R02: Functions of Ubiquitin and SUMO in Protein Phase Separation and Aggregation

The post-translational modifiers ubiquitin and SUMO, two small, conserved proteins known for their ability to modulate protein–protein interactions by becoming covalently attached to other proteins, target many proteins undergoing phase separation (PS) and have been implicated in the formation of pathogenic aggregates. Ubiquitin and SUMO principally act by means of defined protein–protein interaction domains or motifs. However, they also directly modulate the biophysical properties of their target proteins. Using biochemical and biophysical approaches in combination with multi-scale molecular simulations based on polymer physics concepts, we will systematically investigate how ubiquitin and SUMO affect the propensity of targets containing intrinsically disordered regions (IDRs) to undergo PS and aggregation. Based on a set of model target proteins relevant to neurodegenerative diseases, we will examine the influence of ubiquitin and SUMO on PS, aggregation and the properties of the resulting condensates and aggregates. We will consider parameters such as the degree of modification, the arrangement of the ubiquitin/SUMO units, their stoichiometry, and their interplay with IDRs within the targets. In parallel, we will design the hierarchy of coarse-grained (CG) models capable of accurately reproducing the behaviour of globular and disordered domains as well as linear ubiquitin and SUMO chains. These models will help us probe the influence of distinct features of the targets and modifiers on PS and aggregation, providing molecular-level insight into the corresponding in vitro measurements and instructing further experiments through adjustment of relevant parameters. By back-mapping these coarser models to higher-resolution models, we will identify specific protein sequences to investigate experimentally. In the longer term, the outcome of these in vitro and in silico studies will generate predictions to feed into cell biological experiments that address the impact of ubiquitin and SUMO on disease-linked proteins in vivo. At the same time, we aim to generalize our modelling approach for simulating different types of modified proteins.