Exploring the landscape of ectodomain shedding by quantitative protein terminomics.
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ABSTRACT:
SUBMITTER: Tsumagari K
PROVIDER: S-EPMC7995609 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Ectodomain shedding is a proteolytic process that regulates level and function of membrane proteins. By quantitative protein terminomics consisting of N- and C-terminal peptide enrichment, TMT-labeling and mass spectrometry, we quantified protein termini in the cultured media across 10 human cell lines and identified cleavage sites on membrane proteins downregulated upon broad-spectrum metalloprotease inhibitor treatment.
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Project description:Despite intense interest in the proteolysis of the ?-Amyloid Precursor Protein (APP) in Alzheimer's disease, how the normal processing of this type I receptor-like glycoprotein is physiologically regulated remains ill-defined. In recent years, several candidate protein ligands for APP, including F-spondin, Reelin, ?1 Integrin, Contactins, Lingo-1, and Pancortin, have been reported. However, a cognate ligand for APP that regulates its processing by ?- or ?-secretase has yet to be widely confirmed in multiple laboratories. Here, we developed new assays in an effort to confirm a role for one or more of these candidate ligands in regulating APP ectodomain shedding in a biologically relevant context. A comprehensive quantification of APPs? and APPs?, the immediate products of secretase processing, in both non-neuronal cell lines and primary neuronal cultures expressing endogenous APP yielded no evidence that any of these published candidate ligands stimulate ectodomain shedding. Rather, Reelin, Lingo-1, and Pancortin-1 emerged as the most consistent ligands for significantly inhibiting ectodomain shedding. These findings led us to conduct further detailed analyses of the interactions of Reelin and Lingo-1 with APP.
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Project description:The membrane PTK7 pseudokinase, a component of both the canonical and noncanonical/planar cell polarity Wnt pathways, modulates cell polarity and motility in biological processes as diverse as embryo development and cancer cell invasion. To determine the individual proteolytic events and biological significance of the ectodomain shedding in the PTK7 function, we used highly invasive fibrosarcoma HT1080 cells as a model system. Current evidence suggested a likely link between PTK7 shedding and cell invasion in our HT1080 cell model system. We also demonstrated that in HT1080 cells the cleavage of the PTK7 ectodomain by an ADAM proteinase was coupled with the membrane type-1 matrix metalloproteinase (MT1-MMP) cleavage of the PKP(621)↓LI site in the seventh Ig-like domain of PTK7. Proteolytic cleavages led to the generation of two soluble, N-terminal and two matching C-terminal, cell-associated fragments of PTK7. This proteolysis was a prerequisite for the intramembrane cleavage of the C-terminal fragments of PTK7 by γ-secretase. γ-Secretase cleavage was predominantly followed by the efficient decay of the resulting C-terminal PTK7 fragment via the proteasome. In contrast, in HT1080 cells, which overexpressed the C-terminal PTK7 fragment, the latter readily entered the nucleus. Our data imply that therapeutic inhibition of PTK7 shedding may be used to slow cancer progression.
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