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Directed Blocking of TGF-? Receptor I Binding Site Using Tailored Peptide Segments to Inhibit its Signaling Pathway.

ABSTRACT: Background:TGF-? isoforms play crucial roles in diverse cellular processes. Therefore, targeting and inhibiting TGF-? signaling pathway provides a potential therapeutic opportunity. TGF-? isoforms bind and bring the receptors (T?RII and T?RI) together to form a signaling complex in an ordered manner. Objectives:Herein, an antagonistic variant of TGF-? (AnT?) has been designed and prepared to inhibit the formation of signaling complex and consequently its signaling pathway. This TGF-? homodimeric variant contains intact T?RII binding sites and blocked T?RI binding sites by substituting three peptide segments. So, AnT? could only bind to T?RII, but prevent binding and recruitment of T?RI to form a signaling complex. Materials and Methods:A reliable model of AnT? was built and re?ned using molecular dynamics (MD) simulation, followed by investigating the interactions of AnT? with the receptors using in silico docking studies. After expression of disulfide-linked AnT? in a SHuffle strain and purification of the protein using affinity chromatography, its biological activity was evaluated using Mink lung epithelial cells (Mvl Lu). Results:No meaningful significant changes in AnT? structure were observed when compared with the native protein. Based on the docking analysis, AnT? binds to T?RII similar to TGF-? and its binding to T?RI was diminished considerably which was consistent with our design purpose. Cell-based bioassay indicated that AnT? could modulate TGF-?-induced cell growth inhibition. Conclusions:Our analysis suggests that the antagonistic potency of AnT? can be used as an anti-TGF? signaling factor in the future perspectives.

SUBMITTER: Sepehri S

PROVIDER: S-EPMC7461711 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Directed Blocking of TGF-β Receptor I Binding Site Using Tailored Peptide Segments to Inhibit its Signaling Pathway.

Sepehri Sepideh S Arab S Shahriar SS Behmanesh Mehrdad M H Sajedi Reza R

Iranian journal of biotechnology 20200101 1

<h4>Background</h4>TGF-β isoforms play crucial roles in diverse cellular processes. Therefore, targeting and inhibiting TGF-β signaling pathway provides a potential therapeutic opportunity. TGF-β isoforms bind and bring the receptors (TβRII and TβRI) together to form a signaling complex in an ordered manner.<h4>Objectives</h4>Herein, an antagonistic variant of TGF-β (AnTβ) has been designed and prepared to inhibit the formation of signaling complex and consequently its signaling pathway. This TG ...[more]

PMID: 32884960

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Project description:Hydroxysafflor yellow A (HSYA) is an active ingredient of Carthamus tinctorius L.. This study aimed to evaluate the effects of HSYA on transforming growth factor-β1 (TGF-β1)-induced changes in proliferation, migration, differentiation, and extracellular matrix accumulation and degradation in human fetal lung fibroblasts (MRC-5), to explore the mechanisms whereby HSYA may alleviate pulmonary fibrosis. MRC-5 cells were incubated with various doses of HSYA and/or the TGF-β receptor type I kinase inhibitor SB431542 and then stimulated with TGF-β1. Cell proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium inner salt assay. Cell migration was detected by wound-healing assay. Protein levels of α-smooth muscle actin (α-SMA), collagen I α 1 (COL1A1), and fibronectin (FN) were measured by immunofluorescence. Protein levels of matrix metalloproteinase-2, tissue inhibitor of matrix metalloproteinase-1, tissue inhibitor of matrix metalloproteinase-2, TGF-β type II receptor (TβRII), and TGF-β type I receptor were detected by western blotting. TβRII knockdown with siRNA interfered with the inhibitory effect of HSYA on α-SMA, COL1A1, and FN expression, and TGF-β1-induced Sma and Mad protein (Smad), and extracellular signal-regulated kinase/mitogen-activated protein kinase signaling pathway activation. The antagonistic effect of HSYA on the binding of fluorescein isothiocyanate-TGF-β1 to MRC-5 cell cytoplasmic receptors was measured by flow cytometry. HSYA significantly suppressed TGF-β1-induced cell proliferation and migration. HSYA could antagonize the binding of FITC-TGF-β1 to MRC-5 cell cytoplasmic receptors. Also HSYA inhibited TGF-β1-activated cell expression of α-SMA, COL1A1, and FN and phosphorylation level of Smad2, Smad3, and ERK by targeting TβRII in MRC-5 cells. These findings suggest that TβRII might be the target responsible for the inhibitory effects of HSYA on TGF-β1-induced pathological changes in pulmonary fibrosis.

Dataset Information

Directed Blocking of TGF-? Receptor I Binding Site Using Tailored Peptide Segments to Inhibit its Signaling Pathway.

Publications

Directed Blocking of TGF-β Receptor I Binding Site Using Tailored Peptide Segments to Inhibit its Signaling Pathway.

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