Understanding ubiquitin chain interactions and mechanisms of deubiquitinases
Date
2020
Authors
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Journal ISSN
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Publisher
University of Delaware
Abstract
In the past 15 years following the recognition of Ciechanover, Hershko, and Rose’s discovery of ubiquitin in signaling protein degradation, we have made much progress in understanding ubiquitination and deubiquitination as critical cellular signaling processes. Ubiquitination is catalyzed by a cascade of three enzymes (E1, E2 and E3) resulting in site- and linkage-specific ubiquitination of target protein. Deubiquitinases (DUBs) counter the ubiquitin ligases’ activity and reverse this process. It is increasingly realized that DUBs, in addition to linkage specificity to polyubiquitin chains, also exhibit target protein and site specificity. Understanding the source of linkage specificity is critical to understanding cellular signaling pathways including DNA damage response, immune response, and cell cycle progression. Dysregulation of the ubiquitin pathway enzymes are often associated with human diseases like cancer and neurological disorder. ☐ In Chapter 2, I detail the development and utilization of di- and tri-ubiquitin probes/ substrates in understanding DUB activity and specificity. I prepared both terminal and internal warhead-containing chemically linked diubiquitin probes. I generated a new terminal fluorophore-containing diubiquitin probe of K11, K48 and K63 linkages resistant to internal cleavage by DUBs. I also successfully generated hybrid triubiquitin probes bearing a chemical linkage through a non-cleavable or Michael acceptor-containing linker. Finally I created a heterotypic branched triubiquitin chain probes resistant to internal cleavage and containing a terminal warhead. These di-and tri-ubiquitin probes enabled extensive interrogation the linkage specificity of DUBs and polyUb chain reader proteins. ☐ In Chapter 3, I present proteomic study and identification of DUBs and reader proteins specific for branched ubiquitin probes from HEK-293T cell lysates. Branched ubiquitin chains represent a new frontier in understanding of polyubiquitin signaling in the cell with very little known about the roles and functions. These probes show some branched linkage specific to pathways like DNA damage response and mitophagy. While certain ubiquitin binding domains were shown to exhibit specificity to branched triubiquitin chains. ☐ In Chapter 4, I discuss the crystal structure of a USP family DUB, USP9X, catalytic core as well as the structural elements responsible for recognition of polyubiquitin chains of different linkages. Using polyubiquitin probes discussed in Chapter 1, we investigated the linkage dependent endo/exo cleavage selectivity of USP9X towards different polyubiquitin chains. Furthermore, we explore K11 diubiquitin binding dynamics to USP9X catalytic core through high resolution mass spectrometry demonstrating the power of activity-based polyubiquitin probes. ☐ Parts of chapter 2 and most of chapter 4 are adapted from texts and figures published in Paudel et. al. (2019). PNAS. 116(15) 7288-7297. Parts of chapter 2 also contain adapted text and figures from Gui et. al. (2019) Ref Mod Chem. Mol Sci Chem. 14672.
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Keywords
Branched chain ubiquitin, Deubiquitinase, Polyubiquitin chains, Proteomics, Ubiquitin