Intrinsic effects of ubiquitin and SUMO on protein phase separation

Abstract

Proteins can prefer self-interaction over solvent-interaction, which leads to the formation of protein-enriched biomolecular condensates. This phenomenon is known as phase separation, which controls cellular organization and proteostasis. Aberrant phase separation can result in the formation of cytotoxic aggregates that are hallmarks of several neurodegenerative diseases. The post-translational modifiers (PTM) ubiquitin and SUMO can control phase separation by mediating protein-protein interactions (classical effect), but also by directly changing the biophysical properties of proteins undergoing phase separation (intrinsic effect). As this intrinsic effect is rather unexplored, I performed an in vitro screening to investigate how PTMs alter phase separation of several internally disordered regions (IDRs) derived from neurodegenerative disease-relevant hallmark proteins. I could show that both ubiquitin and SUMO2 generally enhance condensate formation, while SUMO1 has an ambivalent role that depends on the targeted IDR and can act both as an enhancer, but strikingly also as an inhibitor of phase separation. By combining biochemistry methods, structural mass-spectrometry and mammalian cell culture, I was able to reveal how the SUMO1 N-terminus and β2-sheet inhibit phase separation of the TDP-43 low-complexity domain (LCD) by blocking its self-assembly site. In contrast, I discovered that the SUMO2 N-terminus can switch from a cis-interaction with its surface to a trans-interaction on another molecule when being attached to a self-assembling protein, which explains the unspecific effect of enhancing condensate formation by SUMO2. Lastly, I started to investigate the physiological consequences of ubiquitylation and SUMOylation on TDP-43 in a cellular context. I was able to establish a high-throughput platform for screening the effects of distinct PTM-deficiencies on TDP-43 LLPS. Furthermore, I studied the effect of TDP-43 mutants deficient in self-interaction on ubiquitylation and SUMOylation. In summary, these experiments can contribute to an understanding of how ubiquitin and SUMO engage with aberrant TDP-43 phase separation.