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Understanding the Functions of DUBs

Cells are composed of a large collection of highly dynamic signaling networks. Controlling the net flow of information through these networks is a small protein called ubiquitin (Ub). Through covalent attachment of the Ub C-terminus to the ε-amino group of substrate lysines (Figure 1A), Ub regulates the function, stability, and localization of numerous proteins. Approximately 5% of the human genome is dedicated to the installation, removal, and processing of information embedded in Ub modifications.

The removal of Ub modifications is carried out by deubiquitinases/DUBs, of which the human genome encodes ~100 enzymes. Little is known about their substrates or how they achieve selectivity. Identifying physiologically relevant substrates is difficult due to the sheer number of possibilities. Proteomics studies, for example, have identified ~19,000 ubiquitination sites on 5,000 different proteins. There is also tremendous complexity within the types of Ub chains a DUB can target for disassembly. Ub has eight amino groups (Met1, Lys6, Lys11, Lys27, Lys29, Lys33, Lys48, and Lys63), each of which can be used to build a single (homotypic) or mixed (heterotypic) chain (Figure 1B). Our objectives are to identify substrates of DUBs, understand how they are selectively processed, and elucidate the biological consequences of this activity.  

Our Approach

Our approach to understanding the activity of DUBs is multifaceted. We leverage expertise in synthetic chemistry to build well-defined Ub conjugates. These tools are then used to interrogate the biochemical activity and mechanisms of DUBs. Biophysical methods (e.g., NMR spectroscopy, fluorescence spectroscopy, calorimetry, small-angle X-ray scattering, crystallography, and molecular dynamics) play an integral role in our efforts. On the foundation of biochemical and biophysical studies, we develop a comprehensive picture of enzyme function through cell biological and proteomic studies. 

Understanding the Functions of DUBs: Project
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