Identification of novel natural substrates of fibroblast activation protein-alpha by differential degradomics and proteomics

Zhang, HE; Hamson, EJ; Koczorowska, MM; Tholen, S; Chowdhury, S; Bailey, CG; Lay, AJ; Twigg, SM; Lee, Q; Roediger, B; Biniossek, ML; O'Rourke, MB; McCaughan, GW; Keane, FM; Schilling, O; Gorrell, MD

Identification of novel natural substrates of fibroblast activation protein-alpha by differential degradomics and proteomics

Zhang, HE Hamson, EJ Koczorowska, MM Tholen, S Chowdhury, S Bailey, CG Lay, AJ Twigg, SM Lee, Q Roediger, B Biniossek, ML O'Rourke, MB

McCaughan, GW Keane, FM Schilling, O Gorrell, MD

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Publication Type:: Journal Article
Citation:: Molecular and Cellular Proteomics, 2019, 18 (1), pp. 65 - 85
Issue Date:: 2019-01-01

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Field	Value	Language
dc.contributor.author	Zhang, HE	en_US
dc.contributor.author	Hamson, EJ	en_US
dc.contributor.author	Koczorowska, MM	en_US
dc.contributor.author	Tholen, S	en_US
dc.contributor.author	Chowdhury, S	en_US
dc.contributor.author	Bailey, CG	en_US
dc.contributor.author	Lay, AJ	en_US
dc.contributor.author	Twigg, SM	en_US
dc.contributor.author	Lee, Q	en_US
dc.contributor.author	Roediger, B	en_US
dc.contributor.author	Biniossek, ML	en_US
dc.contributor.author	O'Rourke, MB https://orcid.org/0000-0002-0552-2962	en_US
dc.contributor.author	McCaughan, GW	en_US
dc.contributor.author	Keane, FM	en_US
dc.contributor.author	Schilling, O	en_US
dc.contributor.author	Gorrell, MD	en_US
dc.date.issued	2019-01-01	en_US
dc.identifier.citation	Molecular and Cellular Proteomics, 2019, 18 (1), pp. 65 - 85	en_US
dc.identifier.issn	1535-9476	en_US
dc.identifier.uri	http://hdl.handle.net/10453/138066
dc.description.abstract	© 2019 Zhang et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc. Fibroblast activation protein-alpha (FAP) is a cell-surface transmembrane-anchored dimeric protease. This unique, constitutively active serine protease has both dipeptidyl aminopeptidase and endopeptidase activities and can hydrolyze the post-proline bond. FAP expression is very low in adult organs but is upregulated by activated fibroblasts in sites of tissue remodeling, including fibrosis, atherosclerosis, arthritis and tumors. To identify the endogenous substrates of FAP, we immortalized primary mouse embryonic fibroblasts (MEFs) from FAP gene knockout embryos and then stably transduced them to express either enzymatically active or inactive FAP. The MEF secretomes were then analyzed using degradomic and proteomic techniques. Terminal amine isotopic labeling of substrates (TAILS)-based degradomics identified cleavage sites in collagens, many other extracellular matrix (ECM) and associated proteins, and lysyl oxidase-like-1, CXCL-5, CSF-1, and C1qT6, that were confirmed in vitro. In addition, differential metabolic labeling coupled with quantitative proteomic analysis also implicated FAP in ECM-cell interactions, as well as with coagulation, metabolism and wound healing associated proteins. Plasma from FAP-deficient mice exhibited slower than wild-type clotting times. This study provides a significant expansion of the substrate repertoire of FAP and provides insight into the physiological and potential pathological roles of this enigmatic protease.	en_US
dc.relation.ispartof	Molecular and Cellular Proteomics	en_US
dc.relation.isbasedon	10.1074/mcp.RA118.001046	en_US
dc.subject.classification	Biochemistry & Molecular Biology	en_US
dc.subject.mesh	Cell Line	en_US
dc.subject.mesh	Fibroblasts	en_US
dc.subject.mesh	Animals	en_US
dc.subject.mesh	Humans	en_US
dc.subject.mesh	Mice	en_US
dc.subject.mesh	Gelatinases	en_US
dc.subject.mesh	Serine Endopeptidases	en_US
dc.subject.mesh	Amino Acid Oxidoreductases	en_US
dc.subject.mesh	Macrophage Colony-Stimulating Factor	en_US
dc.subject.mesh	Membrane Proteins	en_US
dc.subject.mesh	Cell Culture Techniques	en_US
dc.subject.mesh	Proteomics	en_US
dc.subject.mesh	Substrate Specificity	en_US
dc.subject.mesh	Chemokine CXCL5	en_US
dc.subject.mesh	Adipokines	en_US
dc.subject.mesh	Gene Knockout Techniques	en_US
dc.subject.mesh	Protein Interaction Maps	en_US
dc.subject.mesh	Proteolysis	en_US
dc.title	Identification of novel natural substrates of fibroblast activation protein-alpha by differential degradomics and proteomics	en_US
dc.type	Journal Article
utslib.citation.volume	1	en_US
utslib.citation.volume	18	en_US
pubs.embargo.period	Not known	en_US
pubs.organisational-group	/University of Technology Sydney
pubs.organisational-group	/University of Technology Sydney/Faculty of Science
utslib.copyright.status	open_access
pubs.issue	1	en_US
pubs.publication-status	Published	en_US
pubs.volume	18	en_US

Abstract:

© 2019 Zhang et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc. Fibroblast activation protein-alpha (FAP) is a cell-surface transmembrane-anchored dimeric protease. This unique, constitutively active serine protease has both dipeptidyl aminopeptidase and endopeptidase activities and can hydrolyze the post-proline bond. FAP expression is very low in adult organs but is upregulated by activated fibroblasts in sites of tissue remodeling, including fibrosis, atherosclerosis, arthritis and tumors. To identify the endogenous substrates of FAP, we immortalized primary mouse embryonic fibroblasts (MEFs) from FAP gene knockout embryos and then stably transduced them to express either enzymatically active or inactive FAP. The MEF secretomes were then analyzed using degradomic and proteomic techniques. Terminal amine isotopic labeling of substrates (TAILS)-based degradomics identified cleavage sites in collagens, many other extracellular matrix (ECM) and associated proteins, and lysyl oxidase-like-1, CXCL-5, CSF-1, and C1qT6, that were confirmed in vitro. In addition, differential metabolic labeling coupled with quantitative proteomic analysis also implicated FAP in ECM-cell interactions, as well as with coagulation, metabolism and wound healing associated proteins. Plasma from FAP-deficient mice exhibited slower than wild-type clotting times. This study provides a significant expansion of the substrate repertoire of FAP and provides insight into the physiological and potential pathological roles of this enigmatic protease.

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http://hdl.handle.net/10453/138066