Proteases are crucial components of life. They regulate numerous biological pathways, such blood coagulation, neurotransmission, cell proliferation and apoptosis. They are also found to be involved in the development cancer, Alzheimer’s and many infectious diseases. Proteases, as a post-translational modification enzyme, regulate biological processes through proteolysis. And like many other enzymes, one of the key features of proteases is their substrate specificity, which is tightly controlled by specific interactions between the amino acid sequences of the substrates and their binding pockets. It’s important to characterize proteases’ specificities as it is a key to understand their biological functions. This work focused on the development and applications of a mass spectrometry-based technology, named Multiplex Substrate Profiling by Mass Spectrometry (MSP-MS), to characterize the specificities of proteases, and the design of fluorescent substrates and inhibitors based on protease specificity profiles.
Chapter II demonstrates the application of MSP-MS in uncovering the substrate specificity of a bacterial protease, Pd_dinase, which is a putative C1B-like cysteine protease commonly secreted by the human gut commensal Parabacteroidetes distasonis. We designed and synthesized a potent protease inhibitor glycine-arginine-AOMK based on its specificity profile. Furthermore, we revealed that Pd_dinase hydrolyzes several human antimicrobial peptides, such as β-defensin 2 and keratin-derived antimicrobial peptides, indicating that it may be secreted into the extracellular milieu to assist in gut colonization by inactivation of host antimicrobial peptides.
Chapter III presents the study to extend the application of the MSP-MS technology in studying complex biological samples. In this study, we isolated bovine chromaffin granules (CGs) and quantified the endogenous proteins and peptides through proteomics and peptidomics. In addition, we performed degradation assays to profile proteolytic processing of proneuropeptides and MSP-MS assays to characterize proteas activities. With comprehensive profile of proteases, substrates and proteolytic specificities, we were able to discover six catalytically active proteases and assign their activities to some of the cleavages on proneuropeptides.
Chapter IV presents the study of developing quantitative Multiplex Substrate Profiling by Mass Spectrometry (qMSP-MS) method by combining the quantitative power of tandem mass tags (TMTs) with our previously established peptide cleavage assay, MSP-MS. We validated the method with papain, a well-characterized cysteine peptidase and uncovered the substrate specificity of two minimally characterized intramembrane rhomboid proteases. We further showed that activity from multiple peptidases in complex biological samples can be deciphered, including secretions from lung cancer cell lines. Discovery of the protease specificity at the site of the disease highlights the potential for qMSP-MS to guide the development of protease-activating drugs for cancer and infectious disease.
|Advisor:||O'Donoghue, Anthony J., Komives, Elizabeth A.|
|Commitee:||Hook, Vivian, Molinski, Tadeusz F., Wang, Wei|
|School:||University of California, San Diego|
|Department:||Chemistry and Biochemistry|
|School Location:||United States -- California|
|Source:||DAI-B 82/6(E), Dissertation Abstracts International|
|Subjects:||Chemistry, Biochemistry, Molecular biology|
|Keywords:||Mass spectrometry, Protease, Proteolysis, Proteomics, Substrate profiling, Substrate specificity|
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