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Gelatinase B/MMP-9 cleaves cytosolic substrates and thus decreases general T cell responses to intracellular (matrix) proteins Bénédicte Cauwe, Erik Martens, Paul Proost, Nele Berghmans, Chris Dillen and Ghislain Opdenakker - Rega Institute for Medical Research, Department of Microbiology and Immunology, University of Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium Background The action radius of matrix metalloproteinases or MMPs extends from massive extracellular matrix (ECM) degradation to the regulatory proteolysis of secreted cytokines, protein hormones, chemokines and a kaleidoscope of membrane-bound molecules. However, although many instances exist in which cells disintegrate, often in conjunction with induction of MMPs, the intracellular MMP substrate repertoire or degradome remains relatively unexplored. Objectives The aim of the present study was to develop straightforward, inexpensive and broadly applicable technology to identify novel intracellular MMP targets. In addition, the question was raised whether the proteolytic modification of intracellular proteins alters the immunogenicity of released intracellular contents. Methods Gelatinase B/MMP-9 was used as a model enzyme for the cleavage of THP-1 cytosolic proteins. In the first dimension of multidimensional degradomics, ion exchange chromatography separated the THP-1 proteins by their net charge and/or isoelectric point (pI) followed by cleavage of the proteins by MMP-9. In the second dimension, the cytosolic proteins were separated by molecular weight on SDS-PAGE and potential substrates were identified by Edman degradation or LC-MS/MS (1). To evaluate the effect of proteolysis by MMP-9 on the immunogenicity of the intracellular protein pool, mice were immunized twice with THP-1 cytosol in complete Freund’s adjuvant. Lymph node T cells were isolated and stimulated with MMP-9-cleaved or intact THP-1 cytosol. Proliferation was assessed by measuring incorporated 3H-thymidine. Results By means of this unbiased multidimensional degradomics technology platform, 100-200 MMP-9 candidate substrates were isolated, of which 69 were identified, revealing many novel MMP-9 (candidate) substrates from the intracellular matrix (ICM) such as actin, tubulin, gelsolin, moesin, ezrin, Arp2/3 complex subunits, filamin B and stathmin,… In addition, about 2/3 of the identified substrates were described as autoantigens in multiple autoimmune conditions and in cancer (e.g. annexin I, nucleolin, citrate synthase, HMGB1, -enolase, histidyl-tRNA synthetase, HSP27, HSP90, snRNP D3). Remarkably, a significantly lower T cell proliferation was observed after stimulation with cleaved vs. intact intracellular proteins in the presence of antigen-presenting cells. Conclusion Multidimensional degradomics technology is a valuable tool for high-throughput identification of novel MMP substrates. Proteolysis by MMP-9 decreased the immunogenicity of the intracellular contents, suggesting that MMPs may contribute to the dampening of inflammation by the clearance of toxic and immunogenic burdens of intracellular (matrix) proteins released after extensive necrosis or tissue injury. (1)Cauwe et al., Multidimensional degradomics identifies systemic autoantigens and intracellular matrix proteins as novel gelatinase B/MMP-9 substrates’, Integr. Biol., 2009, DOI: 10.1039/b904701h.