Development of a Chimeric Protein BiPPB-mIFNγ-tTβRII for Improving the Anti-Fibrotic Activity in Vivo by Targeting Fibrotic Liver and Dual Inhibiting the TGF-β1/Smad Signaling Pathway

IF 1.9 4区生物学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY

The Protein Journal Pub Date : 2023-09-10 DOI:10.1007/s10930-023-10147-z

Yixin Dong, Xiaohua Wang, Liming Xu, Xin Li, Haibing Dai, Xu Mao, Yanhui Chu, Xiaohuan Yuan, Haifeng Liu

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Abstract

Excessive production of transforming growth factor β1 (TGF-β1) in activated hepatic stellate cells (aHSCs) promotes liver fibrosis by activating the TGF-β1/Smad signaling pathway. Thus, specifically inhibiting the pro-fibrotic activity of TGF-β1 in aHSCs is an ideal strategy for treating liver fibrosis. Overexpression of platelet-derived growth factor β receptor (PDGFβR) has been demonstrated on the surface of aHSCs relative to normal cells in liver fibrosis. Interferon-gamma peptidomimetic (mIFNγ) and truncated TGF-β receptor type II (tTβRII) inhibit the TGF-β1/Smad signaling pathway by different mechanisms. In this study, we designed a chimeric protein by the conjugation of (1) mIFNγ and tTβRII coupled via plasma protease-cleavable linker sequences (FNPKTP) to (2) PDGFβR-recognizing peptide (BiPPB), namely BiPPB-mIFNγ-tTβRII. This novel protein BiPPB-mIFNγ-tTβRII was effectively prepared using Escherichia coli expression system. The active components BiPPB-mIFNγ and tTβRII were slowly released from BiPPB-mIFNγ-tTβRII by hydrolysis using the plasma protease thrombin in vitro. Moreover, BiPPB-mIFNγ-tTβRII highly targeted to fibrotic liver tissues, markedly ameliorated liver morphology and fibrotic responses in chronic liver fibrosis mice by both inhibiting the phosphorylation of Smad2/3 and inducing the expression of Smad7. Meanwhile, BiPPB-mIFNγ-tTβRII markedly reduced the deposition of collagen fibrils and expression of fibrosis-related proteins in acute liver fibrosis mice. Furthermore, BiPPB-mIFNγ-tTβRII showed a good safety performance in both liver fibrosis mice. Taken together, BiPPB-mIFNγ-tTβRII improved the in vivo anti-liver fibrotic activity due to its high fibrotic liver-targeting potential and the dual inhibition of the TGF-β1/Smad signaling pathway, which may be a potential candidate for targeting therapy on liver fibrosis.

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嵌合蛋白BiPPB-mIFNγ-tTβRII的开发通过靶向纤维化肝和双重抑制TGF-β1/Smad信号通路来提高体内抗纤维化活性。

活化的肝星状细胞（aHSC）过度产生转化生长因子β1（TGF-β1）通过激活TGF-β1/Smad信号通路促进肝纤维化。因此，特异性抑制aHSC中TGF-β1的促纤维化活性是治疗肝纤维化的理想策略。相对于肝纤维化中的正常细胞，血小板衍生生长因子β受体（PDGFβR）在aHSC表面的过度表达已得到证实。干扰素γ肽模拟物（mIFNγ）和截短的TGF-β受体II型（tTβRII）通过不同机制抑制TGF-β1/Smad信号通路。在本研究中，我们通过将（1）mIFNγ和tTβRII通过血浆蛋白酶可裂解接头序列（FNPKTP）偶联到（2）PDGFβR-识别肽（BiPPB），即BiPPB-mIFNβ-tTβRII，设计了一种嵌合蛋白。利用大肠杆菌表达系统有效地制备了新型蛋白BiPPB-mIFNγ-tTβRII。在体外用血浆蛋白酶凝血酶水解BiPPB-mIFNγ-tTβRII，从中缓慢释放出活性成分BiPPB-mPIFNγ和tTβRII。此外，BiPPB-mIFNγ-tTβRII高度靶向纤维化肝组织，通过抑制Smad2/3的磷酸化和诱导Smad7的表达，显著改善了慢性肝纤维化小鼠的肝脏形态和纤维化反应。同时，BiPPB-mIFNγ-tTβRII显著降低急性肝纤维化小鼠胶原原纤维的沉积和纤维化相关蛋白的表达。此外，BiPPB-mIFNγ-tTβRII在两种肝纤维化小鼠中都表现出良好的安全性。总之，BiPPB-mIFNγ-tTβRII由于其高纤维化肝靶向潜力和对TGF-β1/Smad信号通路的双重抑制而提高了体内抗肝纤维化活性，这可能是肝纤维化靶向治疗的潜在候选药物。

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来源期刊

The Protein Journal 生物-生化与分子生物学

CiteScore

5.20

自引率

0.00%

发文量

审稿时长

12 months

期刊介绍： The Protein Journal (formerly the Journal of Protein Chemistry) publishes original research work on all aspects of proteins and peptides. These include studies concerned with covalent or three-dimensional structure determination (X-ray, NMR, cryoEM, EPR/ESR, optical methods, etc.), computational aspects of protein structure and function, protein folding and misfolding, assembly, genetics, evolution, proteomics, molecular biology, protein engineering, protein nanotechnology, protein purification and analysis and peptide synthesis, as well as the elucidation and interpretation of the molecular bases of biological activities of proteins and peptides. We accept original research papers, reviews, mini-reviews, hypotheses, opinion papers, and letters to the editor.