E3 ubiquitin ligases are key regulatory enzymes of the ubiquitination pathway as they are responsible for substrate specificity. This thesis aimed at deciphering the molecular mechanisms through which two different E3 ligases, Nedd4 and Rabex-5, exert their activity. Nedd4 is the prototype for HECT-E3 ligase while Rabex-5, containing an A20 zinc finger domain (ZnF_A20) instead of a canonical RING, could be defined as an atypical RING-E3 ligase. In the case of Nedd4, we provided the first crystal structure of the catalytic intermediate of HECTNedd4~Ub in complex with Ub non-covalently bound to the UBD present in the N-lobe of HECTNedd4. Our structure represents the next step of the transfer of UbD from the catalytic cysteine of E2 to the one of E3 in which the UbD C-terminal tail is in an extended conformation primed for catalysis. Our data strongly supports the sequential addition model proposed for HECT proteins. Within this study we also clarified some aspects of Rabex-5 as E3 ligase. By yeast-two-hybrid, GST-pull-down assays and ITC analysis, we identified specific E2 partners, Ube2D and Ube2E families, that bind Rabex-5 only when in their active Ub-loaded state. Performing in vitro auto-ubiquitination assay and disulfide stability assay we confirmed that ZnF_A20 is the minimal domain responsible for the catalytic activity. To obtain the structure of the Rabex-51-74:E2-Ub complex, we tested, unfortunately without success, crystallization trials and SAXS analysis with various samples. We also analyzed Rabex-5 catalytic activity towards on H-Ras, which is the unique substrate of Rabex-5 so far identified, and we disproved that H-Ras is a Rabex-5 substrate. To identify candidate substrates we profiled 20.000 human proteins using a microarray-based ubiquitination screening. A list of 67 proteins represent the most statistically stringent and conservative estimate for Rabex-5 substrates that we are going to validate in the nearest future, starting from the ones involved in the endocytic pathway.
UNDERSTANDING THE CATALYTIC MECHANISMS OF UBIQUITIN-E3 LIGASES / E. Valentini ; Marina Mapelli (internal advisor), David Komander (external advisor) ; supervisor: S. Polo. DIPARTIMENTO DI ONCOLOGIA ED EMATO-ONCOLOGIA, 2016 Mar 18. 27. ciclo, Anno Accademico 2015. [10.13130/valentini-eleonora_phd2016-03-18].
UNDERSTANDING THE CATALYTIC MECHANISMS OF UBIQUITIN-E3 LIGASES
E. Valentini
2016
Abstract
E3 ubiquitin ligases are key regulatory enzymes of the ubiquitination pathway as they are responsible for substrate specificity. This thesis aimed at deciphering the molecular mechanisms through which two different E3 ligases, Nedd4 and Rabex-5, exert their activity. Nedd4 is the prototype for HECT-E3 ligase while Rabex-5, containing an A20 zinc finger domain (ZnF_A20) instead of a canonical RING, could be defined as an atypical RING-E3 ligase. In the case of Nedd4, we provided the first crystal structure of the catalytic intermediate of HECTNedd4~Ub in complex with Ub non-covalently bound to the UBD present in the N-lobe of HECTNedd4. Our structure represents the next step of the transfer of UbD from the catalytic cysteine of E2 to the one of E3 in which the UbD C-terminal tail is in an extended conformation primed for catalysis. Our data strongly supports the sequential addition model proposed for HECT proteins. Within this study we also clarified some aspects of Rabex-5 as E3 ligase. By yeast-two-hybrid, GST-pull-down assays and ITC analysis, we identified specific E2 partners, Ube2D and Ube2E families, that bind Rabex-5 only when in their active Ub-loaded state. Performing in vitro auto-ubiquitination assay and disulfide stability assay we confirmed that ZnF_A20 is the minimal domain responsible for the catalytic activity. To obtain the structure of the Rabex-51-74:E2-Ub complex, we tested, unfortunately without success, crystallization trials and SAXS analysis with various samples. We also analyzed Rabex-5 catalytic activity towards on H-Ras, which is the unique substrate of Rabex-5 so far identified, and we disproved that H-Ras is a Rabex-5 substrate. To identify candidate substrates we profiled 20.000 human proteins using a microarray-based ubiquitination screening. A list of 67 proteins represent the most statistically stringent and conservative estimate for Rabex-5 substrates that we are going to validate in the nearest future, starting from the ones involved in the endocytic pathway.File | Dimensione | Formato | |
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