0000000000142577
AUTHOR
Tomas Koudelka
LC–MS Based Cleavage Site Profiling of the Proteases ADAM10 and ADAM17 Using Proteome-Derived Peptide Libraries
A Disintegrin and Metalloproteinase 10 (ADAM10) and ADAM17 catalyze ectodomain shedding of a number of cell surface proteins important for embryonic development and tissue homeostasis. Changes in the expression levels or dysregulated proteolytic activity of ADAM10 and ADAM17 have been shown to play important roles in multiple diseases such as inflammation, cancer, and neurodegenerative disorders. Despite the well documented substrate repertoire of ADAM10 and ADAM17, little is known about their cleavage site specificity. We optimized Q-PICS (Quantitative Proteomics for the Identification of Cleavage Sites) to elucidate the cleavage site specificity of recombinant murine ADAM10 and ADAM17. Tw…
Ectodomain shedding of CD99 within highly conserved regions is mediated by the metalloprotease meprin β and promotes transendothelial cell migration.
The adhesion molecule CD99 is essential for the transendothelial migration of leukocytes. In this study, we used biochemical and cellular assays to show that CD99 undergoes ectodomain shedding by the metalloprotease meprin β and subsequent intramembrane proteolysis by γ-secretase. The cleavage site in CD99 was identified by mass spectrometry within an acidic region highly conserved through different vertebrate species. This finding fits perfectly to the unique cleavage specificity of meprin β with a strong preference for aspartate residues and suggests coevolution of protease and substrate. We hypothesized that limited CD99 cleavage by meprin β would alter cellular transendothelial migratio…
Metalloproteases meprin α and meprin β are C- and N-procollagen proteinases important for collagen assembly and tensile strength.
Type I fibrillar collagen is the most abundant protein in the human body, crucial for the formation and strength of bones, skin, and tendon. Proteolytic enzymes are essential for initiation of the assembly of collagen fibrils by cleaving off the propeptides. We report that Mep1a −/− and Mep1b −/− mice revealed lower amounts of mature collagen I compared with WT mice and exhibited significantly reduced collagen deposition in skin, along with markedly decreased tissue tensile strength. While exploring the mechanism of this phenotype, we found that cleavage of full-length human procollagen I heterotrimers by either meprin α or meprin β led to the generation of mature collagen molecules that s…
Phosphorylation of meprin β controls its cell surface abundance and subsequently diminishes ectodomain shedding
Meprin β is a zinc-dependent metalloprotease exhibiting a unique cleavage specificity with strong preference for acidic amino acids at the cleavage site. Proteomic studies revealed a diverse substrate pool of meprin β including the interleukin-6 receptor (IL-6R) and the amyloid precursor protein (APP). Dysregulation of meprin β is often associated with pathological conditions such as chronic inflammation, fibrosis, or Alzheimer's disease (AD). The extracellular regulation of meprin β including interactors, sheddases, and activators has been intensively investigated while intracellular regulation has been barely addressed in the literature. This study aimed to analyze C-terminal phosphorylat…
Metalloprotease meprin β is activated by transmembrane serine protease matriptase-2 at the cell surface thereby enhancing APP shedding.
Increased expression of metalloprotease meprin β is associated with fibrotic syndromes and Alzheimer's disease (AD). Hence, regulation of meprin activity might be a suitable strategy for the treatment of these conditions. Meprin β is a type 1 transmembrane protein, but can be released from the cell surface by ectodomain shedding. The protease is expressed as an inactive zymogen and requires proteolytic maturation by tryptic serine proteases. In the present study, we demonstrate, for the first time, the differences in the activation of soluble and membrane bound meprin β and suggest transmembrane serine protease 6 [TMPRSS6 or matriptase-2 (MT2)] as a new potent activator, cleaving off the pr…