Design and Evaluation of a Polypeptide that Mimics the Integrin Binding Site for EDA Fibronectin to Block Profibrotic Cell Activity
Fibrosis is characterized by excessive production of disorganized collagen- and fibronectin-rich extracellular matrices (ECMs) and is driven by the persistence of myofibroblasts within tissues. A key protein contributing to myofibroblast differentiation is extra domain A fibronectin (EDA-FN). We sou...
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doaj-92f278b8b39b4fedba1a0666ed18b48e2021-02-05T00:03:45ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-02-01221575157510.3390/ijms22041575Design and Evaluation of a Polypeptide that Mimics the Integrin Binding Site for EDA Fibronectin to Block Profibrotic Cell ActivityLin Zhang0Hongyu Yan1Yifan Tai2Yueming Xue3Yongzhen Wei4Kai Wang5Qiang Zhao6Shufang Wang7Deling Kong8Adam C. Midgley9Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, Nankai University, Tianjin 300071, ChinaKey Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, Nankai University, Tianjin 300071, ChinaKey Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, Nankai University, Tianjin 300071, ChinaKey Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, Nankai University, Tianjin 300071, ChinaKey Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, Nankai University, Tianjin 300071, ChinaKey Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, Nankai University, Tianjin 300071, ChinaKey Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, Nankai University, Tianjin 300071, ChinaKey Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, Nankai University, Tianjin 300071, ChinaKey Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, Nankai University, Tianjin 300071, ChinaKey Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, Nankai University, Tianjin 300071, ChinaFibrosis is characterized by excessive production of disorganized collagen- and fibronectin-rich extracellular matrices (ECMs) and is driven by the persistence of myofibroblasts within tissues. A key protein contributing to myofibroblast differentiation is extra domain A fibronectin (EDA-FN). We sought to target and interfere with interactions between EDA-FN and its integrin receptors to effectively inhibit profibrotic activity and myofibroblast formation. Molecular docking was used to assist in the design of a blocking polypeptide (antifibrotic 38-amino-acid polypeptide, AF38Pep) for specific inhibition of EDA-FN associations with the fibroblast-expressed integrins α<sub>4</sub>β<sub>1</sub> and α<sub>4</sub>β<sub>7</sub>. Blocking peptides were designed and evaluated in silico before synthesis, confirmation of binding specificity, and evaluation in vitro. We identified the high-affinity EDA-FN C-C′ loop binding cleft within integrins α<sub>4</sub>β<sub>1</sub> and α<sub>4</sub>β<sub>7</sub>. The polypeptide with the highest predicted binding affinity, AF38Pep, was synthesized and could achieve specific binding to myofibroblast fibronectin-rich ECM and EDA-FN C-C′ loop peptides. AF38Pep demonstrated potent myofibroblast inhibitory activity at 10 µg/mL and was not cytotoxic. Treatment with AF38Pep prevented integrin α<sub>4</sub>β<sub>1</sub>-mediated focal adhesion kinase (FAK) activation and early signaling through extracellular-signal-regulated kinases 1 and 2 (ERK1/2), attenuated the expression of pro-matrix metalloproteinase 9 (MMP9) and pro-MMP2, and inhibited collagen synthesis and deposition. Immunocytochemistry staining revealed an inhibition of α-smooth muscle actin (α-SMA) incorporation into actin stress fibers and attenuated cell contraction. Increases in the expression of mRNA associated with fibrosis and downstream from integrin signaling were inhibited by treatment with AF38Pep. Our study suggested that AF38Pep could successfully interfere with EDA-FN C-C′ loop-specific integrin interactions and could act as an effective inhibitor of fibroblast of myofibroblast differentiation.https://www.mdpi.com/1422-0067/22/4/1575blocking peptidemolecular dockingfibrosisantifibroticEDA fibronectinintegrin α4β1 |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Lin Zhang Hongyu Yan Yifan Tai Yueming Xue Yongzhen Wei Kai Wang Qiang Zhao Shufang Wang Deling Kong Adam C. Midgley |
spellingShingle |
Lin Zhang Hongyu Yan Yifan Tai Yueming Xue Yongzhen Wei Kai Wang Qiang Zhao Shufang Wang Deling Kong Adam C. Midgley Design and Evaluation of a Polypeptide that Mimics the Integrin Binding Site for EDA Fibronectin to Block Profibrotic Cell Activity International Journal of Molecular Sciences blocking peptide molecular docking fibrosis antifibrotic EDA fibronectin integrin α4β1 |
author_facet |
Lin Zhang Hongyu Yan Yifan Tai Yueming Xue Yongzhen Wei Kai Wang Qiang Zhao Shufang Wang Deling Kong Adam C. Midgley |
author_sort |
Lin Zhang |
title |
Design and Evaluation of a Polypeptide that Mimics the Integrin Binding Site for EDA Fibronectin to Block Profibrotic Cell Activity |
title_short |
Design and Evaluation of a Polypeptide that Mimics the Integrin Binding Site for EDA Fibronectin to Block Profibrotic Cell Activity |
title_full |
Design and Evaluation of a Polypeptide that Mimics the Integrin Binding Site for EDA Fibronectin to Block Profibrotic Cell Activity |
title_fullStr |
Design and Evaluation of a Polypeptide that Mimics the Integrin Binding Site for EDA Fibronectin to Block Profibrotic Cell Activity |
title_full_unstemmed |
Design and Evaluation of a Polypeptide that Mimics the Integrin Binding Site for EDA Fibronectin to Block Profibrotic Cell Activity |
title_sort |
design and evaluation of a polypeptide that mimics the integrin binding site for eda fibronectin to block profibrotic cell activity |
publisher |
MDPI AG |
series |
International Journal of Molecular Sciences |
issn |
1661-6596 1422-0067 |
publishDate |
2021-02-01 |
description |
Fibrosis is characterized by excessive production of disorganized collagen- and fibronectin-rich extracellular matrices (ECMs) and is driven by the persistence of myofibroblasts within tissues. A key protein contributing to myofibroblast differentiation is extra domain A fibronectin (EDA-FN). We sought to target and interfere with interactions between EDA-FN and its integrin receptors to effectively inhibit profibrotic activity and myofibroblast formation. Molecular docking was used to assist in the design of a blocking polypeptide (antifibrotic 38-amino-acid polypeptide, AF38Pep) for specific inhibition of EDA-FN associations with the fibroblast-expressed integrins α<sub>4</sub>β<sub>1</sub> and α<sub>4</sub>β<sub>7</sub>. Blocking peptides were designed and evaluated in silico before synthesis, confirmation of binding specificity, and evaluation in vitro. We identified the high-affinity EDA-FN C-C′ loop binding cleft within integrins α<sub>4</sub>β<sub>1</sub> and α<sub>4</sub>β<sub>7</sub>. The polypeptide with the highest predicted binding affinity, AF38Pep, was synthesized and could achieve specific binding to myofibroblast fibronectin-rich ECM and EDA-FN C-C′ loop peptides. AF38Pep demonstrated potent myofibroblast inhibitory activity at 10 µg/mL and was not cytotoxic. Treatment with AF38Pep prevented integrin α<sub>4</sub>β<sub>1</sub>-mediated focal adhesion kinase (FAK) activation and early signaling through extracellular-signal-regulated kinases 1 and 2 (ERK1/2), attenuated the expression of pro-matrix metalloproteinase 9 (MMP9) and pro-MMP2, and inhibited collagen synthesis and deposition. Immunocytochemistry staining revealed an inhibition of α-smooth muscle actin (α-SMA) incorporation into actin stress fibers and attenuated cell contraction. Increases in the expression of mRNA associated with fibrosis and downstream from integrin signaling were inhibited by treatment with AF38Pep. Our study suggested that AF38Pep could successfully interfere with EDA-FN C-C′ loop-specific integrin interactions and could act as an effective inhibitor of fibroblast of myofibroblast differentiation. |
topic |
blocking peptide molecular docking fibrosis antifibrotic EDA fibronectin integrin α4β1 |
url |
https://www.mdpi.com/1422-0067/22/4/1575 |
work_keys_str_mv |
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