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Abstract

Here, I present a comprehensive assay termed UbiReal for monitoring each step of the Ub signaling cascade, including linkage-specific polyUb ligation and deubiquitination. Next, I use bioinformatic tools and recombinant protein expression to identify additional, HECT-like ligases from human and plant pathogens, and validate these ligases using UbiReal. I apply structural biology and biochemical techniques to elucidate the mechanism of Ub ligation by the bacterial HECT-like ligases, and capture a rare glimpse of a linkage-specific polyUb ligation interface. I leverage insights from the linkage-specific polyUb interface to engineer an NleL specific for K6-linked polyUb, and show that similar rules influence the ligation of K6-linked polyUb in humans. Finally, through analysis of a family of deubiquitinating enzymes in bacteria, I demonstrate the utility of the deubiquitinase LotA as a tool for detecting the presence of K6-linked polyUb. Collectively, this dissertation provides the critical tools for understanding K6-linked polyUb biology, and in particular sets the stage for elucidating the enigmatic role of K6-linked polyUb at the host-pathogen interface.

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