GST pull-down联合质谱技术鉴定与calponin-3相互作用的蛋白质

Identification of Calponin-3-interacting Proteins by Using GST Pull-down Combined With Mass Spectrometry Techniques

人(Homo sapiens)源calponin-3蛋白由CNN3基因编码,是一种与应激纤维相关肌动蛋白结合的蛋白质。研究表明其参与伤口愈合、细胞收缩、生物发育、细胞融合以及癌细胞侵袭转移等生物过程。鉴于calponin-3生物学功能的多样性,了解其在细胞中的相互作用信息,将有助于深入探讨其执行生物功能的分子机制。本研究通过构建人源calponin-3的重组原核表达质粒pGEX-4T-CNN3,优化重组蛋白的原核表达诱导条件,获得GST-calponin-3融合蛋白。并通过GST pull-down实验“垂钓”人胚肾上皮细胞293t中直接或间接与calponin-3结合的靶蛋白。经由LC-MS/MS质谱分析,共鉴定到11个潜在的与calponin-3相互作用靶蛋白。进而对这些蛋白质进行GO(gene ontology)功能分析,发现其主要参与细胞黏附、催化活性、结构分子活性和翻译调控等生物过程。其中,靶蛋白Actin参与了阿尔茨海默病、钙黏着蛋白信号通路、Rho GTPase调节细胞骨架、亨廷顿病、趋化因子和细胞因子信号转导途径介导炎症以及Wnt信号通路等。这些结果表明calponin-3可能通过这些途径发挥重要生物学功能。因此,本研究从相互作用蛋白质的角度,为深入阐明calponin-3的生物学功能提供实验基础。

Human(Homo sapiens)calponin-3,encoded by CNN3,is an actin-binding protein that associates with stress fibers.Studies have shown that it is involved in a range of biological processes,including wound healing,cell contractility,biological development,cell fusion and cancer cell invasion.Considering the diversity of biological functions of calponin-3,understanding the interactions information of calponin-3 will help to explore its molecular mechanisms for performing biological functions.The pGEX-4 T-CNN3,a recombinant prokaryotic expression plasmid for expression of human calponin-3,was successfully constructed,and the induced conditions for expressing fusion protein were optimized.Subsequently,the purified GST-calponin-3 fusion protein was used to screen of its target proteins from embryonic kidney epithelial cells 293 t of human by GST pull-down assay and 11 potential interacting proteins were identified.These target proteins were further analyzed with GO(gene ontology)functional tool and the result revealed that these proteins are mainly involved in cell adhesion,catalytic activity,structural molecular activity,and translation management activities.Pathway analysis further revealed that Actin(a calponin-3 interacting protein)is involved in Alzheimer′s disease,cadherin signaling pathway,Rho GTPase regulation of cytoskeleton,Huntington′s disease,chemokine and cytokine signaling pathways-mediated inflammation,Wnt signaling pathways and other pathways.These data suggest that calponin-3 may play important roles in biological function through these pathways mentioned above,and this study will provide a basis for further elucidation of biological functions of calponin-3.