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Gelatinase B/MMP-9 cleaves intracellular substrates and thus decreases general T cell responses to cytosol 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 Objectives The action radius of matrix metalloproteinases or MMPs is not restricted to massive extracellular matrix (ECM) degradation but extends to the proteolysis of secreted cytokines and membrane-bound receptors and adhesion molecules. 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. The aims of the present study were to identify novel intracellular MMP targets and to answer the question whether the proteolytic modification of intracellular proteins alters the immunogenicity of released intracellular contents. Methods Multidimensional degradomics technology was developed by the integration of broadly available biotechniques and applied to THP-1 cytosol using gelatinase B/MMP-9 as a model enzyme. In the first dimension, 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, potential substrates were separated by molecular weight on SDS-PAGE. 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 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, stathmin,… About 2/3 of the identified substrates were described as systemic autoantigens in one or multiple autoimmune conditions. Remarkably, a significantly lower T cell proliferation was observed in the presence of cleaved vs. intact cytosol. 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 and tissue injury.