Structural and Functional Studies of Prokaryotic Enzymes Which Manipulate the Ubiquitin Landscape

Michael Jeffrey Sheedlo, Purdue University

Abstract

Ubiquitin is a small eukaryotic protein that can be covalently linked to other proteins through a three enzyme cascade resulting in the formation of an isopeptide bond between the C-terminus of ubiquitin and the amino group of a lysine side chain present on that substrate. This modification results in a variety of different outcomes, most notably leading to the degradation of tagged proteins through either the 26S proteasome or the lysosome, earning ubiquitin the moniker “the kiss of death”. Though protein degradation is highly reliant on ubiquitination, it is now known to function in a variety of different cellular processes. Over the past 10 years, a host of prokaryotic enzymes which manipulate this pathway have emerged, demonstrating the ability of a pathogen to participate in these decidedly eukaryotic signaling cascades. The studies presented here attempt to shed light onto the structural arrangement and functionality of this family of enzymes, initially focusing on a family of deubiquitinating enzymes from the pathogenic bacterium Legionella pneumophila, known as the SidE family. Reported here is the domain architecture of a deubiquitinating module from this family of large proteins, constituting the first structure of a prokaryotic enzyme of this kind. From these studies this family of effectors was found to utilize a novel binding mode, one that brings glutamine-40 of ubiquitin to the forefront of these interactions rather than isoleucine-44, significantly deviating from known eukaryotic binding modes. In conjunction with these studies it was found that the deubiquitinating module also displays specificity for the ubiquitin-like protein Nedd8, utilizing a similar mode of recognition in which the glutamine-40 patch acts as molecular answer to the problem of recognizing two distinct proteins within the same family. Further investigations into the ubiquitin chain type preference has yielded results which demonstrate that the SidE family has a distinct preference for lysine-63 ubiquitin linkages, suggesting that these deubiquitinases modulate the lysosomal degradation pathways of the host organism. The scope of the project was then broadened to include enzymes from Escherichia coli, Salmonella typhimurium, and Chlamydia trachomatis in an effort to understand the commonalities that arise within this family of enzymes with an emphasis on determining the mechanisms of ubiquitin recognition in light of the discoveries made within the SidE family. These studies suggested a divergence in ubiquitin recognition within the prokaryotic family of deubiquitinases with the Salmonella typhimurium virulence factor, SseL, likely exhibiting a similar mode of recognition as observed in the SidE family and the Escherichia coli enzyme, ElaD, resembling the canonical ubiquitin interacting modes observed in many eukaryotic enzymes. Additional studies were conducted to dissect the activities of other modules within the SidE family. To this end, the previously characterized ubiquitin ligase domain of the SidE family was found to function with a novel mechanism, harnessing the high energy molecule, NAD, to accomplish this task. The manner of this mechanism appears to be a hybrid between that of classical ubiquitination and ADP-ribosylation, an unexpected occurrence within the world of ubiquitin. These observations, when taken together, will likely induce a paradigm shift in the ubiquitination field, opening the door for only the second known mechanism discovered for this process in the last 40 years.

Degree

Ph.D.

Advisors

Das, Purdue University.

Subject Area

Biochemistry

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