The human genome encodes approximately 100 deubiquitinating enzymes (also known as DUBs). These enzymes regulate a broad swath of cell and organismal biology by removing the small protein ubiquitin (Ub) from target proteins or trimming Ub oligomers. Despite the importance of DUBs, there are fundamental gaps in our knowledge regarding how they work. The family of DUBs known as the Ub C-terminal hydrolases (UCHs) embodies this situation. Biochemical data suggests UCHs catalyze the removal of small C-terminal adducts from Ub, whereas data from cellular studies implicates these enzymes in the disassembly of Ub oligomers. Recently, our laboratory developed a straightforward chemical approach towards the synthesis of a wide array of ubiquitin oligomers. Using these oligomers to probe the function of DUBs, we discovered two members of the UCH family, UCH37 and UCHL3, selectively hydrolyze Ub chains in which a single Ub subunit is modified with two Ub molecules through two lysine residues (herein referred to as branched Ub chains). This activity is unprecedented, as the capacity of UCH37 and UCHL3 to dismantle other defined Ub oligomers has not been observed and the function of branched Ub chains is entirely unknown. Considering the importance of UCHL3 and UCH37 in cellular differentiation, development, and motility, our results suggest branched Ub chains play far more important roles in biology than ever appreciated. In this application, we propose to uncover the mechanism by which UCHs selectively hydrolyze branched Ub chains and test this activity in the context of a pathway regulated by UCH37. The proposed work is divided into three specific aims. In the first aim, we will expand the repertoire of chemically synthesized Ub chains to investigate the kinetics and selectivity of chain disassembly. In the second aim, we will structurally characterize branched Ub chains and their interactions with UCHs. Together with the studies proposed in aim 1, these investigations will lead to working model for the function of UCH37 and UCHL3.
In aim 3, we will put this model to the test with regards to UCH37. A number of tumors (e.g., cervical, hepatocellular, and esophageal) display abnormally high levels of UCH37. We hypothesize that UCH37 promotes tumorigenesis by disrupting a critical regulator of cellular migration, i.e., branched Ub chains. The mechanistic insights gained from our proposed studies have excellent potential to be translated into the development of new drugs to fight cancer.

Public Health Relevance

This proposal aims to develop chemical tools to understand the physiological role of a family of deubiquitinases referred to as ubiquitin C-terminal hydrolases (UCHs). Our recent discovery that UCHs selectively hydrolyze a unique set of ubiquitin modifications suggests new opportunities for therapeutic intervention, especially considering these enzymes regulate myriad biological pathways and are important drug targets in cancers and neurodegenerative diseases.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Synthetic and Biological Chemistry B Study Section (SBCB)
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Gerratana, Barbara
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University of Wisconsin Madison
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Chang, Lin Hui; Strieter, Eric R (2018) Reprogramming a Deubiquitinase into a Transamidase. ACS Chem Biol 13:2808-2818
Du, Jiale; Strieter, Eric R (2018) A fluorescence polarization-based competition assay for measuring interactions between unlabeled ubiquitin chains and UCH37•RPN13. Anal Biochem 550:84-89
Bowerman, Samuel; Rana, Ambar S J B; Rice, Amy et al. (2017) Determining Atomistic SAXS Models of Tri-Ubiquitin Chains from Bayesian Analysis of Accelerated Molecular Dynamics Simulations. J Chem Theory Comput 13:2418-2429
Crowe, Sean O; Rana, Ambar S J B; Deol, Kirandeep K et al. (2017) Ubiquitin Chain Enrichment Middle-Down Mass Spectrometry Enables Characterization of Branched Ubiquitin Chains in Cellulo. Anal Chem 89:4428-4434
Rana, Ambar S J B; Ge, Ying; Strieter, Eric R (2017) Ubiquitin Chain Enrichment Middle-Down Mass Spectrometry (UbiChEM-MS) Reveals Cell-Cycle Dependent Formation of Lys11/Lys48 Branched Ubiquitin Chains. J Proteome Res 16:3363-3369
Whedon, Samuel D; Markandeya, Nagula; Rana, Ambar S J B et al. (2016) Selenocysteine as a latent bioorthogonal electrophilic probe for deubiquitylating enzymes. J Am Chem Soc :
Crowe, Sean O; Pham, Grace H; Ziegler, Jacob C et al. (2016) Subunit-Specific Labeling of Ubiquitin Chains by Using Sortase: Insights into the Selectivity of Deubiquitinases. Chembiochem 17:1525-31
Pham, Grace H; Rana, Ambar S J B; Korkmaz, E Nihal et al. (2016) Comparison of native and non-native ubiquitin oligomers reveals analogous structures and reactivities. Protein Sci 25:456-71
Pham, Grace H; Strieter, Eric R (2015) Peeling away the layers of ubiquitin signaling complexities with synthetic ubiquitin-protein conjugates. Curr Opin Chem Biol 28:57-65
Strieter, Eric R; Andrew, Trisha L (2015) Restricting the ? Torsion Angle Has Stereoelectronic Consequences on a Scissile Bond: An Electronic Structure Analysis. Biochemistry 54:5748-56

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