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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
- Source :
- Mol Cell Proteomics
- Publication Year :
- 2018
-
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.
- Subjects :
- Proteomics
Proteome
Quantitative proteomics
Biochemistry
Analytical Chemistry
Substrate Specificity
Thrombospondin 1
03 medical and health sciences
Matrix Metalloproteinase 10
Stable isotope labeling by amino acids in cell culture
Cell Line, Tumor
KLK7
medicine
Chymotrypsin
Humans
Amino Acid Sequence
Cell adhesion
Molecular Biology
030304 developmental biology
Ovarian Neoplasms
0303 health sciences
Chemistry
Hydrolysis
Research
030302 biochemistry & molecular biology
Cancer
Terminal amine isotopic labeling of substrates
medicine.disease
Cell biology
Enzyme Activation
Gene Ontology
Culture Media, Conditioned
Proteolysis
Female
Kallikreins
Benjamin Cravatt III
Peptides
Subjects
Details
- ISSN :
- 15359484
- Volume :
- 18
- Issue :
- 5
- Database :
- OpenAIRE
- Journal :
- Molecularcellular proteomics : MCP
- Accession number :
- edsair.doi.dedup.....37e04e2635809a74bacfc64c6689aa88