Study of the TRIM-E2 interaction specific determinants and identification of TRIM-associated DUBs

Germana Meroni

One of the specific requirements for an E3 ligase is the interaction with the ubiquitin-charged E2. The important role of E2 enzymes in determining ubiquitin chain length and topology, hence the final fate of the modified substrates, is nowadays well accepted.

The objective of this project is to understand E3-E2 pair selection and its determinants for the TRIM proteins studied within this network (TRIM8, 17, 19, 25, 28, 32, 33, 41, 63 and Malin). The ultimate goal is to explore novel strategies of drug design and screening to interfere with specific E3 activities.

ESR10 will test functional interactions of the above TRIM proteins and E2 enzymes using autoubiquitination assays with panels of E2s provided by PO2 (Ubiquigent) and test physical interactions by performing MBP pull-down assays. To define the RING domain-E2 interaction surfaces, he/she will use modelling analyses combining alignment of RING primary sequences and threading on solved structures of isolated RING fingers and/or RING-E2 complexes to predict the residues responsible for the specific binding. Site-specific mutagenesis coupled with the above assays to reveal either loss or specificity alteration of the binding will be also employed. Structural information will be essential in understanding the manner in which potential small molecules inhibitors might mimic the most important binding elements of the native ligands guiding the strategies for class selection and optimization of compounds.

As a further objective, the ESR will also employ mid-throughput screening assays to identify the DUBs implicated in the TRIM-associated disease pathways under study within the consortium. Using the cellular system developed by the other beneficiaries siRNA DUBs libraries will be tested for destabilization of the disease-relevant TRIM protein substrates. Identified DUBs will be validated with in vitro and in the above mentioned cellular systems.

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