Identificazione di interattori della nuova E3 ubiquitin ligasi HectPH1 in D. discoideum

Ubiquitination is a molecular mechanism through which the cell is able to regulate many biological processes, among them the major one is protein turnover. This process requires the sequential action of three enzymes that in the end will ubiquitinate their substrates, leading to proteasomal degradation or non-proteolytic pathways. Although this system has been extensively investigated over the years, much information is still missing in terms of interactors/substrates of the ubiquitinating enzymes as well as their role in modulating signalling pathways (e.g. mTORC2-AKT pathway). The aim of this research is to better characterize the role of the ubiquitin system in regulating the mTORC2 pathway using the model organism Dictyostelium discoideum . Indeed, the newly discovered E3 ubiquitin ligase HectPH1 (orthologue of human HERC1) has been found as key regulator of chemotaxis during early development, modulating mTORC2 pathway, in the D. discoideum mutant strain HSB1HectPH1-. The original mutant strain, HSB1, harbours a point mutation in the piaA gene, which encodes for the TORC2 component Pia/Rictor, impairing chemotaxis and development. The subsequent mutation of HectPH1 is able to rescue the WT phenotype, by reactivating the AKT pathway bypassing mTORC2, that remains inactive. For this purpose, Co-IP followed by two-dimensional electrophoresis and MALDI-ToF Mass Spectrometry has been used to identify interactors of the HECT domain of HectPH1 enzyme in HSB1HectPH1- cell exogenously overexpressing a GFP-fused form of this domain. Proteomics analysis provided many putative interactors, that have been subjected to Co-IP followed by Western Blot analysis to verify their interaction. Among them, the most relevant ones we identified are Myosin II Heavy Chain, ABP34, Actin, Histone H4, Hsp90 and RacE, providing strong evidences for HectPH1 regulating both mTORC2 pathway and the actin cytoskeleton itself. Moreover, starting from data obtained from human HERC1, we validated the interaction of HectPH1 with Pia/Rictor in D. discoideum, strongly contributing to confirm its role in directly regulating mTORC2 activity. Vice versa, we also tested our findings in human cell line (293T), validating in human the interaction between HERC1 and myosin heavy chain II, Histone H3 and H4 and Rictor. In the end, we wanted to investigate the role of ACA (adenylyl cyclase A) in regulating the mTORC2 pathway in HSB1HectPH1- cells. Using CRISPR/Cas9 technique we generated stable ACA-null cells that will be further characterized. For the first time we were able to identify interactors of a large E3 ubiquitin ligase and demonstrate the importance of the ubiquitin-proteasome system in modulating the mTORC2 pathway, key regulator of chemotaxis. In particular, we found partners both at the signal transduction (Pia/Rictor, RacE) and at the downstream level (actin cytoskeleton), laying the foundations for the molecular study of HectPH1 in regulating this signal transduction pathway. Nevertheless, our work confirms D. discoideum as an excellent model organism to investigate the mTORC2 signalling as well as the ubiquitin proteasome system.