Cellular and Molecular Life Sciences (CMLS), Jan, 2013, Vol.70(1), p.309(25)
Byline: Tamara Jefferson (1), Ulrich Keller (2,3), Caroline Bellac (3), Verena V. Metz (4), Claudia Broder (1), Jana Hedrich (5), Anke Ohler (6), Wladislaw Maier (7), Viktor Magdolen (8), Erwin Sterchi (9), Judith S. Bond (10), Arumugam Jayakumar (11), Heiko Traupe (12), Athena Chalaris (1), Stefan Rose-John (1), Claus U. Pietrzik (7), Rolf Postina (4), Christopher M. Overall (3), Christoph Becker-Pauly (1,13) Keywords: Meprin; ADAM10; Metalloproteases; Proteomics; TAILS; Degradome Abstract: The in vivo roles of meprin metalloproteases in pathophysiological conditions remain elusive. Substrates define protease roles. Therefore, to identify natural substrates for human meprin [alpha] and [beta] we employed TAILS (terminal amine isotopic labeling of substrates), a proteomics approach that enriches for N-terminal peptides of proteins and cleavage fragments. Of the 151 new extracellular substrates we identified, it was notable that ADAM10 (a disintegrin and metalloprotease domain-containing protein 10)--the constitutive [alpha]-secretase--is activated by meprin [beta] through cleavage of the propeptide. To validate this cleavage event, we expressed recombinant proADAM10 and after preincubation with meprin [beta], this resulted in significantly elevated ADAM10 activity. Cellular expression in murine primary fibroblasts confirmed activation. Other novel substrates including extracellular matrix proteins, growth factors and inhibitors were validated by western analyses and enzyme activity assays with Edman sequencing confirming the exact cleavage sites identified by TAILS. Cleavages in vivo were confirmed by comparing wild-type and meprin.sup.-/- mice. Our finding of cystatin C, elafin and fetuin-A as substrates and natural inhibitors for meprins reveal new mechanisms in the regulation of protease activity important for understanding pathophysiological processes. Author Affiliation: (1) Institute of Biochemistry, Christian-Albrechts-University, 24118, Kiel, Germany (2) Institute of Molecular Health Sciences, Swiss Federal Institute of Technology Zurich, ETH Hoenggerberg, HPM D24, Zurich, Switzerland (3) Departments of Oral Biological and Medical Sciences and Biochemistry and Molecular Biology, Centre for Blood Research, University of British Columbia, Vancouver, BC, Canada (4) Institute of Pharmacy and Biochemistry, Johannes Gutenberg-University, Mainz, Germany (5) Institute of Physiology and Pathophysiology, University Medical Center, Johannes Gutenberg-University, Mainz, Germany (6) Cell and Matrix Biology, Johannes Gutenberg University, Mainz, Germany (7) Institute of Pathobiochemistry, University Medical Center, Johannes Gutenberg-University, Mainz, Germany (8) Clinical Research Unit, Department of Obstetrics and Gynecology, Technical University of Munich, Munich, Germany (9) Institute of Biochemistry and Molecular Medicine, University of Bern, Bern, Switzerland (10) Department of Biochemistry and Molecular Biology, The Pennsylvania State University College of Medicine, Hershey, PA, USA (11) Department of Experimental Therapeutics, M.D. Anderson Cancer Center, The University of Texas, Houston, TX, USA (12) Department of Dermatology, University Hospital Munster, Munster, Germany (13) Unit for Degradomics of the Protease Web, Christian-Albrechts-University, Rudolf-Hober-Str. 1, 24118, Kiel, Germany Article History: Registration Date: 23/07/2012 Received Date: 17/04/2012 Accepted Date: 23/07/2012 Online Date: 01/09/2012 Article note: Electronic supplementary material The online version of this article (doi: 10.1007/s00018-012-1106-2) contains supplementary material, which is available to authorized users.
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