Interactions des sous-unités protéasomales et de la protéine Tat du VIH-1

Etant la source centrale de production de peptides antigéniques, le protéasome, un complexe multiprotéique qui est un constituant majeur de la voie protéolytique non-lysosomale, semble être une des cibles principales pour les virus. En réponse à une production d'interféron-y lors d'une infection virale, les nouveaux immunoprotéasomes produisent des peptides provenant de la dégradation de protéines virales qui vont alors interagirent avec les molécules de classes I du CMH afin de déclencher une réponse cytosolique en vue d'éliminer les cellules infectées. La réponse immunitaire cellulaire à une infection par le VIH est inhabituelle. Ce phénomène serait dû à la protéine Tat du VIH. Un des buts précis de ce projet était de démontrer les interactions entre les différentes sous-unités alpha du protéasome 20S qui n'ont pas été prédictes auparavant dans l'étude de la structure crystal du protéasome 20S de levure. A l'aide de la technique du double hybride nous avons pu montrer que la sous-unité alpha7 intéragit fortement avec la sous-unité alpha4 ainsi qu'avec les 5 autres sous-unités alpha. Ces résultats ont été également justifié par l'interaction des sous-unités alpha-radiomarquées, aux protéines de fusions GST-alpha. L'autre but de ce projet était de déterminer quelles sous-unités du protéasome 20S interagissaient avec la protéine Tat. Utilisant la technique de chromatographie d'affinité nous avons pu montrer que la protéine Tat intéragissait avec les sous-unités alpha4 et alpha7 ainsi qu'avec 8 pro-sous-unités bêta. En outre, la protéine Tat inhibe partiellement l'interaction entre ces deux sous-unités. D'autre part la protéine Tat inhibe l'activité chymotrypsine du protéasome 20S in vivo. La protéine Tat peut ainsi, d'une part, interférer avec l'assemblage du protéasome 20S et, d'autre part, inhiber la production de peptides antigéniques et donc leurs présentations aux molécules de classes I des cellules infectéesCLERMONT FD-BCIU Sci.et Tech. (630142101) / SudocSudocFranceF

Fanconi anemia A protein participates in nucleolar homeostasis maintenance and ribosome biogenesis

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A yeast-based assay identifies drugs that interfere with immune evasion of the Epstein-Barr virus

Epstein-Barr virus (EBV) is tightly associated with certain human cancers, but there is as yet no specific treatment against EBV-related diseases. The EBV-encoded EBNA1 protein is essential to maintain viral episomes and for viral persistence. As such, EBNA1 is expressed in all EBV-infected cells, and is highly antigenic. All infected individuals, including individuals with cancer, have CD8+ T cells directed towards EBNA1 epitopes, yet the immune system fails to detect and destroy cells harboring the virus. EBV immune evasion depends on the capacity of the Gly-Ala repeat (GAr) domain of EBNA1 to inhibit the translation of its own mRNA in cis, thereby limiting the production of EBNA1-derived antigenic peptides presented by the major histocompatibility complex (MHC) class I pathway. Here we establish a yeast-based assay for monitoring GAr-dependent inhibition of translation. Using this assay we identify doxorubicin (DXR) as a compound that specifically interferes with the GAr effect on translation in yeast. DXR targets the topoisomerase-II–DNA complexes and thereby causes genomic damage. We show, however, that the genotoxic effect of DXR and various analogs thereof is uncoupled from the effect on GAr-mediated translation control. This is further supported by the observation that etoposide and teniposide, representing another class of topoisomerase-II–DNA targeting drugs, have no effect on GAr-mediated translation control. DXR and active analogs stimulate, in a GAr-dependent manner, EBNA1 expression in mammalian cells and overcome GAr-dependent restriction of MHC class I antigen presentation. These results validate our approach as an effective high-throughput screening assay to identify drugs that interfere with EBV immune evasion and, thus, constitute candidates for treating EBV-related diseases, in particular EBV-associated cancers

Isoginkgetin derivative IP2 enhances the adaptive immune response against tumor antigens

International audienceAbstract The success of cancer immunotherapy relies on the induction of an immunoprotective response targeting tumor antigens (TAs) presented on MHC-I molecules. We demonstrated that the splicing inhibitor isoginkgetin and its water-soluble and non-toxic derivative IP2 act at the production stage of the pioneer translation products (PTPs). We showed that IP2 increases PTP-derived antigen presentation in cancer cells in vitro and impairs tumor growth in vivo. IP2 action is long-lasting and dependent on the CD8 + T cell response against TAs. We observed that the antigen repertoire displayed on MHC-I molecules at the surface of MCA205 fibrosarcoma is modified upon treatment with IP2. In particular, IP2 enhances the presentation of an exon-derived epitope from the tumor suppressor nischarin. The combination of IP2 with a peptide vaccine targeting the nischarin-derived epitope showed a synergistic antitumor effect in vivo. These findings identify the spliceosome as a druggable target for the development of epitope-based immunotherapies

The nascent polypeptide-associated complex (NAC) controls translation initiation in cis by recruiting nucleolin to the encoding mRNA

Protein aggregates and abnormal proteins are toxic and associated with neurodegenerative diseases. There are several mechanisms to help cells get rid of aggregates but little is known on how cells prevent aggregate-prone proteins from being synthesised. The EBNA1 of the Epstein-Barr virus (EBV) evades the immune system by suppressing its own mRNA translation initiation in order to minimize the production of antigenic peptides for the major histocompatibility (MHC) class I pathway. Here we show that the emerging peptide of the disordered glycine-alanine repeat (GAr) within EBNA1 dislodges the nascent polypeptide-associated complex (NAC) from the ribosome. This results in the recruitment of nucleolin to the GAr-encoding mRNA and suppression of mRNA translation initiation in cis. Suppressing NAC alpha (NACA) expression prevents nucleolin from binding to the GAr mRNA and overcomes GAr-mediated translation inhibition. Taken together, these observations suggest that EBNA1 exploits a nascent protein quality control pathway to regulate its own rate of synthesis that is based on sensing the nascent GAr peptide by NAC followed by the recruitment of nucleolin to the GAr-encoding RNA sequence

A fluorine scan of a tubulin polymerization inhibitor isocombretastatin A-4: Design, synthesis, molecular modelling, and biological evaluation

A novel series of tubulin polymerization inhibitors, based on fluorinated derivatives of isocombretastatin A-4 was synthesized with the goal of evaluating the effect of these compounds on the proliferative activity. The introduction of fluorine atom was performed on the phenyl ring or at the linker between the two aromatic rings. The modification of isoCA-4 by introduction of difluoromethoxy group at the para-position (3i) and substitution of the two protons of the linker by two fluorine atoms (3m), produced the most active compounds in the series, with IC50 values of 0.15–2.2 nM (3i) and 0.1–2 nM (3m) respectively, against a panel of six cancer cell lines. Compounds 3i and 3m had greater antiproliferative activity in comparison with references CA-4 or isoCA-4, the presence of fluorine group leads to a significant enhancement of the antiproliferative activity. Molecular docking studies indicated that compounds 3i and 3m occupy the colchicine binding site of tubulin. Evaluation of cytotoxicity in Human noncancer cells indicated that the compounds 3i and 3m were practically ineffective in quiescent peripheral blood lymphocytes, and may have a selective antiproliferative activity against cancer cells. Analyses of cell cycle distribution, and morphological microtubules organization showed that compound 3m induced G2/M phase arrest and, dramatically disrupted the microtubule network

Design, synthesis and biological evaluation of quinoline-2-carbonitrile-based hydroxamic acids as dual tubulin polymerization and histone deacetylases inhibitors

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