Defining the regulatory determinants in substrate catalysis by biochemical, biophysical, and kinetic studies for the development of specific small-molecule inhibitors of ubiquitin specific proteases 7 and 17

Nicole M Hjortland, Purdue University

Abstract

Ubiquitination is an important post-translational modification involved in maintaining cellular homeostasis by regulating many delicate cellular processes, including the cell-cycle, membrane protein trafficking, endocytosis and apoptosis. Ubiquitin Specific Proteases (USPs) remove ubiquitin modifications from protein substrates to reverse the signal imposed by the ubiquitination. Perturbations in the expression levels of USPs has been implicated in many types of cancers where patients show significant elevation in cellular levels of specific USPs. This suggests that targeting specific upregulated members of the USP family in specific disease states would be ideal for the development of personalized anti-cancer therapeutics. Each member of the USP family is composed of a conserved catalytic domain flanked by a variety of other domains that make the function of each USP unique. These flanking domains are important for substrate recognition, regulation, and function. The conserved catalytic domains of the USPs share high structural similarities, and within two regions of the catalytic domain they also share high sequence similarity. These regions are referred to as the Cys and His boxes, and they contain the catalytic triad composed of a cysteine, histidine, and aspartate residues. These structural and sequence similarities make the task of inhibiting specific members of the USP family substantially more difficult. Therefore, it is essential that we thoroughly characterize not only the enzymatic activity of the catalytic domain of the specific USP of interest; but also the structure and function of the flanking domains that make the USP unique. Along these lines, this dissertation describes the enzymatic and biochemical characterization of two USP family members, USP17 and USP7. USP17 is a cytokine-inducible USP that is required for cell-cycle progression through the G1-S and G2-M checkpoints. No structural information specific to USP17 is available, but cellular studies have determined that USP17 contains the conserved catalytic domain at the N-terminus and two hyaluronan binding motifs at the C-terminus. Cellular characterization of USP17 has led to the identification of three substrates that USP17 deubiquitinates: the Ras Converting Enzyme 1 (RCE1), the Cell Division Cycle 25A (CDC25A) phosphatase, and a component of the Sin3 co-repressor complex SDS3. Under normal cellular conditions, USP17 is only expressed during the cell-cycle checkpoints and if USP17 is expressed at other times, cellular proliferation ceases. However, the upregulation of USP17 has been implicated in both breast and prostate cancer, and it is thought that the persistent overexpression of USP17 applies continuous stress on the cell-cycle through perturbation of the Ras and CDC25A signaling pathways. As such, USP17 is an important target for inhibition leading to the development of anti-cancer therapeutics. (Abstract shortened by ProQuest.)

Degree

Ph.D.

Advisors

Mescar, Purdue University.

Subject Area

Biochemistry|Biophysics

COinS