PMID- 15280543 OWN - NLM STAT- publisher DA - 20040811 IS - 0027-8424 VI - 101 IP - 32 DP - 2004 Aug 10 TI - A proteomic approach for the discovery of protease substrates. PG - 11785-11790 AB - Standardized, comprehensive platforms for the discovery of protease substrates have been extremely difficult to create. Screens for protease specificity are now frequently based on the cleavage patterns of peptide substrates, which contain small recognition motifs that are required for the cleavage of the scissile bond within an active site. However, these studies do not identify in vivo substrates, nor can they lead to the definition of the macromolecular features that account for the biological specificity of proteases. To use properly folded proteins in a proteomic screen for protease substrates, we used 2D difference gel electrophoresis and tandem MS to identify substrates of an apoptosis-inducing protease, granzyme B. We confirmed the cleavage of procaspase-3, one of the key substrates of this enzyme, and identified several substrates that were previously unknown, as well as the cleavage site for one of these substrates. We were also able to observe the kinetics of substrate cleavage and cleavage product accumulation by using the 2D difference gel electrophoresis methodology. "Protease proteomics" may therefore represent an important tool for the discovery of the native substrates of a variety of proteases. AD - Department of Medicine, Divisions of Oncology and Metabolism, Siteman Cancer Center and Proteomics Center, Washington University School of Medicine, St. Louis, MO 63110. AU - Bredemeyer AJ AU - Lewis RM AU - Malone JP AU - Davis AE AU - Gross J AU - Townsend RR AU - Ley TJ LA - ENG PT - JOURNAL ARTICLE DEP - 20040727 TA - Proc Natl Acad Sci U S A JID - 7505876 EDAT- 2004/07/29 05:00 MHDA- 2004/07/29 05:00 PHST- 2004/Jul/27 [aheadofprint] AID - 10.1073/pnas.0402353101 [doi] AID - 0402353101 [pii] PST - ppublish SO - Proc Natl Acad Sci U S A 2004 Aug 10;101(32):11785-11790. Epub 2004 Jul 27. PMID- 15220480 OWN - NLM STAT- completed DA - 20040708 DCOM- 20040730 IS - 0027-8424 VI - 101 IP - 27 DP - 2004 Jul 6 TI - Activity-based probes for the proteomic profiling of metalloproteases. PG - 10000-5 AB - Metalloproteases (MPs) are a large and diverse class of enzymes implicated in numerous physiological and pathological processes, including tissue remodeling, peptide hormone processing, and cancer. MPs are tightly regulated by multiple posttranslational mechanisms in vivo, hindering their functional analysis by conventional genomic and proteomic methods. Here we describe a general strategy for creating activity-based proteomic probes for MPs by coupling a zinc-chelating hydroxamate to a benzophenone photocrosslinker, which promote selective binding and modification of MP active sites, respectively. These probes labeled active MPs but not their zymogen or inhibitor-bound counterparts and were used to identify members of this enzyme class up-regulated in invasive cancer cells and to evaluate the selectivity of MP inhibitors in whole proteomes. Interestingly, the matrix metalloproteinase inhibitor GM6001 (ilomastat), which is currently in clinical development, was found to also target the neprilysin, aminopeptidase, and dipeptidylpeptidase clans of MPs. These results demonstrate that MPs can display overlapping inhibitor sensitivities despite lacking sequence homology and stress the need to evaluate MP inhibitors broadly across this enzyme class to develop agents with suitable target selectivities in vivo. Activity-based profiling offers a powerful means for conducting such screens, as this approach can be carried out directly in whole proteomes, thereby facilitating the discovery of disease-associated MPs concurrently with inhibitors that selectively target these proteins. AD - The Skaggs Institute for Chemical Biology and Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. FAU - Saghatelian, Alan AU - Saghatelian A FAU - Jessani, Nadim AU - Jessani N FAU - Joseph, Arul AU - Joseph A FAU - Humphrey, Mark AU - Humphrey M FAU - Cravatt, Benjamin F AU - Cravatt BF LA - eng PT - Journal Article DEP - 20040625 PL - United States TA - Proc Natl Acad Sci U S A JID - 7505876 RN - 0 (Dipeptides) RN - 0 (GM 6001) RN - EC 3.4.- (Metalloproteases) RN - EC 3.4.24.11 (Neprilysin) SB - IM MH - Dipeptides/pharmacology MH - Human MH - Melanoma/enzymology MH - Metalloproteases/*analysis/antagonists & inhibitors MH - Neprilysin/antagonists & inhibitors MH - *Proteomics MH - Support, Non-U.S. Gov't MH - Support, U.S. Gov't, P.H.S. EDAT- 2004/06/29 05:00 MHDA- 2004/07/31 05:00 PHST- 2004/Jun/25 [aheadofprint] AID - 10.1073/pnas.0402784101 [doi] AID - 0402784101 [pii] PST - ppublish SO - Proc Natl Acad Sci U S A 2004 Jul 6;101(27):10000-5. Epub 2004 Jun 25. PMID- 15118097 OWN - NLM STAT- completed DA - 20040505 DCOM- 20040622 IS - 0027-8424 VI - 101 IP - 18 DP - 2004 May 4 TI - Membrane protease proteomics: Isotope-coded affinity tag MS identification of undescribed MT1-matrix metalloproteinase substrates. PG - 6917-22 AB - By proteolytic modification of low abundant signaling proteins and membrane receptors, proteases exert potent posttranslational control over cell behavior at the postsecretion level. Hence, substrate discovery is indispensable for understanding the biological role of proteases in vivo. Indeed, matrix metalloproteinases (MMPs), long associated with extracellular matrix degradation, are increasingly recognized as important processing enzymes of bioactive molecules. MS is now the primary proteomic technique for detecting, identifying, and quantitating proteins in cells or tissues. Here we used isotopecoded affinity tag labeling and multidimensional liquid chromatography inline with tandem MS to identify MDA-MB-231 breast carcinoma cell proteins shed from the cell surface or the pericellular matrix and extracellular proteins that were degraded or processed after transfection with human membrane type 1-MMP (MT1-MMP). Potential substrates were identified as those having altered protein levels compared with the E240A inactive MT1-MMP mutant or vector transfectants. New substrates were biochemically confirmed by matrix-assisted laser desorption ionization-time-of-flight MS and Edman sequencing of cleavage fragments after incubation with recombinant soluble MT1-MMP in vitro. We report many previously uncharacterized substrates of MT1-MMP, including the neutrophil chemokine IL-8, secretory leukocyte protease inhibitor, pro-tumor necrosis factor alpha, death receptor-6, and connective tissue growth factor, indicating that MT1-MMP is an important signaling protease in addition to its traditionally ascribed roles in pericellular matrix remodeling. Moreover, the high-throughput and quantitative nature of isotope-coded affinity tag labeling combined with tandem MS sequencing is a previously undescribed degradomic screen for protease substrate discovery that should be generally adaptable to other classes of protease for exploring proteolytic function in complex and dynamic biological contexts. AD - Department of Biochemistry and Molecular Biology, Centre for Blood Research and Canadian Institutes of Health Research Group in Matrix Dynamics, University of British Columbia, Vancouver, BC, Canada V6T 1Z3. FAU - Tam, Eric M AU - Tam EM FAU - Morrison, Charlotte J AU - Morrison CJ FAU - Wu, Yi I AU - Wu YI FAU - Stack, M Sharon AU - Stack MS FAU - Overall, Christopher M AU - Overall CM LA - eng PT - Journal Article DEP - 20040426 PL - United States TA - Proc Natl Acad Sci U S A JID - 7505876 RN - 0 (CTNNBIP1 protein, human) RN - 0 (Cell Cycle Proteins) RN - 0 (Chemokines) RN - 0 (Cytokines) RN - 0 (Repressor Proteins) RN - EC 3.4.24 (Metalloendopeptidases) RN - EC 3.4.24.- (membrane-type 1 matrix metalloproteinase) SB - IM MH - Cell Cycle Proteins MH - Chemokines/metabolism MH - Cytokines/metabolism MH - Human MH - Metalloendopeptidases/chemistry/*metabolism MH - Proteomics MH - Repressor Proteins MH - Spectrum Analysis, Mass MH - Staining and Labeling MH - Substrate Specificity/physiology MH - Support, Non-U.S. Gov't EDAT- 2004/05/01 05:00 MHDA- 2004/06/23 05:00 PHST- 2004/Apr/26 [aheadofprint] AID - 10.1073/pnas.0305862101 [doi] AID - 0305862101 [pii] PST - ppublish SO - Proc Natl Acad Sci U S A 2004 May 4;101(18):6917-22. Epub 2004 Apr 26.