Protease degradomics: mass spectrometry discovery of protease substrates and the CLIP-CHIP, a dedicated DNA microarray of all human proteases and inhibitors
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C.M. Overall
Abstract
The biological role of most proteases in vivo is largely unknown. Therefore, to develop robust techniques to analyze the protease degradome in cells and tissues and to elucidate their substrate degradomes we have developed a dedicated and complete human protease and inhibitor microarray that we have called the CLIP-CHIP. Oligonucleotides (70-mers) for identifying all 715 human proteases, inactive homologs and inhibitors were spotted in triplicate onto glass slides with a dedicated subarray containing oligonucleotides for specific human breast carcinoma genes. Initial analyses revealed the elevated expression of a number of proteases in invasive ductal cell carcinoma including ADAMTS17, carboxypeptidases A5 and M, tryptasegamma and matriptase-2. Matrix metalloproteinases (MMPs) showed a restricted expression pattern in both normal and cancerous breast tissues with most expressed at low levels. However, of the several MMPs expressed in significant quantities, the carcinoma samples showed only slightly elevated amounts other than for MMP-28 which was strongly elevated. To discover new protease substrates we developed a novel yeast twohybrid approach we term inactive catalytic domain capture (ICDC). Here, an inactive mutant protease catalytic domain lacking the propeptide was used as a yeast two hybrid bait to screen a human fibroblast cDNA library for interactor proteins as a substrate trap. Wntinduced signaling protein-2 (WISP-2) was identified by ICDC and was biochemically confirmed as a new MMP substrate. In another approach we used isotopecoded affinity tag (ICAT) labeling with tandem mass spectrometry to quantitate the levels of secreted or shed extracellular proteins in MDAMB-231 breast carcinoma cell cultures in the presence or absence of membrane type 1-MMP (MT1-MMP) overexpression. By this proteomic approach we identified and biochemically confirmed that IL-8, the serine protease inhibitor SLPI, the death receptor-6, proTNFα and CTGF are novel substrates of MT1-MMP. The utility and quantitative nature of ICAT with MS/MS analysis as a new screen for protease substrate discovery based on detection of cleaved or shed substrate products should be readily adaptable to other classes of protease for assessing proteolytic function in a cellular context.
Copyright © 2004 by Walter de Gruyter GmbH & Co. KG
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Articles in the same Issue
- Highlight: 3rd General IPS Meeting/International Conference on Protease inhibitors
- Colon cancer: genomics and apoptotic events
- Interaction of calpastatin with calpain: a review
- Cathepsin L and Arg/Lys aminopeptidase: a distinct prohormone processing pathway for the biosynthesis of peptide neurotransmitters and hormones
- Searching for the most effective screening system to identify cell-active inhibitors of β-secretase
- Accumulation of mini-plasmin in the cerebral capillaries causes vascular invasion of the murine brain by a pneumotropic influenza A virus: implications for influenza-associated encephalopathy
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- Human cathepsin F: expression in baculovirus system, characterization and inhibition by protein inhibitors
- Proteinases participating in the processing and activation of prolegumain in primary cultured rat macrophages
- Human kallikrein 6 activity is regulated via an autoproteolytic mechanism of activation/inactivation
- Growth phase-dependent production of a cell wall-associated elastinolytic cysteine proteinase by Staphylococcus epidermidis
- Evidence for an interaction between leptin, T cell costimulatory antigens CD28, CTLA-4 and CD26 (dipeptidyl peptidase IV) in BCG-induced immune responses of leptin- and leptin receptor-deficient mice
- Characterisation of a highly specific, endogenous inhibitor of cysteine protease from Staphylococcus epidermidis, a new member of the staphostatin family
- Identification of cysteine protease inhibitors that belong to cystatin family 1 in the cellular slime mold Dictyostelium discoideum
- High molecular weight kininogen as substrate for cathepsin B
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- The crystal structure of human dipeptidyl peptidase IV (DPPIV) complex with diprotin A
- Metalloproteases with EGF, CUB, and thrombospondin-1 domains function in molting of Caenorhabditis elegans
