Nedd4 is the prototype member of the HECT E3 ubiquitin ligase family involved in signalling and cancer and is the subject of this work. In humans, there are nine members of this family that are implicated in a range of biological processes such as endocytosis, protein transport, viral budding, signalling, cellular growth and proliferation. Among them Nedd4 is involved in the intracellular trafficking of the epidermal growth factor receptor (EGFR), either directly or indirectly through its action on endocytic adaptors such as eps15 and epsins. In this thesis we elucidated the mechanisms that regulate the activity of this E3 ligase. We addressed this issue with two approaches: i) protein interaction studies, crystallization analysis and mutagenesis approach were used to determine the intra-molecular interaction between ubiquitin and the HECT domain and between the C2 and the HECT domains and their biological relevance ii) mass spectrometry analysis was conducted on Nedd4 purified from cells in order to identify post-translational modifications induced by EGF stimulation. We provided structural data on the ubiquitin-binding domain present in the HECT domain of the ligase and we demonstrated that it is critically required for the processivity of the enzyme. In addition, we found that the intra-molecular interaction between C2 and HECT domain maps very close to the ubiquitin-binding site in the HECT, inhibiting its activity. Finally, we identified an additional level of complexity exerted by post-translational modifications (ubiquitination and phosphorylation) in the context of the EGFR signalling cascade. The results described in this thesis suggest a model whereby in resting condition the ligase is inactive due to an intra-molecular interaction that limits the ubiquitin binding ability and the activity of the ligase; ubiquitination and phosphorylation induced by EGF stimulation may regulate the release of this auto-inhibition, leading to ligase activation and its membrane relocalization via the displaced C2 domain.
|Titolo:||REGULATION OF THE E3 LIGASE NEDD4|
|Autori interni:||MARI, SARA|
|Relatore:||POLO, SIMONA LAURA ANNA|
|Data di pubblicazione:||4-mar-2013|
|Settore Scientifico Disciplinare:||Settore BIO/10 - Biochimica|
|Citazione:||REGULATION OF THE E3 LIGASE NEDD4 ; supervisor: S.Polo. - Milano : Università degli studi di Milano. UNIVERSITA' DEGLI STUDI DI MILANO, 2013 Mar 04. ((24. ciclo, Anno Accademico 2012.|
|Appare nelle tipologie:||Tesi di dottorato|
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