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The substrate degradome of meprin metalloproteases reveals an unexpected proteolytic link between meprin ? and ADAM10.

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 ? and ? 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 ?-secretase-is activated by meprin ? through cleavage of the propeptide. To validate this cleavage event, we expressed recombinant proADAM10 and after preincubation with meprin ?, 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(-/-) 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.

SUBMITTER: Jefferson T

PROVIDER: S-EPMC3535375 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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The substrate degradome of meprin metalloproteases reveals an unexpected proteolytic link between meprin β and ADAM10.

Jefferson Tamara T Auf dem Keller Ulrich U Bellac Caroline C Metz Verena V VV Broder Claudia C Hedrich Jana J Ohler Anke A Maier Wladislaw W Magdolen Viktor V Sterchi Erwin E Bond Judith S JS Jayakumar Arumugam A Traupe Heiko H Chalaris Athena A Rose-John Stefan S Pietrzik Claus U CU Postina Rolf R Overall Christopher M CM Becker-Pauly Christoph C

Cellular and molecular life sciences : CMLS 20120901 2

The in vivo roles of meprin metalloproteases in pathophysiological conditions remain elusive. Substrates define protease roles. Therefore, to identify natural substrates for human meprin α and β 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 protei ...[more]

PMID: 22940918

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Project description:The substantial rise in multidrug-resistant bacterial infections is a current global imperative. Cumulative efforts to characterize antimicrobial resistance in bacteria has demonstrated the spread of six families of multidrug efflux pumps, of which resistance-nodulation-cell division (RND) is the major mechanism of multidrug resistance in Gram-negative bacteria. RND is composed of a tripartite protein assembly and confers resistance to a range of unrelated compounds. In the major enteric pathogen Campylobacter jejuni, the three protein components of RND are posttranslationally modified with N-linked glycans. The direct role of N-linked glycans in C. jejuni and other bacteria has long been elusive. Here, we present the first detailed account of the role of N-linked glycans and the link between N-glycosylation and antimicrobial resistance in C. jejuni We demonstrate the multifunctional role of N-linked glycans in enhancing protein thermostability, stabilizing protein complexes and the promotion of protein-protein interaction, thus mediating antimicrobial resistance via enhancing multidrug efflux pump activity. This affirms that glycosylation is critical for multidrug efflux pump assembly. We present a generalized strategy that could be used to investigate general glycosylation system in Campylobacter genus and a potential target to develop antimicrobials against multidrug-resistant pathogens.IMPORTANCE Nearly all bacterial species have at least a single glycosylation system, but the direct effects of these posttranslational protein modifications are unresolved. Glycoproteome-wide analysis of several bacterial pathogens has revealed general glycan modifications of virulence factors and protein assemblies. Using Campylobacter jejuni as a model organism, we have studied the role of general N-linked glycans in the multidrug efflux pump commonly found in Gram-negative bacteria. We show, for the first time, the direct link between N-linked glycans and multidrug efflux pump activity. At the protein level, we demonstrate that N-linked glycans play a role in enhancing protein thermostability and mediating the assembly of the multidrug efflux pump to promote antimicrobial resistance, highlighting the importance of this posttranslational modification in bacterial physiology. Similar roles for glycans are expected to be found in other Gram-negative pathogens that possess general protein glycosylation systems.

Dataset Information

The substrate degradome of meprin metalloproteases reveals an unexpected proteolytic link between meprin ? and ADAM10.

Publications

The substrate degradome of meprin metalloproteases reveals an unexpected proteolytic link between meprin β and ADAM10.

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