tailieunhanh - Báo cáo khoa học: A novel 2D-based approach to the discovery of candidate substrates for the metalloendopeptidase meprin

In the past, protease-substrate finding proved to be rather haphazard and was executed byin vitro cleavage assays using singly selected targets. In the present study, we report the first protease proteomic approach applied to meprin, an astacin-like metalloendopeptidase, to determine physiological substrates in a cell-based system of Madin–Darby canine kidney epithelial cells. | ễFEBS Journal A novel 2D-based approach to the discovery of candidate substrates for the metalloendopeptidase meprin Daniel Ambort1 Daniel Stalder2 Daniel Lottaz1 Maya Huguenin1 Beatrice Oneda1 Manfred Heller2 and Erwin E. Sterchi1 1 Institute of Biochemistry and Molecular Medicine University of Berne Switzerland 2 Department of ClinicalResearch University Hospital Berne Switzerland Keywords astacin family image analysis Madin-Darby canine kidney cells meprin protease proteomics Correspondence E. E. Sterchi Institute of Biochemistry and Molecular Medicine University of Berne Buhlstrasse 28 CH-3012 Berne Switzerland Fax 41 31 631 3737 Tel 41 31 631 4199 E-mail Received 14 April2008 revised 8 July 2008 accepted 10 July 2008 doi In the past protease-substrate finding proved to be rather haphazard and was executed by in vitro cleavage assays using singly selected targets. In the present study we report the first protease proteomic approach applied to meprin an astacin-like metalloendopeptidase to determine physiological substrates in a cell-based system of Madin-Darby canine kidney epithelial cells. A simple 2D lEF SDS PAGE-based image analysis procedure was designed to find candidate substrates in conditioned media of Madin-Darby canine kidney cells expressing meprin in zymogen or in active form. The method enabled the discovery of hitherto unkown meprin substrates with shortened non-trypsin-generated N- and C-terminally truncated cleavage products in peptide fragments upon LC-MS MS analysis. Of 22 17 nonredundant candidate substrates identified the proteolytic processing of vinculin lysyl oxidase collagen type V and annexin A1 was analysed by means of immunoblotting validation experiments. The classification of substrates into functional groups may propose new functions for meprins in the regulation of cell homeostasis and the extracellular environment and in innate immunity respectively. The astacin-like .

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