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University of Washington

1. Whedon, Sam. Chemical strategies for investigation of deubiquitinases.

Degree: PhD, 2019, University of Washington

Regulation of protein structure and function by post-translational modification is a key mechanism in cellular homeostasis. Among known modifications the small protein ubiquitin is unique in the breadth of functions it directs. Regulation of protein ubiquitylation is the function of more than 600 ligases, and ~100 deubiquitinases. Dysregulated ubiquitylation is involved in infection, inflammation, neurodegeneration, metabolic syndromes and cancer. Therapeutic intervention in these conditions benefits from characterization of substrate-specific ligases and deubiquitinases. Substrate specificity is documented among deubiquitinases, but lags behind knowledge of ligases. In the interest of characterizing specificity of cysteine protease deubiquitinases we have developed chemical methods for site-specific ubiquitylation and electrophile incorporation. We began by developing a ubiquitin-derived electrophile through installation of a C-terminal selenocysteine residue, and orthogonal oxidative conversion to dehydroalanine. Upon validation of the electrophile we expanded our substrate scope to a ubiquitylated peptide, with which we captured the deubiquitinase USP15. In order to access ubiquitylated lysine residues in protein regions inaccessible by native chemical ligation we undertook the synthesis of two selenazolidine amino acids for amber suppression. While conducting amber suppression selections we concurrently pursued semi-synthesis of p53, in order to introduce a C-terminal ubiquitin electrophile. Purification of the semi-synthetic protein proved materially intensive, prompting development of cleavable affinity handles for ligation product purification. A strategy for incorporation at glycine and incorporation at glutamine were developed. A glycine-derived biotin handle proved amenable to reduction by Zn, which furnishes a native glycine, and aromatic thiols, which formed novel site-specific conjugates at the previously modified glycine. Incorporation of a pendant flag tag at glutamine enabled immunoprecipitation followed by traceless tag removal with Zn. Advisors/Committee Members: Chatterjee, Champak (advisor).

Subjects/Keywords: dehydroalanine; deubiquitinase; selenocysteine; ubiquitin; Organic chemistry; Chemistry

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APA (6th Edition):

Whedon, S. (2019). Chemical strategies for investigation of deubiquitinases. (Doctoral Dissertation). University of Washington. Retrieved from http://hdl.handle.net/1773/43327

Chicago Manual of Style (16th Edition):

Whedon, Sam. “Chemical strategies for investigation of deubiquitinases.” 2019. Doctoral Dissertation, University of Washington. Accessed March 20, 2019. http://hdl.handle.net/1773/43327.

MLA Handbook (7th Edition):

Whedon, Sam. “Chemical strategies for investigation of deubiquitinases.” 2019. Web. 20 Mar 2019.

Vancouver:

Whedon S. Chemical strategies for investigation of deubiquitinases. [Internet] [Doctoral dissertation]. University of Washington; 2019. [cited 2019 Mar 20]. Available from: http://hdl.handle.net/1773/43327.

Council of Science Editors:

Whedon S. Chemical strategies for investigation of deubiquitinases. [Doctoral Dissertation]. University of Washington; 2019. Available from: http://hdl.handle.net/1773/43327

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