Identification of Deubiquitinases (DUBs) associated with TRIM18/MID1

Ubiquitination is a post-translation modification process crucial to control protein degradation, localization, and activity. Tripartite Motif (TRIM) proteins participate in the ubiquitination process behaving as E3 ubiquitin ligases, responsible for the specific recognition of the substrate to be ubiquitinated. In the opposite process, Deubiquitinating enzymes (DUBs) can deconjugate the ubiquitin from the protein target. The antagonism of DUBs and E3s is essential to maintain protein homeostasis and signaling in cells. In this project, we focused on TRIM18 (also named MID1) that when mutated causes the Xlinked form of Opitz G/BBB Syndrome (XLOS). MID1 controls the ubiquitin-mediated proteasomal degradation of the catalytic subunit of PP2A (PP2AC), one of the major phosphatases in the cell. Although MID1 mutations lead to an increase in PP2AC levels, the exact mechanism remains unclear. The main objective of this project was to find DUBs that work in conjunction with MID1 rescuing the increase of PP2Ac level observed upon its mutations. We specifically silenced 24 DUBs and analyzed the protein abundance of PP2AC. A decrease in the protein target levels will be indicative of a suitable DUB candidate to further study. We also made the opposite assay, overexpressing the same 24 DUBs, in this case, the DUBs overexpression should increase the PP2Ac protein. From both screenings, we found USP8 as a good candidate, which we confirmed in further assays, to modulate the PP2AC levels. Consistently with the regulation of PP2AC, USP8 overexpression alters 4E-BP1 phosphorylation levels, affecting the mTOR pathway. To conclude USP8/MID1 is a functional pair controlling the degradative fate of PP2AC. Furthermore, we noticed that in Mouse Embryo Fibroblasts from Mid1 KO, the Usp8 protein levels were up regulated. We found that USP8 levels are decreased in both the cytoplasmic and nuclear fractions when MID1 was overexpressed, recovering the USP8 levels when using the ΔRING form of MID1 (the non-catalytic form). Moreover, the nuclear fraction of PP2AC decreased when MID1 was overexpressed and increased when USP8 was overexpressed. To conclude we discovered a new DUB/TRIM pair that works in a highly coordinated manner. MID1 controls the levels of USP8 in the cytoplasm and nucleus, and both MID1/USP8 control the levels of PP2AC, a mutual substrate, in the nucleus fraction. These findings will be relevant in basic knowledge and for the future investigation of potential therapeutic.