Structural and biochemical studies on the regulation of the ubiquitin C-terminal hydrolase family of deubiquitinating enzymes

David Allen Boudreaux, Purdue University

Abstract

Ubiquitination is a post translational modification with roles in nearly all major biological applications. Some of the processes linked to ubiquitination include cell cycle regulation and division, modulation of cell surface receptors, transcriptional regulation, DNA repair, regulation of the immune response and biogenesis of organelles. The diversity of ubiquitination stems from the possibility to mono- or polyubiquitinate targets using one of the seven internal Lys residues of ubiquitin. The different chain types allow for multiple signaling mechanisms using only the ubiquitin moiety. The ubiquitin signal is tightly regulated by the enzymes that conjugate and remove the Ub tag from the target protein. Ubiquitin is covalently attached in an ATP-dependent manner through the E1-E2-E3 enzyme cascade. Deubiquitinating enzymes (DUBs) are responsible for the termination of the ubiquitin signal by removing and recycling the Ub monomer. There are five classes of DUBs based upon their proteolytic domains. My thesis work has mainly focused on the ubiquitin C-terminal hydrolase (UCH) family of DUBs. The biological role and substrates for many of these enzymes has still not been elucidated. I have worked to characterize these enzymes through structural and biochemical methods to advance our basic understanding of these proteins. My initial goal was to determine if UCHL1 was a true deubiquitinating enzyme. Previous studies have shown UCHL1 demonstrates activity with Ub-substrate analogs. However, the previously solved crystal structure of the enzyme contained a misaligned catalytic site incapable of activity. After solving the structure of UCHL1 bound to a suicide substrate inhibitor, I was able to conclude that UCHL1 is a true deubiquitinating enzyme. Our structure revealed that UCHL1 is regulated by ubiquitin binding which causes a novel rearrangement of side chains to produce a functional, active enzyme. Mutational and kinetic data also indicated that the distal site was important for binding in the other members of the UCH subfamily. These results indicated a potential drug target site for the UCH family of enzymes located at the distal binding site, which was tested using a simple peptide fragment as an inhibitor. The last phase of my dissertation focused on investigating the mechanism of ubiquitination. It has been speculated that a conserved glutamine residue located in the active site acts to stabilize the transition state of the reaction. However, this has never been demonstrated experimentally for these enzymes. Mutating the glutamine to an alanine led to an approximately 30-fold loss in activity, mainly through a decrease in the turnover number, for all three structurally characterized UCH enzymes. However, this seemed like a small loss for a residue that is supposed to stabilize the transition state. Subsequent analysis of the structures revealed this residue was involved in a special bond referred to as a C–H···O hydrogen bond. This bond may serve to increase the electron density around the imidazole ring of the histidine residue, thus increasing its ability to act as a general base. This data may reveal that the conserved glutamine residue serves a different role than previous believed.

Degree

Ph.D.

Advisors

Das, Purdue University.

Subject Area

Biochemistry

COinS