Our long-term goal is to create a suite of robust and quantitative tools and technologies for dissecting proteolytic signaling pathways, and to apply them near-term to prioritize and test the importance of caspase cleavages in apoptosis. There are >500 proteases in humans. However, until recently there were no unbiased proteomic approaches for determining which intact proteins are cleaved in cells or extracts, or the exact site of the cleavage. The current R01 grant (R01 GM081051) allowed us to develop a first generation positive enrichment N-terminomics technology for direct site-specific labeling of free N-termini produced by proteases in cells, extracts, and serum. Key to this technology was the use of an engineered peptide ligase, called subtiligase which has now been distributed to >30 laboratories. Currently we have identified >1400 proteins cleaved by caspases, cysteine-class proteases, that are activated in the apoptotic process and specifically cleave after aspartic acid. This vast data set more than doubles the number of known caspase targets and tells us what can be cut, but not how fast or how conserved in phylogeny.
In Specific Aim 1, we will use quantitative selected reaction monitoring (SRM) approaches to determine for the first time for intact proteins in a cell the fundamental rate constants (kcat/KM) for individual caspase substrates. This will allow us to rank them by the catalytic rates at which they are cleaved by specific caspases in human cells and extracts.
In Specific Aim 2, we plan to determine how evolutionarily conserved these substrates are through the characterization of caspase cleaved products from primitive to advanced metazoans: worm, fly, mouse and human. We hypothesize that critical nodes are likely those cleaved most rapidly and most highly conserved.
In Specific Aim 3, we will test this hypothesis by site-specific proteolysis of selected individual targets. We will use a new genetically encoded highly specific split-TEV protease (the """"""""SNIPer"""""""") developed in our laboratory, which can be activated by a small molecule and directed to cleave a single target. This will allow us to test if proteolysis of selected nodes is indeed capable of initiating apoptosis similar to how many cancer drugs target an individual caspase substrate. The identification and confirmation of critical nodes in apoptosis will provide insight into the fundamental process of cellular deconstruction and could serve to identify targets for new cancer drugs.
Specific Aim 1 : Development of the global quantitative methods for kinetic analysis of kcat/KM for intact caspase substrates in extracts and cells Specific Aim 2: Examination of the conservation of caspase cleavage sites and substrates in model metazoans.
Specific Aim 3 : Site-specific proteolysis of a selected set of conserved and rapidly cleaved substrates using the SNIPer technology.

Public Health Relevance

The proposed work will aid in the determination of which targets, biological pathways and processes are most rapidly and consistently involved in apoptosis. Identification of the most critical apoptotic nodes would draw the attention of cancer biologists and drug discovery scientists interested in triggering apoptosis in cancer cells. Thus, in addition to mapping the process of apoptosis and developing novel technology, the proposed work may identify new drug targets for the development of cancer therapies.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM081051-06A1
Application #
8370887
Study Section
Enabling Bioanalytical and Imaging Technologies Study Section (EBIT)
Program Officer
Edmonds, Charles G
Project Start
2007-04-01
Project End
2016-07-31
Budget Start
2012-08-01
Budget End
2013-07-31
Support Year
6
Fiscal Year
2012
Total Cost
$299,611
Indirect Cost
$99,611
Name
University of California San Francisco
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
094878337
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Julien, Olivier; Zhuang, Min; Wiita, Arun P et al. (2016) Quantitative MS-based enzymology of caspases reveals distinct protein substrate specificities, hierarchies, and cellular roles. Proc Natl Acad Sci U S A 113:E2001-10
Hill, Maureen E; MacPherson, Derek J; Wu, Peng et al. (2016) Reprogramming Caspase-7 Specificity by Regio-Specific Mutations and Selection Provides Alternate Solutions for Substrate Recognition. ACS Chem Biol 11:1603-12
Seaman, J E; Julien, O; Lee, P S et al. (2016) Cacidases: caspases can cleave after aspartate, glutamate and phosphoserine residues. Cell Death Differ 23:1717-26
Morgan, Charles W; Diaz, Juan E; Zeitlin, Samantha G et al. (2015) Engineered cellular gene-replacement platform for selective and inducible proteolytic profiling. Proc Natl Acad Sci U S A 112:8344-9
Wiita, Arun P; Seaman, Julia E; Wells, James A (2014) Global analysis of cellular proteolysis by selective enzymatic labeling of protein N-termini. Methods Enzymol 544:327-58
Julien, Olivier; Kampmann, Martin; Bassik, Michael C et al. (2014) Unraveling the mechanism of cell death induced by chemical fibrils. Nat Chem Biol 10:969-76
Wiita, Arun P; Hsu, Gerald W; Lu, Chuanyi M et al. (2014) Circulating proteolytic signatures of chemotherapy-induced cell death in humans discovered by N-terminal labeling. Proc Natl Acad Sci U S A 111:7594-9
Morgan, Charles W; Julien, Olivier; Unger, Elizabeth K et al. (2014) Turning on caspases with genetics and small molecules. Methods Enzymol 544:179-213
Wiita, Arun P; Ziv, Etay; Wiita, Paul J et al. (2013) Global cellular response to chemotherapy-induced apoptosis. Elife 2:e01236
Crawford, Emily D; Seaman, Julia E; Agard, Nick et al. (2013) The DegraBase: a database of proteolysis in healthy and apoptotic human cells. Mol Cell Proteomics 12:813-24

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