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 protein 10)—the constitutive α-secretase—is activated by meprin β through cleavage of the propeptide. To validate this cleavage event, we expressed recombinant proADAM10 and after preincubation with meprin β, 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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00018-012-1106-2) contains supplementary material, which is available to authorized users.

Background: Meprin ␤ cleaves the amyloid precursor protein.Results: Meprin ␤-mediated cleavage of the amyloid precursor protein leads to an increase of amyloid ␤ production and to the generation of an N-terminal truncated amyloid ␤ variant. Conclusion: Meprin ␤ can generate N-terminal truncated amyloid ␤ peptides. Significance: Our data indicate that meprin ␤ is a novel protease in amyloid ␤ generation.

Identification of physiologically relevant substrates is still the most challenging part in protease research for understanding the biological activity of these enzymes. The zinc-dependent metalloprotease meprin ␤ is known to be expressed in many tissues with functions in health and disease. Here, we demonstrate unique interactions between meprin ␤ and the amyloid precursor protein (APP). Although APP is intensively studied as a ubiquitously expressed cell surface protein, which is involved in Alzheimer disease, its precise physiological role and relevance remain elusive. Based on a novel proteomics technique termed terminal amine isotopic labeling of substrates (TAILS), APP was identified as a substrate for meprin ␤. Processing of APP by meprin ␤ was subsequently validated using in vitro and in vivo approaches. N-terminal APP fragments of about 11 and 20 kDa were found in human and mouse brain lysates but not in meprin ␤ ؊/؊ mouse brain lysates. Although these APP fragments were in the range of those responsible for caspase-induced neurodegeneration, we did not detect cytotoxicity to primary neurons treated by these fragments. Our data demonstrate that meprin ␤ is a physiologically relevant enzyme in APP processing.

Ectodomain shedding at the cell surface is a major mechanism to regulate the extracellular and circulatory concentration or the activities of signaling proteins at the plasma membrane. Human meprin β is a 145-kDa disulfide-linked homodimeric multidomain type-I membrane metallopeptidase that sheds membrane-bound cytokines and growth factors, thereby contributing to inflammatory diseases, angiogenesis, and tumor progression. In addition, it cleaves amyloid precursor protein (APP) at the β-secretase site, giving rise to amyloidogenic peptides. We have solved the X-ray crystal structure of a major fragment of the meprin β ectoprotein, the first of a multidomain oligomeric transmembrane sheddase, and of its zymogen. The meprin β dimer displays a compact shape, whose catalytic domain undergoes major rearrangement upon activation, and reveals an exosite and a sugar-rich channel, both of which possibly engage in substrate binding. A plausible structure-derived working mechanism suggests that substrates such as APP are shed close to the plasma membrane surface following an "N-like" chain trace. P hysiological processes in the extracellular milieu and the circulation require finely tuned concentrations of signal molecules such as cytokines, growth factors, receptors, adhesion molecules, and peptidases. Many of these proteins are synthesized as type-I membrane protein variants or precursors consisting of a glycosylated N-terminal ectoprotein, a transmembrane helix, and a Cterminal cytosolic tail. Their localization at the cell surface restricts their field of action to autocrine or juxtacrine processes. However, to act at a distance in paracrine, synaptic, or endocrine events, they have to be released from the plasma membrane into the extracellular space as soluble factors through "protein ectodomain shedding" (1, 2). This entails limited proteolysis and is a major posttranslational regulation mechanism that affects 2-4% of the proteins on the cell surface, occurs at or near the plasma membrane (3), and apparently follows a common release mechanism (2). It may also proteolytically inactivate proteins to terminate their function on the cell surface (4). Peptidases engaged in such processing are "sheddases" and the most studied transmembrane sheddases are members of the adamalysin/ADAMs (4, 5) and matrix-metalloproteinase (MMP) (6) families within the metzincin clan of metallopeptidases (MPs) (7-9). These include ADAM

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