Date of Award

2021

Document Type

Thesis

Degree Name

Master of Science (M.S.)

Department

Pharmaceutical and Chemical Sciences

First Advisor

Joseph S. Harrison

First Committee Member

William K. Chan

Second Committee Member

Jerry W. Tsai

Abstract

In most eukaryotic organisms, the ubiquitination pathway is one of the most important and versatile signaling systems in use. It is integral to processes such as protein degradation and homeostasis, DNA repair cell cycle regulation, signaling and regulation, epigenetics, and many more. Ubiquitin (Ub) is a short polypeptide of 8.6 kDa, 76 residues that functions as a reversible post-translation modification (PTM). It furthermore contains 7 different lysine residues (K6, K11, K27, K29, K33, K48, K63), all of which can form isopeptide linkages with one another to link individual Ub moieties to form unique polyUb chains onto substrates. The type of polyUb chain a substrate gets labeled with can determine the subsequent activity of that substrate.

Substrate ubiquitination is achieved through an enzymatic cascade. First, an E1-activating enzyme activates a free Ub moiety. Then Ub is transferred onto an E2-conjugating enzyme, and finally an E3 ligase interacts with both substrate and E2~Ub complex to facilitate Ub transfer onto a substrate. Within this scheme, the E2-enzyme acts as a master manipulator in that, it controls when, where and how a ubiquitin chain is transferred onto a substrate.

Irregular activity of E2-conjugating enzyme has been implicated in a wide variety of diseases such as cancer, neurodegenerative diseases, muscular dystrophy, genetic azoospermia and more. While attempts have been made to inhibit other ubiquitination cascade enzymes such as E3 ligases and E1-activating enzymes, there is a strikingly small number of inhibitors specifically targeting E2 enzymes mainly due to the high degree of structural conservation that exists among members of the E2 enzyme family.

In this work, we introduce 3 novel linked-domain protein inhibitors of the E2-conjugating enzyme Ube2D. We covalently attached either UHRF1 RING domain or an affinity optimized U-box domain, with UHRF1 UBL domain or UbvD1.1 (A ubiquitin variant specific for Ube2D), through a glycine-serine linker, producing 3 unique inhibitors: Ring-UBL (RU), U-box-UBL (UU), and U-box-UbvD1.1 (UUD1.1).

In this way, we attempt to specifically inhibit Ube2D for two purposes : 1) While Ube2D can interact with the largest number of E3 ligases and facilitate the largest number of polyUb chains, very little is known about cellular phenotypes specifically associated with Ube2D; 2) We want to establish whether targeting the E2 enzyme in general can be utilized as a viable therapeutic treatment for cancer.

We show that all three inhibitors are able to inhibit ubiquitin assays using Ube2D and using ITC we measured binding affinities of UUD1.1 (5 nM) > UUWT (300 nM) > RU WT (3 µM). Furthermore, we found that all inhibitors could prevent E1, E3 and backside binding domain interactions simultaneously, which single domain UBL could not. UU and RU showed specificity towards Ube2D when tested against APC/C and Cullin1 E3 ligases and their cognate E2 enzymes. We propose that linking domains in this way, by targeting the backside binding domains of E2 enzymes, could be a strategy that can be standardized and applied to the rest of the E2 enzyme family as well. In vivo testing must now elucidate whether these inhibitors can provide more information about the cellular role of Ube2D and whether it is a viable therapeutic target to treat cancer.

Pages

176

Available for download on Wednesday, August 23, 2023

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