Elucidation of UBQLN2 liquid-liquid phase separation mechanisms via point mutations and domain deletions
Date of Award
Master of Science (MS)
Carlos A. Castañeda
Liquid-Liquid Phase Separation, Protein, UBQLN2
Organization, separation, and cellular regulation are all functions of membraneless organelles (MLOs), which arise by a biophysical phenomenon termed liquid-liquid phase separation (LLPS). By this process, macromolecules in a mixed liquid solution condense together to form liquid droplets within a liquid solution, comparable to oil droplets in water. Some known MLOs formed in cells via LLPS include nucleoli, stress granules, Cajal bodies, and processing-bodies, among other membrane-lacking liquid granules. Previous work has shown that many proteins which compose these liquid compartments also undergo LLPS isolated in vitro, and thus have become model systems to investigate the forces that drive these macromolecules to undergo phase transitions.
Currently, the LLPS field has identified key features of proteins which contribute to phase separation. Included in this are sequences of intrinsic disorder and structured sequences, prion-like regions, oligomerization, and multivalent interactions. In this thesis, the protein of interest, UBQLN2, contains all such features. Additionally, prior work in the Castañeda lab and others has shown that UBQLN2 is recruited to stress granules, and disease-related inclusion bodies. In vitro, UBQLN2 phase separates into spherical liquid droplets in a concentration and temperature-dependent manner. As UBQLN2 exhibits LLPS both in vitro and in vivo, it serves as a model system to uncover, on a molecular level, the driving forces of phase separation.
The studies provided herein, investigate the properties of UBQLN2 phase separation and how they are modified with the introduction of mutations and domain deletions. By identifying how molecular variations modify UBQLN2 LLPS properties, one can identify a molecular code which UBQLN2 follows to drive and modulate its LLPS. Through experimental investigation via turbidity assays, phase diagram construction, microscopy, and self-association studies, we elucidate the molecular foundations of UBQLN2 LLPS.
Here, I propose that UBQLN2 LLPS is driven by “sticker” sequences which contribute to interchain interactions, and that hydrophobic and polar interactions are important sequence-intrinsic features which drive LLPS and control material properties of UBQLN2 droplets. Additionally, I look at UBQLN2 on a domain-by-domain level to uncover how sequence features like structure, disorder, and prion propensity may contribute differently to phase separation. Finally, I propose a method of UBQLN2 purification that potentially incorporates native post-translational modifications (PTMs) to create a more physiologically relevant system for study.
Jones, Holly Brianne, "Elucidation of UBQLN2 liquid-liquid phase separation mechanisms via point mutations and domain deletions" (2020). Theses - ALL. 441.