Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article

Degree

Doctor of Philosophy

Program

Biochemistry

Supervisor

Shaw, Gary S.

Abstract

Ubiquitin (Ub) is a small modifying protein abundant in cells where it serves numerous regulatory roles including immune signaling, transcriptional regulation, and proteostasis. To exert its function, Ub covalently interacts with a series of E1, E2, and E3 enzymes before final substrate modification. Dysregulation of Ub signaling has implications in human maladies such as cancer, autoimmune disorders, and neurodegenerative diseases. In these diseases and associated in cellulo models, modifications to Ub serve an additional role in Ub regulation. Post-translational modifications like acetylation or phosphorylation modulate protein-protein interactions and Ub signaling. To understand how acetylation of Ub alters the central E2 step in the Ub cascade, we used orthogonal translation to synthesize seven unique versions (K6, K11, K27, K29, K33, K48, or K63) of acetylated Ub (acUb) and showed them to be competent in RING and HECT-type ubiquitination experiments. We then created a library of acUb proteins labeled with the CyPet fluorophore, and in conjunction with the YPet-labeled E2 proteins UBE2D1 and UBE2L3, we optimized a Förster Resonance Energy Transfer (FRET) experiment that highlighted how Ub acetylation modulates the catalytic activity of the E1 protein UBA1. We found that Ub acetylation at most sites significantly altered the formation of both the UBE2D1~Ub and UBE2L3~Ub conjugates. The unloading of the UBE2D1~Ub and UBE2L3~Ub conjugates was then evaluated with the acUb proteins in the presence of small nucleophiles. Our findings that both E2 proteins were sensitive to nucleophilic cysteine prompted the use of cysteine-dependent E3 proteins IPAH3 and HUWE1 to study how acUb alters transthiolation from the E2 protein to an E3 ligase. We used Nuclear Magnetic Resonance (NMR) spectroscopy to study conformation of the UBE2L3~Ub and acUb conjugates. Together, these sets of experiments demonstrated that a subset of the acUb proteins were ineffective in supporting E3 function likely through E2~Ub conformational changes and E3-chain building preference. Overall, we provide the first analysis of the complete set of acUb proteins and how they modulate E2-dependent steps in ubiquitination. We expect our data will guide future experiments to understand the full involvement of Ub acetylation in regulating cellular processes.

Summary for Lay Audience

Our bodies are made of tiny compartments called cells. These cells are controlled by a variety of factors, one of which being proteins. One protein called ubiquitin is responsible for the maintenance of our cells. Similar to how a handyman is called upon to build, fix, or destroy, ubiquitin helps guide the body to make new proteins, repair damaged structures, and remove unneeded components. An incapacitated handyman is detrimental to your life, as is an incapacitated ubiquitin. When this protein cannot exert its handyman function, the chance of diseases like cancer, Parkinson’s, or Alzheimer’s increases.

The job of a handyman depends on the function of their vision and their hands: wearing sunglasses or thick gloves might alter how quickly their work is done. Ubiquitin is the same way: changing certain physical aspects has an impact on function. In my thesis, I have optimized a method to make physical changes to ubiquitin, and I created an experiment to examine how fast or slow ubiquitin can function with these changes. I found that most physical changes to ubiquitin mimic what might be expected of a handyman wearing thick gloves: slower overall, but still capable of getting the job done.

The impairment in function caused by the physical changes is expected to cause a problem within cells, but the full effects are still unknown. Can ubiquitin compensate for physical changes to still get the job done? What exactly happens in cells when physical changes incapacitate ubiquitin? There are still many questions that remain to be answered, but my thesis provides an excellent foundation to continue studying physical changes to ubiquitin. It provides information on how the changes alter specific functions that might have unknown roles in various human diseases.

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