The Phase Behavior of Ubqln Proteins and Implications for Protein Quality Control

The ubiquitin-proteasome system (UPS) and autophagy are essential pathways for maintaining protein quality control (PQC) in cells. Misfolded proteins and large aggregates are cleared by UPS and autophagy signaled by ubiquitin (Ub) or polyubiquitin (polyUb) chains. Shuttle proteins facilitate cargo transporting by interacting with both ubiquitin and degradation machineries. Previously, our lab discovered that the shuttle protein Ubiquilin-2 (UBQLN2) is recruited to stress granules in cells and undergoes liquid-liquid phase separation (LLPS) in vitro. LLPS is a biophysical process by which proteins separate themselves from the surrounding aqueous solution by forming protein-rich droplets. The overarching goals of this work are to understand how the UBQLN family of proteins form droplets via phase separation, as well as how UBQLN2 LLPS is regulated by interactions with protein quality control (PQC) proteins, specifically Ub and polyUb chains.Chapter two focused on studying the molecular driving forces of UBQLN2 LLPS. Previously, our lab identified residues of UBQLN2 that promote self-interactions and phase separation, which are called stickers and the sequences separating stickers are called spacers . Here, I systematically introduced all 19 single amino acid substitutions at three stickers and two spacers , leading to a UBQLN2 library of 95 point mutants. By screening for the phase transitions of these proteins and mapping out phase diagrams, we discovered that the LLPS of UBQLN2 is largely driven by hydrophobic amino acids in the sticker positions but not spacers. Amino acid substitutions in the sticker positions significantly changed the shape of the phase diagram as well as the characteristics of the dense phase, which is the protein-rich phase inside the condensates. PolyUb chains of different linkages have different functions in cells. Preliminary data from our lab suggest that K48-linked and K63-linked polyubiquitin chains differentially modulate UBQLN2 LLPS. Chapter three revealed how interactions between UBQLN2 and polyUb chains affect UBQLN2 functionality and its ability to phase separate. The data suggest that polyUb chains of different linkages differentially modulate UBQLN2 LLPS. I found that the compact Ub4 chains (K11 and K48) largely drive disassembly of UBQLN2 condensates, while extended and more flexible Ub4 chains (K63 and M1) promote multi-component LLPS via heterotypic interactions with UBQLN2. The UBQLN family consists of 5 protein members (UBQLN1-4 and UBQLNL), with highly conserved sequences and structures. Besides UBQLN2, UBQLN4 also plays a role in PQC. However, the phase behavior of UBQLN4 is largely unknown, as well as the sequence and structural basis for the phase behavior of the protein. In chapter four, I characterize the LLPS properties of UBQLN4 for the first time and compare its behavior with UBQLN2. I successfully designed, cloned, expressed, and purified various UBQLN4 C-terminal deletion constructs. Turbidity assay data suggest that the LLPS behavior of UBQLN4 constructs is concentration- and temperature-dependent, like UBQLN2, but in a different manner than UBQLN2 equivalent constructs. More stringent studies are needed to unravel the differences in LLPS behavior of different UBQLNs

Role of Solvent Compatibility in the Phase Behavior of Binary Solutions of Weakly Associating Multivalent Polymers

[Image: see text] Condensate formation of biopolymer solutions, prominently those of various intrinsically disordered proteins (IDPs), is often driven by “sticky” interactions between associating residues, multivalently present along the polymer backbone. Using a ternary mean-field “stickers-and-spacers” model, we demonstrate that if sticker association is of the order of a few times the thermal energy, a delicate balance between specific binding and nonspecific polymer–solvent interactions gives rise to a particularly rich ternary phase behavior under physiological circumstances. For a generic system represented by a solution comprising multiassociative scaffold and client polymers, the difference in solvent compatibility between the polymers modulates the nature of isothermal liquid–liquid phase separation (LLPS) between associative and segregative. The calculations reveal regimes of dualistic phase behavior, where both types of LLPS occur within the same phase diagram, either associated with the presence of multiple miscibility gaps or a flip in the slope of the tie-lines belonging to a single coexistence region