First name
Eleonora
Last name
Valentini
Year of Study
Research Center
Thesis Title
Understanding the catalytic mechanisms of ubiquitin-E3 ligases
Thesis Abstract
E3 ubiquitin ligases are regulatory enzymes of the ubiquitination pathway, 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.
For 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 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.
We clarified some aspects of Rabex-5 as E3 ligase. By Y2H, GST-pull-down assays and
ITC analysis, we identified specific E2 partners, Ube2D and Ube2E families, that bind Rabex-
5 only when in active Ub-loaded state. Performing IVT-auto-Ub assay and disulfide stability
assay we confirmed that ZnF_A20 is the minimal domain responsible for catalytic activity. To
obtain the structure of Rabex-51-74:E2-Ub complex, we tested, unfortunately without success,
crystallization trials and SAXS analysis.
We also analyzed Rabex-5 catalytic activity towards on H-Ras, 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. List of 67 proteins represent the most statistically stringent and
conservative estimate for Rabex-5 substrates that we will validate.
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.
For 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 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.
We clarified some aspects of Rabex-5 as E3 ligase. By Y2H, GST-pull-down assays and
ITC analysis, we identified specific E2 partners, Ube2D and Ube2E families, that bind Rabex-
5 only when in active Ub-loaded state. Performing IVT-auto-Ub assay and disulfide stability
assay we confirmed that ZnF_A20 is the minimal domain responsible for catalytic activity. To
obtain the structure of Rabex-51-74:E2-Ub complex, we tested, unfortunately without success,
crystallization trials and SAXS analysis.
We also analyzed Rabex-5 catalytic activity towards on H-Ras, 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. List of 67 proteins represent the most statistically stringent and
conservative estimate for Rabex-5 substrates that we will validate.
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