|
|
Zinc-Dependent Hydrolases: Structure-Function Study of Glutamate Carboxypeptidase II and Histone Deacetylase 6
Škultétyová, Ľubica ; Bařinka, Cyril (advisor) ; Obšil, Tomáš (referee) ; Novák, Petr (referee)
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...
|
|
|
The role of histone deacetylase 6 in murine polyomavirus replication cycle
Vlachová, Štěpánka ; Horníková, Lenka (advisor) ; Saláková, Martina (referee)
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...
|
|
|
Zinc-Dependent Hydrolases: Structure-Function Study of Glutamate Carboxypeptidase II and Histone Deacetylase 6
Škultétyová, Ľubica
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...
|
|
|
Analysis of Histone Deacetylase 6/Kinesin Interactions
Nedvědová, Jana ; Bařinka, Cyril (advisor) ; Pavlíček, Jiří (referee)
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...
|
|
|
Zinc-Dependent Hydrolases: Structure-Function Study of Glutamate Carboxypeptidase II and Histone Deacetylase 6
Škultétyová, Ľubica
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...
|
|
|
Zinc-Dependent Hydrolases: Structure-Function Study of Glutamate Carboxypeptidase II and Histone Deacetylase 6
Škultétyová, Ľubica ; Bařinka, Cyril (advisor) ; Obšil, Tomáš (referee) ; Novák, Petr (referee)
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...
|
guest ::
