The replication cycle of polyomaviruses is, consistently with other viruses, fully dependent on host cells. Not only the cellular replicational and translational mechanisms are important for viruses, but also the virus infection is affected by other cellular proteins. This work is focused on the role of major cytoplasmic deacetylase, histone deacetylase 6 (HDAC6) in replication cycle of murine polyomavirus (MPyV). We showed that the presence of fully functional HDAC6 is essential for successful and productive infection. We found that HDAC6 affects not only early phase, but also late phase of infection. Cells with inhibited, or absent HDAC6 are infected with decreased effectivity and moreover lower amount of infectious viral particles is produced. On the other side, using cells with partially functional HDAC6, either in its deacetylase activity or in ubiquitin-binding activity, leads to increased ability of MPyV to infect those cells. Analysis of levels of early LT antigen and late structural protein VP1 in the infected cells showed, that viral proteins are affected by HDAC6. Our data suggest, that in the replication cycle of MPyV mainly the ubiquitin-binding domain of HDAC6 is required and the role of this domain in protein metabolism and degradation. In the second part of diploma project, we...

Intracellular transport is provided by two major types of molecular motors kinesins and cytoplasmic dynein. Kinesin-1 is a molecular motor that transports molecules and organelles along microtubule tracks anterogradely. Specific protein-protein interactions are required to activate kinesin-1 as the free kinesin exist in an autoinhibited state. The activation of kinesin-1 induces its conformational change, enables microtubule binding and ATP hydrolysis necessary for the directional cargo transport. HDAC6 is a multifunctional protein composed of several domains. It plays an important role in many microtubule dependent processes as HDAC6 is a major tubulin deacetylase. It has been shown that HDAC6 manipulation (inhibition/genetic ablation) affects transport along microtubules but the exact mechanisms are unknown. The effect can be caused either by deacetylation microtubules or direct interaction with molecular motors. This thesis is focused on characterization of interactions between kinesin-1 and HDAC6 that have not been described so far. To this end, we expressed and purified various constructs of kinesin-1 and HDAC6 and tested their interactions by microscale thermophoresis (MST) and hydrogen deuterium exchange (HDX) to determine affinity and interaction sites, respectively. MST data revealed that...

Avant le début de ma thèse, le laboratoire avait découvert et caractérisé HDAC6, une Histone Déacétylase atypique qui possède deux domaines déacétylases et peut interagir directement avec l'ubiquitine, grâce à son domaine ZnF-UBP. De plus, le laboratoire avait montré que HDAC6 interagit avec UFD3/PLAP, un régulateur du recyclage de l'ubiquitine, et p97/VCP, un orthologue murin de la chaperonne de levure Cdc48p. Cependant, aucune fonction biologique dans la voie d'ubiquitination des protéines n'était connue pour HDAC6. Nous avons tout d'abord observé que la surexpression de HDAC6 ralenti la dégradation des protéines poly-ubiquitinées, via son ZnF-UBP, son domaine de liaison à l'ubiquitine. Grâce à une série d'expériences, nous avons pu montrer que les complexes HDAC6-p97/VCP régulent directement la stabilité des protéines poly-ubiquitinées. L'accumulation intracellulaire de protéines poly-ubiquitinées peut être toxique pour les cellules si aucune réponse cellulaire n'est engagée. En réalité, une telle accumulation active le facteur de transcription Heat Shock Factor 1 (HSF1) afin de promouvoir la survie de la cellule. Grâce à ces considérations, nous avons découvert que HDAC6 contrôle la réponse cellulaire à l'accumulation de protéines poly-ubiquitinées et avons disséqué les mécanismes impliqués dans ce contrôle. Nous avons trouvé qu'en l'absence de stress, HDAC6 et HSF1 sont en complexes avec p97/VCP et HSP90. Cependant, lorsque la concentration intracellulaire en protéines poly-ubiquitinées augmente, comme lors d'une inhibition du protéasome, HDAC6 est re-larguée du complexe de manière ubiquitine et ZnF-UBP dépendante. Un tel re-largage permet ensuite à p97/VCP d'activer HSF1 et d'engager la cellule dans la réponse au stress.

Mouse polyomavirus (MPyV) capsid is composed of the main capsid protein VP1 and minor capsid proteins VP2 and VP3. Minor proteins are not essential capsid assembly, but they are key for efficient viral infection. The first part of this thesis studies the modifications of VP2 and VP3, the deamidation of Asn at 253 of VP2 (137 of VP3) and N-terminal acetylation of Ala of VP3, which could be the cause of double bands for VP2 and VP3 on SDS-PAGE. Mutated genomes of MPyV N253D (Asn to Asp) and N253E (Asn to Glu) simulating deamidation and A117V (Ala to Val) with reduced acetylation were prepared previously. We prepared three isolations of the mutant viruses and we confirmed that the deamidation is the cause of the double bands. Mutant viruses were compared to the wild type in terms of efficiency of infection, but the role of deamidation could not be proven. Virus A117V is noninfectious either due to lowered acetylation or the substitution of amino acid at this position. This thesis also studies the role of -tubulin acetylation in the infection of MPyV. The role of -tubulin acetylation in viral infection is being investigated to find new antiviral strategies. Acetylation rises after MPyV infection, but this is not due to a change in mRNA expression of tubulin acetylating (TAT1) or deacetylating enzyme...

Zinc-binding proteins represent approximately one tenth of the proteome and a good portion of them are zinc-dependent hydrolases. This thesis focuses on biochemical and structural characterization of glutamate carboxypeptidase II (GCPII) and histone deacetylase 6 (HDAC6), two members of the zinc-dependent metallohydrolase superfamily. We describe here their interactions with natural substrates and inhibitors. GCPII is a homodimeric membrane protease catalyzing hydrolytic cleavage of glutamate from the neurotransmitter N-acetylaspartylglutamate (NAAG) and dietary folates in the central and peripheral nervous systems and small intestine, respectively. This enzyme is associated with several neurological disorders and also presents an ideal target for imaging and treatment of prostate cancer. GCPII inhibitors typically consist of a zinc-binding group (ZBG) linked to an S1' docking moiety (a glutamate moiety or its isostere). As such, these compounds are highly hydrophilic molecules therefore unable to cross the blood-brain barrier and this hampers targeting GCPII to the central nervous system. Different approaches are adopted to alter the S1' docking moiety of the existing inhibitors. As a part of this thesis, we present different strategies relying on replacement of the canonical P1' glutamate residue...