Membrane protease proteomics: Isotope-coded affinity tag MS identification of undescribed MT1–matrix metalloproteinase substrates

Abstract

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 ␣, 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.I n all living organisms, proteases exert high-order posttranslational control over a diverse range of cellular functions. Altered protease expression and substrate proteolysis are pivotal elements in the pathogenesis of many diseases (1) with 53 hereditary genetic diseases of proteolysis recognized (2). Indeed, proteases represent Ϸ10% of current drug targets (3). Elucidating the substrate repertoire of a protease is critical to understanding its biological role, but given the large number of proteases (Ͼ553) present in the human genome (2), this is a daunting task. Innovative approaches using combinatorial or positional scanning libraries of fluorogenic (4) and inhibitory peptides (5) and oriented (6) and phage display (7) peptide libraries can determine consensus protease cleavage sequences. However, bioinformatic identification of proteins containing these sequences followed by biochemical and in vivo validation of proteolytic susceptibility has led to the identification of relatively few biologically relevant new substrates. This is not s...

Towards third generation matrix metalloproteinase inhibitors for cancer therapy

“…For this, the MMP substrate degradome ) must be identified. Since biologically relevant substrates might differ from theoretical activities inferred from in vitro experiments -'just because it can, does not mean it does' -the best method of substrate discovery is to identify protease-cleaved substrates in complex milieus Tam et al, 2004). Similarly, detecting cleavage fragments of known substrates confirms proteolysis in vivo and might be useful as cancer biomarkers.…”

“…Similarly, detecting cleavage fragments of known substrates confirms proteolysis in vivo and might be useful as cancer biomarkers. Recognising this, proteomic approaches have been developed to rapidly identify new protease substrates (Bredemeyer et al, 2004;Overall et al, 2004;Tam et al, 2004;Butler and Overall, 2006).…”

Stable isotopic labeling in proteomics

“…Upon comparing a control cell population with one under oxidative stress, ICAT-tagged peptide couples showing altered ratios may point to in vivo modified cysteines [56]. ICAT labeling has also been used to study protein processing by matrix metalloproteases (e.g., [57,58]). Here, the levels of secreted and shed extracellular proteins are quantified by ICAT using cell lines that typically over-express the metalloprotease of interest.…”

Finding the needles in the haystack: mapping constitutive proteolytic events in vivo

“…In fact, a number of new studies have recently been reported that make use of system-wide proteomic methods to carry out unbiased searches for substrates of specific proteases [5][6][7]. These methods take advantage of isotopic labels to compare pools of proteins that either have or have not been exposed to a protease of interest.…”