Design and Development of Novel Chemical Biology Tools for Investigating Deubiquitinating Enzymes
Abstract
Ubiquitination, also known as ubiquitylation, is a major type of post-translational modification (PTM), which is closely associated with numerous cellular processes, such as protein degradation, intracellular trafficking, cell signaling, and DNA damage response. Substrate proteins can be monoubiquitinated at one or multiple lysine residues or further polyubiquitinated at one of the eight possible sites (seven lysines and N-terminal methionine) of ubiquitin (Ub) itself. Thus, various ubiquitin polymers (poly-Ub) with distinct linkage types will be produced and they possess different topological characteristics and cellular functions. Deubiquitinases (DUBs), also called deubiquitinating enzymes, can remove Ub from target proteins and regulate most Ub-dependent processes. The human genome encodes ~100 DUBs, which are essential for ubiquitin homeostasis, protein stability, and a wide range of signaling pathways. Consistent with this, their misregulations have been implicated in the pathogenesis of many human diseases, including chronic inflammatory diseases and various types of cancer. In contrast to the increasing interest in deubiquitinases study, ubiquitin conjugates that allow quantitative analyses of DUB activities and high-throughput screening (HTS) of DUB inhibitors, as well as poly-Ub with various linkage types for DUB structural and functional studies are not readily accessible.
A straightforward and readily adoptable approach that employed ubiquitin-activating enzyme (E1)-catalyzed amidation reaction to append ubiquitin C-terminus with multiple functionalities for further syntheses of various Ub-based conjugates, as well as poly-Ub mimics with high efficiency was developed. With this facile method, Ub-based fluorescent substrates with distinct flexible linkages, which could mimic the native isopeptide bond in physiological DUB substrates, were produced and tested with one common DUB, UCH-L3 (ubiquitin carboxyl-terminal hydrolase isozyme L3). Moreover, desirable poly-Ub with diverse linkage types could also be readily generated, in combination with thiol-ene coupling (TEC) chemistry, thereby providing abundant versatile substrates for DUB specificity study. A novel luminescence-based experiment for high-throughput screening (HTS) was also developed and applied to the discovery of ubiquitin specific peptidase 2 (USP2) inhibitors for cancer therapy. Altogether, these novel chemical biology tools allow accelerated studies of DUBs that have pivotal roles in numerous cellular pathways, but whose investigations have been hampered by the difficulty of access to suitable substrates.
Subject
deubiquitinasesubiquitination
ubiquitin functionalization
drug discovery
polyubiquitin chains
deubiquitinase substrates
Citation
Wang, Xiaoyan (2016). Design and Development of Novel Chemical Biology Tools for Investigating Deubiquitinating Enzymes. Doctoral dissertation, Texas A&M University. Available electronically from https : / /hdl .handle .net /1969 .1 /191967.