title: Proteomic analysis of silenced cathepsin B expression suggests non-proteolytic cathepsin B functionality.
abstract: Cathepsin B (CTSB) is a lysosomal endo- and exopeptidase that is also secreted in high amounts by malignant and non-malignant cells. We determined the effect of CTSB on the tumor cell secretome by shRNA-mediated silencing of CTSB mRNA expression and subsequent proteomic LC-MS/MS analysis of the cell supernatants. We identified significant protein changes of 17 secreted or shed proteins. Notably, we found a general reduction in protein abundance of ADAM10 substrates and lysosomal proteins. We corroborated reduced amounts of soluble ADAM10 (sADAM10) and soluble APP (sAPP) in the two cancer cell lines MDA-MB-231 and U2OS by immunoblotting. Interestingly, reductions in sADAM10 and sAPP could be reversed by re-introducing a catalytically inactive variant of CTSB, suggesting a formerly unknown non-catalytic function of the protease.
authors.affiliation: Institute of Molecular Medicine and Cell Research, University of Freiburg, D-79104 Freiburg, Germany; Faculty of Biology, University of Freiburg, D-79104 Freiburg, Germany.
authors.forename: Florian Christoph
authors.initials: FC
authors.lastname: Sigloch
authors.affiliation: Institute of Molecular Medicine and Cell Research, University of Freiburg, D-79104 Freiburg, Germany.
authors.forename: Julia Daniela
authors.initials: JD
authors.lastname: Knopf
authors.affiliation: Institute of Molecular Medicine and Cell Research, University of Freiburg, D-79104 Freiburg, Germany.
authors.forename: Juliane
authors.initials: J
authors.lastname: Weißer
authors.affiliation: Institute of Molecular Medicine and Cell Research, University of Freiburg, D-79104 Freiburg, Germany; Faculty of Biology, University of Freiburg, D-79104 Freiburg, Germany; Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg, D-79104 Freiburg, Germany.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Gomez-Auli
authors.affiliation: Institute of Molecular Medicine and Cell Research, University of Freiburg, D-79104 Freiburg, Germany.
authors.forename: Martin Lothar
authors.initials: ML
authors.lastname: Biniossek
authors.affiliation: Institute of Molecular Medicine and Cell Research, University of Freiburg, D-79104 Freiburg, Germany.
authors.forename: Agnese
authors.initials: A
authors.lastname: Petrera
authors.affiliation: Institute of Molecular Medicine and Cell Research, University of Freiburg, D-79104 Freiburg, Germany; BIOSS Centre for Biological Signaling Studies, University of Freiburg, Freiburg, Germany. Electronic address: oliver.schilling@mol-med.uni-freiburg.de.
authors.forename: Oliver
authors.initials: O
authors.lastname: Schilling
mesh_terms: D000072197:ADAM10 Protein / Q000235:genetics / Q000378:metabolism; D053829:Amyloid Precursor Protein Secretases / Q000235:genetics / Q000378:metabolism; D016564:Amyloid beta-Protein Precursor / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D002401:Cathepsin B / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D009369:Neoplasms / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D040901:Proteomics / Q000379:methods; D034622:RNA Interference; D015398:Signal Transduction; D053719:Tandem Mass Spectrometry; D013997:Time Factors; D014162:Transfection
chemical_list: C545487:APP protein, human; D016564:Amyloid beta-Protein Precursor; D008565:Membrane Proteins; D053829:Amyloid Precursor Protein Secretases; C533912:CTSB protein, human; D002401:Cathepsin B; D000072197:ADAM10 Protein; C491236:ADAM10 protein, human
keywords: ADAM10; CTSB; LC-MS/MS; Non-catalytic function; Secretome