Date of Award

Winter 12-22-2021

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

Advisor(s)

Castañeda, Carlos A.

Keywords

Liquid-liquid phase separation, Oligomerization, Phase diagram, Polyubiquitin chains, Protein quality control, UBQLN

Subject Categories

Biochemistry | Biochemistry, Biophysics, and Structural Biology | Biophysics | Chemistry | Life Sciences | Physical Sciences and Mathematics

Abstract

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.

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Open Access

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