Project

Discipline

Metalloproteinase; Meprin; Intestinal epithelial cell; Brush border membrane; Cell adhesion; Protein interaction; Proteomics

Lead
Lay summary
Meprins (meprin? and meprin?) are proteinases that belong to the Astacin family of metalloproteinases. They are abundantly expressed at the membrane of intestinal epithelial cells facing the intestinal lumen. Membrane-bound and soluble meprin activity is finely regulated, but can be destructive if unchecked, misplaced, and/or inappropriately activated. The function of meprins in health and disease is not clear and subject of intensive research. Meprins are involved in cell migration, colon cancer, and inflammatory bowel diseases. Meprinß knockout mice are less susceptible to inflammatory bowel disease than wild-type mice and we know that in compromised tissues, meprins may be re-distributed to the cytosol and the basolateral membrane of epithelial cells thus causing damage to these cells.The aims of the project are:1) to investigate how meprins alter cell-cell and cell-matrix interactions by proteolytic processing of specific proteins involved in these processes.2) to identify proteins within cells that interact with membrane-bound meprin?3) to investigate the role of membrane-bound meprin in signalling events through the proteolytic cleavage and thus activation of memrane receptors and/or their ligands.Experimental design:Proteolytic processing of specific proteins will be studied in cultured epithelial cells and intestinal tissue explants and the functional consequences of this processing will be determined in cell adhesion and cell migration assays. A novel protein-protein interaction screen will be used to identify intracellular proteins that interact with the cytosolic domain of meprinß. The ability of meprin? to activate epidermal growth factor receptor (EGFR) in colorectal cancer cells will be studied in epithelial cell cultures. Expected value of the proposed project:Data obtained will enhance our understanding of meprin substrates by providing knowledge about specific protein substrates including those involved in cell-cell / cell-matrix interactions, as well as proteins that interact with meprinß and include membrane receptors and intracellular signalling molecules. This proposal has close links to a research project supported by the EU (http://www.ibdase.org) of which the PI is a partner.

Direct link to Lay Summary

Last update: 21.02.2013

Active participation

Number	Title	Start	Funding scheme

1. Zusammenfassung / SummaryBackground:Meprina and ß are membrane-bound and secreted metalloproteases of the astacin family, and are abundantly expressed at the apical membrane of intestinal epithelial cells. Membrane-bound and soluble meprin activity is finely regulated, but can be destructive if unchecked, misplaced, and/or inappropriately activated. We have learnt a lot about structure, biosynthesis and secretion, activation, expression and regulation of meprina and ß subunits but we do not know much about the function of meprin in health and disease. We have begun to explore the role of meprin in cell migration, cancer, and inflammatory bowel diseases. From previous studies we know that meprins are involved in processing of cell adhesion and ECM molecules. We also know that meprinß knockout mice are less susceptible to inflammatory bowel disease induced by DSS than wild-type mice and we also know that in compromised tissue, meprin is re-distributed to the cytosol and the basolateral membrane of epithelial cells.Hypothesis: Redistribution of apical meprins to the cytosol and the basolateral membrane of polarised epithelial cells is damaging to the cells. We propose that new interactions of meprins with proteins in the cytosol, the basolateral membrane and the basal lamina cause injury and this leads to or aggravates disruption of the mucosal epithelium. This will lead to new insights into mechanisms that increase permeability of the intestinal barrier in colorectal cancer and inflammatory bowel disease and will ultimately open up ways for new therapies.Specific Aims:1)Investigate processig of cell-cell and cell-matrix adhesion proteins by meprinß in epithelial cells2)Identify intracellular binding partners of meprinß3)Determine if epidermal growth factor receptor (EGFR) is activated by a meprina-mediated pathway in colorectal cancer cells4)Identify substrates of meprins in the intestinal epitheliumExperimental Design:In cell lines (either meprinß-transfected or endogenously expressing meprinß) study processing of cell-cell (e.g. E-cadherin) and cell-matrix (e.g. tenascin-C) interactions. Identify breakdown products. Expand these studies to organ cultured mucosal explants from wild-type and meprin knock-out mice and to human intestine (healthy and inflamed). Cell adhesion and cell migration assays in cell cultures; structural (macroscopic, microscopic) and functional (biosynthetic) studies in organ culture to assess consequences of the hydrolysis of cell-cell and cell-matrix proteins. Determine if meprinß aggravates damage to the epithelium when this is challenged (e.g. by hypoxia/reoxygenation). Use a novel protein-protein interaction screen to identify intracellular proteins that interact with the cytosolic domain of meprinß.Investigate the ability of meprina to activate epidermal growth factor receptor (EGFR) in colorectal cancer cells. Transwell cultures and co-cultures in combination with biosynthetic labelling with [35S]-methionine, immunoisolation SDS-PAGE and fluorography. ELISAs for the analysis of growth factors and growth factor shedding by mepirna. Functional studies to assess cell proliferation, differentiation and migration.Proteomics/degradomics analyses using Terminal Amine Isotope Labelling of Substrates (TAILS) to identify the global meprina, meprinß and meprina/ß degradome in intestinal mucosa.Expected Value of the proposed Project:Data obtained will enhance our understanding of meprin substrates by providing knowledge about specific protein substrates including those involved in cell-cell / cell-matrix interactions, as well as proteins that interact with meprinß and include membrane receptors and intracellular signalling molecules.The proteomics/degradomics data will identify hitherto unknown substrates of meprins and will provide the basis for further focussed studies in the context of colorectal cancer and inflammatory bowel disease.This proposal has close links to the EU / FP7 IBDase project (http://www.ibdase.org) of which the PI is a partner. The IBDase project addresses the etiology and pathogenesis of Inflammatory Bowel Disease (IBD), a multifactorial disease influenced by environmental factors in a background of complex genetic susceptibility. The project envisages a multidisciplinary approach for innovative diagnosis and therapy focused on mucosal proteases and their inhibitors (P/PIs).