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Integration of Two In-depth Quantitative Proteomics Approaches Determines the Kallikrein-related Peptidase 7 (KLK7) Degradome in Ovarian Cancer Cell Secretome.
Mol Cell Proteomics 2019; 18(5):818-836MC

Abstract

Kallikrein-related peptidase 7 (KLK7) is a serine peptidase that is over expressed in ovarian cancer. In vitro functional analyses have suggested KLK7 to play a cancer progressive role, although monitoring of KLK7 expression has suggested a contradictory protective role for KLK7 in ovarian cancer patients. In order to help delineate its mechanism of action and thereby the functional roles, information on its substrate repertoire is crucial. Therefore, in this study a quantitative proteomics approach-PROtein TOpography and Migration Analysis Platform (PROTOMAP)-coupled with SILAC was used for in-depth analysis of putative KLK7 substrates from a representative ovarian cancer cell line, SKOV-3, secreted proteins. The Terminal Amine Isotopic Labeling of Substrates (TAILS) approach was used to determine the exact cleavage sites and to validate qPROTOMAP-identified putative substrates. By employing these two technically divergent approaches, exact cleavage sites on 16 novel putative substrates and two established substrates, matrix metalloprotease (MMP) 2 and insulin growth factor binding protein 3 (IGFBP3), were identified in the SKOV-3 secretome. Eight of these substrates were also identified on TAILS analysis of another ovarian cancer cell (OVMZ-6) secretome, with a further seven OVMZ-6 substrates common to the SKOV-3 qPROTOMAP profile. Identified substrates were significantly associated with the common processes of cell adhesion, extracellular matrix remodeling and cell migration according to the gene ontology (GO) biological process analysis. Biochemical validation supports a role for KLK7 in directly activating pro-MMP10, hydrolysis of IGFBP6 and cleavage of thrombospondin 1 with generation of a potentially bioactive N-terminal fragment. Overall, this study constitutes the most comprehensive analysis of the putative KLK7 degradome in any cancer to date, thereby opening new avenues for KLK7 research.

Authors+Show Affiliations

From the ‡Queensland University of Technology (QUT), Institute of Health and Biomedical Innovation (IHBI) and School of Biomedical Sciences at the Translational Research Institute, 37 Kent Street, Woolloongabba, Queensland, 4102, Australia; munasinghage.silva@nih.gov.From the ‡Queensland University of Technology (QUT), Institute of Health and Biomedical Innovation (IHBI) and School of Biomedical Sciences at the Translational Research Institute, 37 Kent Street, Woolloongabba, Queensland, 4102, Australia.§Protein Discovery Centre, QIMR Berghofer Medical Research Institute, 300 Herston Road, Herston, Queensland, 4006, Australia.§Protein Discovery Centre, QIMR Berghofer Medical Research Institute, 300 Herston Road, Herston, Queensland, 4006, Australia.From the ‡Queensland University of Technology (QUT), Institute of Health and Biomedical Innovation (IHBI) and School of Biomedical Sciences at the Translational Research Institute, 37 Kent Street, Woolloongabba, Queensland, 4102, Australia.From the ‡Queensland University of Technology (QUT), Institute of Health and Biomedical Innovation (IHBI) and School of Biomedical Sciences at the Translational Research Institute, 37 Kent Street, Woolloongabba, Queensland, 4102, Australia.§Protein Discovery Centre, QIMR Berghofer Medical Research Institute, 300 Herston Road, Herston, Queensland, 4006, Australia.‖Klinische Forschergruppe der Frauenklinik, Klinikum Rechts der Isar, TU München, Munich, Germany.¶Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Victoria, Australia 3800.§Protein Discovery Centre, QIMR Berghofer Medical Research Institute, 300 Herston Road, Herston, Queensland, 4006, Australia.From the ‡Queensland University of Technology (QUT), Institute of Health and Biomedical Innovation (IHBI) and School of Biomedical Sciences at the Translational Research Institute, 37 Kent Street, Woolloongabba, Queensland, 4102, Australia; j.clements@qut.edu.au.

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

30705123

Citation

Silva, Lakmali Munasinghage, et al. "Integration of Two In-depth Quantitative Proteomics Approaches Determines the Kallikrein-related Peptidase 7 (KLK7) Degradome in Ovarian Cancer Cell Secretome." Molecular & Cellular Proteomics : MCP, vol. 18, no. 5, 2019, pp. 818-836.
Silva LM, Kryza T, Stoll T, et al. Integration of Two In-depth Quantitative Proteomics Approaches Determines the Kallikrein-related Peptidase 7 (KLK7) Degradome in Ovarian Cancer Cell Secretome. Mol Cell Proteomics. 2019;18(5):818-836.
Silva, L. M., Kryza, T., Stoll, T., Hoogland, C., Dong, Y., Stephens, C. R., ... Clements, J. A. (2019). Integration of Two In-depth Quantitative Proteomics Approaches Determines the Kallikrein-related Peptidase 7 (KLK7) Degradome in Ovarian Cancer Cell Secretome. Molecular & Cellular Proteomics : MCP, 18(5), pp. 818-836. doi:10.1074/mcp.RA118.001304.
Silva LM, et al. Integration of Two In-depth Quantitative Proteomics Approaches Determines the Kallikrein-related Peptidase 7 (KLK7) Degradome in Ovarian Cancer Cell Secretome. Mol Cell Proteomics. 2019;18(5):818-836. PubMed PMID: 30705123.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Integration of Two In-depth Quantitative Proteomics Approaches Determines the Kallikrein-related Peptidase 7 (KLK7) Degradome in Ovarian Cancer Cell Secretome. AU - Silva,Lakmali Munasinghage, AU - Kryza,Thomas, AU - Stoll,Thomas, AU - Hoogland,Christine, AU - Dong,Ying, AU - Stephens,Carson Ryan, AU - Hastie,Marcus Lachlan, AU - Magdolen,Viktor, AU - Kleifeld,Oded, AU - Gorman,Jeffrey John, AU - Clements,Judith Ann, Y1 - 2019/01/31/ PY - 2018/12/31/received PY - 2020/05/01/pmc-release PY - 2019/2/2/pubmed PY - 2020/1/17/medline PY - 2019/2/2/entrez KW - Degradomics* KW - Extracellular matrix* KW - Kallikrein-related Peptidases KW - Ovarian cancer KW - Proteases* KW - Secretome KW - Substrate identification SP - 818 EP - 836 JF - Molecular & cellular proteomics : MCP JO - Mol. Cell Proteomics VL - 18 IS - 5 N2 - Kallikrein-related peptidase 7 (KLK7) is a serine peptidase that is over expressed in ovarian cancer. In vitro functional analyses have suggested KLK7 to play a cancer progressive role, although monitoring of KLK7 expression has suggested a contradictory protective role for KLK7 in ovarian cancer patients. In order to help delineate its mechanism of action and thereby the functional roles, information on its substrate repertoire is crucial. Therefore, in this study a quantitative proteomics approach-PROtein TOpography and Migration Analysis Platform (PROTOMAP)-coupled with SILAC was used for in-depth analysis of putative KLK7 substrates from a representative ovarian cancer cell line, SKOV-3, secreted proteins. The Terminal Amine Isotopic Labeling of Substrates (TAILS) approach was used to determine the exact cleavage sites and to validate qPROTOMAP-identified putative substrates. By employing these two technically divergent approaches, exact cleavage sites on 16 novel putative substrates and two established substrates, matrix metalloprotease (MMP) 2 and insulin growth factor binding protein 3 (IGFBP3), were identified in the SKOV-3 secretome. Eight of these substrates were also identified on TAILS analysis of another ovarian cancer cell (OVMZ-6) secretome, with a further seven OVMZ-6 substrates common to the SKOV-3 qPROTOMAP profile. Identified substrates were significantly associated with the common processes of cell adhesion, extracellular matrix remodeling and cell migration according to the gene ontology (GO) biological process analysis. Biochemical validation supports a role for KLK7 in directly activating pro-MMP10, hydrolysis of IGFBP6 and cleavage of thrombospondin 1 with generation of a potentially bioactive N-terminal fragment. Overall, this study constitutes the most comprehensive analysis of the putative KLK7 degradome in any cancer to date, thereby opening new avenues for KLK7 research. SN - 1535-9484 UR - https://www.unboundmedicine.com/medline/citation/30705123/Integration_of_Two_In_depth_Quantitative_Proteomics_Approaches_Determines_the_Kallikrein_related_Peptidase_7__KLK7__Degradome_in_Ovarian_Cancer_Cell_Secretome_ L2 - http://www.mcponline.org/cgi/pmidlookup?view=long&pmid=30705123 DB - PRIME DP - Unbound Medicine ER -