MMP26与GRP78在体内体外的相互作用

发布时间：2018-05-04 22:02

本文选题：基质金属蛋白酶26 + 葡萄糖调节蛋白78　；参考：《吉林大学》2017年硕士论文

【摘要】：基质金属蛋白酶家族(MMPs)是一类锌离子(Zn2+)和钙离子(Ca2+)依赖的肽链内切酶。MMPs通过降解细胞外基质(ECM)参与许多生理过程,包括生殖、发育、形态发生和组织重塑以及关节炎、心血管疾病和癌症转移在内的病理情况。基质金属蛋白酶26(MMP-26/endometase/matrilysin-2)是公认的乳腺癌、前列腺癌和其他上皮来源的癌症的生物标志物。MMP-26含有261个氨基酸,主要在胎盘和子宫中表达。MMP-26可以降解多种蛋白质,例如IV型胶原蛋白、纤连蛋白、纤维蛋白原、玻连蛋白、变性胶原蛋白类型I-IV、胰岛素样生长因子。MMP-26在肿瘤细胞中的表达及其广泛的蛋白降解活性的研究,表明MMP-26在肿瘤的发生和发展过程中发挥重要作用。通过免疫学和蛋白质组学等方法发现在敲除MMP-26基因的细胞内热休克蛋白90、葡萄糖调节蛋白78(GRP78)、膜联蛋白V、原肌球蛋白和过氧氧化还原酶II等蛋白表达量明显上调,而α-微管蛋白、半胱氨酸蛋白酶抑制剂SA-III、β-肌动蛋白等蛋白表达量下调。这一现象为我们研究GRP78与MMP-26在细胞内的相互作用提供依据。葡萄糖调节蛋白78(GRP78)又称为免疫球蛋白重链结合蛋(Immunoglobulin heavy chain binding protein,Bip)属于热休克蛋白70(Heat shock protein 70)家族中的一员。GRP78作为分子伴侣,主要功能是参与新合成蛋白质的跨膜迁移以及蛋白质的成熟、折叠和转运,抑制内质网中新合成蛋白质的聚集、调节内质网中钙离子的平衡等。GRP78在正常细胞中低表达或不表达,而在多种恶性肿瘤细胞,如乳腺癌、前列腺癌、卵巢癌、肾癌、胃癌和肺癌等癌症中呈高表达,提示我们GRP78的表达与肿瘤的发生和发展有重要的联系。同时研究发现:过表达GRP78的肿瘤组织对化疗药物有一定程度的耐药作用,而敲除GRP78基因后会明显降低肿瘤细胞对化疗药物的耐药性,因此抑制其基因表达或抑制其功能可作为肿瘤治疗的一种方案。这提示我们可以以GRP78为靶点进行肿瘤治疗,为研究MMP-26与GRP78在体内体外的相互作用提供依据。首先,我们构建体外重组表达GRP78蛋白的重组基因p GEX-4T-1-GRP78,并将其入表达菌BL21(DE3)获得大量可溶表达的GRP78蛋白,并利用GST柱纯化获得GRP78-GST融合蛋白;同时体外重组表达获得cat MMP-26蛋白,因其以包涵体形式表达,通过透析复性恢复其天然构象和生物活性,并通过明胶酶谱法和荧光酶标仪检测复性后cat MMP-26蛋白的活性。随后通过免疫荧光化学实验,荧光显微镜观察发现,细胞内MMP-26与GRP78共同定位于内质网,为研究MMP-26蛋白与GRP78蛋白的相互作用奠定基础;同时利用GST pull down试验研究体外重组表达的GRP78-GFP融合蛋白对MMP-26蛋白的捕获作用证明MMP-26与GRP78在体外的相互作用;同时模拟人体温度37℃,通过免疫印迹试验分析发现体外MMP-26对GRP78蛋白有降解作用;利用GFP亲和层析纯化实验分离获得细胞内表达的MMP-26蛋白,同时获得细胞内与MMP-26有相互作用的GRP78蛋白,证明在细胞内MMP-26与GRP78之间存在相互作用;同时通过培养MMP-26和sh RNA MMP-26稳定表达的Hela细胞,证明细胞内MMP-26对GRP78的蛋白降解作用。综上所述,本论文证明了细胞内MMP-26与GRP78共定位于内质网;在体外及体外MMP-26与GRP78均有相互作用及MMP-26对GRP78的蛋白降解作用。至此研究发现MMP-26与GRP78在体内体外的相互作用,这将为探索MMP-26与GRP78在肿瘤中的作用奠定基础。
[Abstract]:Matrix metalloproteinase family (MMPs) is a class of zinc ions (Zn2+) and calcium ion (Ca2+) dependent peptide endonuclease.MMPs involved in many physiological processes by degradation of extracellular matrix (ECM), including reproduction, development, morphogenesis and tissue remodeling, and pathology of arthritis, heart blood tube disease and cancer metastasis. Matrix metalloproteinase 26 (MMP-26/endometase/matrilysin-2) a recognized biomarker of breast cancer, prostate cancer and other epithelial sources of cancer.MMP-26 contains 261 amino acids, mainly in the placenta and uterus that.MMP-26 can degrade a variety of proteins, such as IV type collagen, fibronectin, fibrinogen, bosinin, denatured collagen The expression of type I-IV and insulin-like growth factor.MMP-26 in the tumor cells and their extensive protein degradation activity have shown that MMP-26 plays an important role in the development and development of the tumor. By immunological and proteomics methods, the MMP-26 protein 90, the glucose regulator protein 78, are detected. GRP78), the expression of annexin V, promyosin and peroxy oxidoreductase II was significantly up-regulated, while the expression of alpha microtubulin, cysteine protease inhibitor SA-III, and beta actin was downregulated. This phenomenon provides a basis for our study of the interaction of GRP78 and MMP-26 in cells. Glucose regulator protein 78 (GRP78) Immunoglobulin heavy chain binding protein (Bip), also known as a member of the heat shock protein 70 (Heat shock protein 70) family, is a member of a member of the family of.GRP78 as a molecular chaperone. The main function is to participate in transmembrane migration of new synthetic proteins and the maturation, folding and transport of egg white matter, and the inhibition of new synthesis in the endoplasmic reticulum. The aggregation of protein and the regulation of the balance of calcium in the endoplasmic reticulum, such as the balance of calcium ions in the endoplasmic reticulum, are low expression or non expression in normal cells, and high expression in cancer cells, such as breast cancer, prostate cancer, ovarian cancer, kidney cancer, gastric cancer and lung cancer, suggests that the expression of.GRP78 has an important relationship with the occurrence and development of the tumor. It is found that the tumor tissue over expression of GRP78 has a certain degree of resistance to chemotherapeutic drugs, while knockout GRP78 gene can obviously reduce the resistance of tumor cells to chemotherapeutic drugs. Therefore, inhibition of its gene expression or inhibition of its function can be used as a scheme for cancer treatment. This suggests that we can target tumor with GRP78 as a target. To provide a basis for studying the interaction of MMP-26 and GRP78 in vivo and in vitro, we first constructed recombinant gene P GEX-4T-1-GRP78 for recombinant expression of GRP78 protein in vitro, and obtained a large amount of soluble GRP78 protein expressed in the expression bacteria BL21 (DE3), and obtained the GRP78-GST fusion protein by the purification of GST column, and the recombinant expression in vitro obtained C. At MMP-26 protein, expressed in inclusion body form, restores its natural conformation and biological activity through dialysis refolding, and detects the activity of cat MMP-26 protein after refolding by gelatinase spectrum method and fluorescence enzyme labeling instrument. Subsequently, the immunofluorescence chemical experiment, fluorescence microscope observation and fluorescence microscopy are observed. The intracellular MMP-26 and GRP78 are located in the endoplasmic reticulum. In order to study the interaction between MMP-26 protein and GRP78 protein, the interaction of MMP-26 and GRP78 in vitro was demonstrated by the acquisition of GRP78-GFP fusion protein expressed by GST pull down in vitro, and at the same time the body temperature was simulated at 37 degrees C, and MMP-26 was found to be G in vitro by immunoblotting test. RP78 protein has a degradation effect; MMP-26 protein expressed in cell is obtained by GFP affinity chromatography, and GRP78 protein interacting with MMP-26 is obtained. It is proved that there is interaction between MMP-26 and GRP78 in the cell, and the Hela cells, which are expressed by MMP-26 and sh RNA, are proved to be stable. The protein degradation of GRP78 by internal MMP-26. In summary, this paper proves that intracellular MMP-26 and GRP78 are Co located in the endoplasmic reticulum; both in vitro and in vitro MMP-26 and GRP78 have interaction and the protein degradation of GRP78 in MMP-26. At the end of this study, the interaction between MMP-26 and GRP78 in vivo and in vitro is found to explore MMP-26 and GRP78. The role of the tumor is the foundation.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R73-3

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