les cellules de mammifères, ce qui en fait des cibles intéressantes pour le développement de drogue. Dans la levure, Saccharomyces cerevisiae, la métacaspase YCA1 est impliquée dans la mort des cellules âgées, la mort des cellules défectueuses dans certaines fonctions biologiques et dans les cellules exposées à différents stress environnementaux. Dans cette étude, une complémentation hétérologue fonctionnelle d'un mutant de la levure déficient en YCA1 par le gène LmjMCA de L. major lors de l'induction de ta mort par stress oxydatif a été évaluée. Nos résultats montrent que LmjMCA peut remplacer YGA1 dans le programme de mort cellulaire chez la levure et que celte fonction dépend de son activité catalytique. De plus, LmjMCA subit une auto clivage comme les caspases mais n'exhibe aucune spécificité pour les substrats des caspases. Au contraire, LmjMCA est active envers des substrats ayant une arginine en position P1, son activité étant détruite suite à des changements de son domaine catalytique par les mutations H147A et C202A ou suite à une inhibition para la leupeptine. Afin d'identifier quels pourraient être les substrats, les inhibiteurs ou les molécules interagissant avec LmjMCA, nous avons entrepris un criblage double-hybride en utilisant des librairies de d'ADNc provenant de différents stades du cycle parasitaire. Plusieurs protéines potentiellement impliquées dans un programme de mort cellulaire ont été identifiées comme interagissant avec LmjMCA. Cependant, l'identification de protéines impliquées dans le transport vésiculaire suggère aussi que LmjMCA pourrait avoir un rôle additionnel dans les différents stades du cycle parasitaire. Résumé destiné à un large public: De nos jours, la leishmaniose est la deuxième plus importante maladie parasitaire après la malaria. Malgré les avancées accomplies dans les stratégies de contrôle, près de deux millions de nouveaux cas apparaissent chaque année. Actuellement, la principale stratégie pour faire face à ce problème épidémiologique consiste en un traitement pharmacologique des personnes infectées. Pourtant, seule une dizaine de médicaments, dont la plupart sont toxiques, est disponible pour traiter la leishmaniose et des cas de résistance émergent dans certains pays endémiques. Il devient donc urgent de mettre au point de nouveaux traitements anti-leishmaniens capables d'éliminer le parasite sans effets indésirables sur le patient. Récemment, des caractéristiques morphologiques et biochimiques de la mort cellulaire programmée (MCP) semblables au processus de l'apoptose chez les mammifères ont été décrites dans Leishmania. Cependant, des gènes codant pour des protéines similaires à celles qui sont impliquées dans l'apoptose, comme les caspases, ne se retrouvent pas dans le génome de Leishmanía major. Néanmoins, les espèces de Leishmanía, aussi bien que d'autres parasites protozoaires responsables des trypanosomiases et de la malaria, possèdent des métacaspases qui sont des protéines homologues aux caspases mais qui ne sont pas présentes chez les mammifères. C'est pourquoi, la caractérisation de la métacaspase de Leishmania (LmjMCA) ainsi que ses mécanismes d'activation pourrait être une piste d'investigation intéressante dans l'identification de nouvelles cibles thérapeutiques dans les voies de signalisation de la MCP des parasites protozoaires. Dans la levure, Saccharomyces cerevisiae, la métacaspase YCA1 est impliquée dans la mort des cellules âgées, la mort des cellules défectueuses dans certaines fonctions biologiques et dans les cellules exposées à différents stress environnementaux. Dans cette étude, une complémentation hétérologue fonctionnelle d'un mutant de la levure déficient en YCA1 par le gène LmjMCA de L major lors de l'induction de la mort par stress oxydatif a été évaluée. Nos résultats montrent que LmjMCA peut remplacer YCA1 dans le programme de mort cellulaire chez la levure et que cette fonction dépend de son activité catalytique. De plus, LmjMCA subit une auto clivage comme les caspases mais n'exhibe aucune spécificité pour les substrats des caspases. Au contraire, LmjMCA est active envers des substrats ayant une arginine en position P1, son activité étant détruite suite à des changements de son domaine catalytique par les mutations H147A et C202A ou suite à une inhibition para la leupeptine. Afin d'identifier quels pourraient être les substrats, les inhibiteurs ou les molécules interagissant avec LmjMCA, nous avons entrepris un criblage double-hybride en utilisant des librairies de d'ADNe provenant de différents stades du cycle parasitaire. Plusieurs protéines potentiellement impliquées dans un programme de mort cellulaire ont été identifiées comme interagissant avec LmjMCA. Cependant, l'identification de protéines impliquées dans le transport vésiculaire suggère aussi que LmjMCA pourrait avoir un rôle additionnel dans les différents stades du cycle parasitaire. Resumen destinado al público en general: La leishmaniasis es la segunda enfermedad parasitaria más importante en el mundo actual. Aproximadamente 2 millones de nuevos casos ocurren cada año a pesar de los avances logrados en el desarrollo de nuevos métodos de control. El tratamiento farmacológico de las personas infectadas es actualmente la principal estrategia de control, sin embargo, menos de una decena de medicamentos se encuentran disponibles en el mercado, la mayoría de ellos son tóxicos, y ya empiezan a encontrarse parásitos resistentes en algunos países endémicos para la leishmaniasis. El desarrollo de nuevos medicamentos capaces de eliminar los parásitos sin producir efectos indeseables en los humanos, es una necesidad inminente. Recientemente, algunas de las características morfológicas y bioquímicas de la muerte celular programada (MCP) similares al proceso de la apoptosis en mamíferos, han sido descritas en parasitos de Leishmania. Sin embargo, genes que codifiquen proteínas similares a aquellas involucradas en la apoptosis, como las caspasas, no se encuentran en el genoma de Leishmania major. AI contrario, las especies de Leishmania, así como de los otros parásitos responsables de la tripanosomiasis y de la malaria, poseen metacaspases, proteínas homologas a las caspases pero que no están presentes en las células de mamíferos. La caracterización de la metacaspasa de L. major y de sus mecanismos de activación constituye, por lo tanto, un área de investigación interesante para la identificación de nuevos blancos terapéuticos en el proceso de MCP de los parásitos protozoarios. En la levadura Saccharomyces cerevisiae, la metacaspasa YCA1 ha sido descrita como implicada en la muerte de células envejecidas, células defectuosas en algunas funciones biológicas, y en células expuestas a diferentes tipos de estrés ambiental. En el presente estudio se evaluó la complementación heteróloga funcional de una levadura mutante deficiente en YCA1 con el gen de metacaspase de L. major (LmjMCA) en la MCP inducida por estrés oxidativo. Nuestros resultados muestran que la LmjMCA puede reemplazarla YCA1 en la MCP de la levadura dependiente de su actividad catalítica y que la LmjMCA se auto-procesa similar a las caspasas. Sin embargo, LmjMCA no reconoce los substratos de caspasas sino substratos con una arginina en ta posición P1. Dicha actividad enzimática fue abolida con la inducción de las mutaciones puntuales H147A y C202A en la díada catalítica de LmjMCA y con la adición de leupeptina, un inhibidor con arginina. Con el fin de identificar proteínas que pudieran funcionar como substratos, inhibidores o moléculas modificadoras de LmjMCA, se aplicó el método de doble-híbrido en levadura con bibliotecas de ADNc provenientes de diferentes estadios del ciclo de vida del parásito. Algunas proteínas potencialmente implicadas. en la MCP del parásito fueron identiñcadas interactuando con LmjMCA. La identificación de otras proteínas involucradas en transporte vesicular sugiere que la LmjMCA podría tener una función biológica adicional en los diferentes estadios del ciclo de vida dei parásito.

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