A KLK4 proteinase substrate capture approach to antagonize PAR1
Abstract Proteinase-activated receptor-1 (PAR1), triggered by thrombin and other serine proteinases such as tissue kallikrein-4 (KLK4), is a key driver of inflammation, tumor invasiveness and tumor metastasis. The PAR1 transmembrane G-protein-coupled receptor therefore represents an attractive targe...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2021-08-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-021-95666-4 |
| _version_ | 1818856089053036544 |
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| author | Eitan Rabinovitch Koishiro Mihara Amiram Sananes Marianna Zaretsky Michael Heyne Julia Shifman Amir Aharoni Morley D. Hollenberg Niv Papo |
| author_facet | Eitan Rabinovitch Koishiro Mihara Amiram Sananes Marianna Zaretsky Michael Heyne Julia Shifman Amir Aharoni Morley D. Hollenberg Niv Papo |
| author_sort | Eitan Rabinovitch |
| collection | DOAJ |
| description | Abstract Proteinase-activated receptor-1 (PAR1), triggered by thrombin and other serine proteinases such as tissue kallikrein-4 (KLK4), is a key driver of inflammation, tumor invasiveness and tumor metastasis. The PAR1 transmembrane G-protein-coupled receptor therefore represents an attractive target for therapeutic inhibitors. We thus used a computational design to develop a new PAR1 antagonist, namely, a catalytically inactive human KLK4 that acts as a proteinase substrate-capture reagent, preventing receptor cleavage (and hence activation) by binding to and occluding the extracellular R41-S42 canonical PAR1 proteolytic activation site. On the basis of in silico site-saturation mutagenesis, we then generated KLK4S207A,L185D, a first-of-a-kind ‘decoy’ PAR1 inhibitor, by mutating the S207A and L185D residues in wild-type KLK4, which strongly binds to PAR1. KLK4S207A,L185D markedly inhibited PAR1 cleavage, and PAR1-mediated MAPK/ERK activation as well as the migration and invasiveness of melanoma cells. This ‘substrate-capturing’ KLK4 variant, engineered to bind to PAR1, illustrates proof of principle for the utility of a KLK4 ‘proteinase substrate capture’ approach to regulate proteinase-mediated PAR1 signaling. |
| first_indexed | 2024-12-19T08:18:57Z |
| format | Article |
| id | doaj.art-1a0212dd91364948b979eba1d92f904e |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-12-19T08:18:57Z |
| publishDate | 2021-08-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-1a0212dd91364948b979eba1d92f904e2022-12-21T20:29:27ZengNature PortfolioScientific Reports2045-23222021-08-0111111310.1038/s41598-021-95666-4A KLK4 proteinase substrate capture approach to antagonize PAR1Eitan Rabinovitch0Koishiro Mihara1Amiram Sananes2Marianna Zaretsky3Michael Heyne4Julia Shifman5Amir Aharoni6Morley D. Hollenberg7Niv Papo8Avram and Stella Goldstein-Goren Department of Biotechnology Engineering, National Institute of Biotechnology in the Negev, Ben-Gurion University of the NegevDepartment of Physiology and Pharmacology, Cumming School of Medicine, University of CalgaryAvram and Stella Goldstein-Goren Department of Biotechnology Engineering, National Institute of Biotechnology in the Negev, Ben-Gurion University of the NegevDepartment of Life Sciences, National Institute of Biotechnology in the Negev, Ben-Gurion University of the NegevAvram and Stella Goldstein-Goren Department of Biotechnology Engineering, National Institute of Biotechnology in the Negev, Ben-Gurion University of the NegevDepartment of Biological Chemistry, The Hebrew University of JerusalemDepartment of Life Sciences, National Institute of Biotechnology in the Negev, Ben-Gurion University of the NegevDepartment of Physiology and Pharmacology, Cumming School of Medicine, University of CalgaryAvram and Stella Goldstein-Goren Department of Biotechnology Engineering, National Institute of Biotechnology in the Negev, Ben-Gurion University of the NegevAbstract Proteinase-activated receptor-1 (PAR1), triggered by thrombin and other serine proteinases such as tissue kallikrein-4 (KLK4), is a key driver of inflammation, tumor invasiveness and tumor metastasis. The PAR1 transmembrane G-protein-coupled receptor therefore represents an attractive target for therapeutic inhibitors. We thus used a computational design to develop a new PAR1 antagonist, namely, a catalytically inactive human KLK4 that acts as a proteinase substrate-capture reagent, preventing receptor cleavage (and hence activation) by binding to and occluding the extracellular R41-S42 canonical PAR1 proteolytic activation site. On the basis of in silico site-saturation mutagenesis, we then generated KLK4S207A,L185D, a first-of-a-kind ‘decoy’ PAR1 inhibitor, by mutating the S207A and L185D residues in wild-type KLK4, which strongly binds to PAR1. KLK4S207A,L185D markedly inhibited PAR1 cleavage, and PAR1-mediated MAPK/ERK activation as well as the migration and invasiveness of melanoma cells. This ‘substrate-capturing’ KLK4 variant, engineered to bind to PAR1, illustrates proof of principle for the utility of a KLK4 ‘proteinase substrate capture’ approach to regulate proteinase-mediated PAR1 signaling.https://doi.org/10.1038/s41598-021-95666-4 |
| spellingShingle | Eitan Rabinovitch Koishiro Mihara Amiram Sananes Marianna Zaretsky Michael Heyne Julia Shifman Amir Aharoni Morley D. Hollenberg Niv Papo A KLK4 proteinase substrate capture approach to antagonize PAR1 Scientific Reports |
| title | A KLK4 proteinase substrate capture approach to antagonize PAR1 |
| title_full | A KLK4 proteinase substrate capture approach to antagonize PAR1 |
| title_fullStr | A KLK4 proteinase substrate capture approach to antagonize PAR1 |
| title_full_unstemmed | A KLK4 proteinase substrate capture approach to antagonize PAR1 |
| title_short | A KLK4 proteinase substrate capture approach to antagonize PAR1 |
| title_sort | klk4 proteinase substrate capture approach to antagonize par1 |
| url | https://doi.org/10.1038/s41598-021-95666-4 |
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