人cubilin蛋白突变体的构建与功能研究

发布时间：2018-01-20 03:21

本文关键词： cubilin 同源模建原核表达亲和层析　出处：《第三军医大学》2006年硕士论文　论文类型：学位论文

【摘要】： 肾小管上皮细胞超负荷重吸收白蛋白可通过多种途经损伤肾组织,最终导致慢性肾功能衰竭[1,2]。Cubilin是分布于肾近曲小管上皮细胞刷状缘负责重吸收白蛋白等大分子物质的吞饮受体[3-5]。在cubilin缺陷的人和狗,均出现大量白蛋白尿,但肾组织无明显病理改变[6-8],也不会进展为慢性肾功能衰竭[9]。我们的研究表明,肾病综合征患者肾小管上皮细胞cubilin表达上调,重吸收白蛋白增加,与肾小管间质MCP-1和RANTES表达增加和炎症细胞浸润的改变相关[10],而反义cubilin RNA,在抑制白蛋白超负荷重吸收的同时,也明显抑制肾小管上皮细胞MCP-1和RANTES的表达[11],提示cublilin在介导白蛋白超负荷引起的肾小管间质损害中具有重要作用。Cubilin全长3623aa,460kD,由N-端,8个EGF样结构和27个CUB结构域组成[12],CUB结构域是主要的功能域,负责和绝大部分的配体结合,其中CUB7-8结构域是白蛋白的结合域,但二者结合的关键氨基酸残基尚未确定[13]。由于蛋白质之间的相互作用是通过分子内部的肽段形成特殊空间结构实现的(如“配体结合口袋”负责受体与配体间的识别与结合),通常只需几个关键功能残基决定其相互作用的效率。针对Cubilin各段生物学功能不同的特性,选取能够与白蛋白结合的CUB7-8功能片段为研究对象,以同源模建方法建立CUB8功能域的三维模型,并以计算机辅助的分子对接方法预测CUB8功能域与白蛋白的候选结合氨基酸残基,通过原核表达野生型和突变型CUB7-8蛋白片段,利用生物传感器技术,测定野生型和突变型CUB7-8蛋白片段与白蛋白的亲和力,以确定cubilin与白蛋白结合的关键氨基酸残基,为进一步研究cubilin的生理功能和病理生理意义奠定基础。实验方法 1.利用NCBI和PDB数据库,搜索CUB8的氨基酸序列及与该片段相似性较高且已经解析三维结构的模板蛋白,再利用insight II工作站对CUB8蛋白片段进行同源模建,然后对得到的CUB8蛋白片段和白蛋白进行分子对接,寻找白蛋白和CUB8的候选结合氨基酸残基。
[Abstract]:Renal tubule epithelial cells overload and reabsorption of albumin can lead to chronic renal failure through multiple ways to damage renal tissue. [Cubilin is a receptor that reabsorbs albumin and other macromolecules in the brush border of renal proximal tubule epithelial cells. [3-5] .A large amount of albuminuria was found in both human and dog with cubilin deficiency, but there were no obvious pathological changes in renal tissue. [6-8], and it doesn't progress to chronic renal failure. [9. Our study showed that the expression of cubilin in renal tubular epithelial cells was up-regulated and the reabsorption of albumin was increased in patients with nephrotic syndrome. Correlated with increased expression of MCP-1 and RANTES in renal tubulointerstitium and changes of inflammatory cell infiltration. [While antisense cubilin RNAs inhibited albumin overload and reabsorption, it also inhibited the expression of MCP-1 and RANTES in renal tubular epithelial cells. [It is suggested that cublilin plays an important role in mediating tubulointerstitial damage induced by albumin overload. Composition of 8 EGF like structures and 27 CUB domains. [12 the cub domain is the main domain responsible for binding to most of the ligands, in which the CUB7-8 domain is the binding domain of albumin, but the key amino acid residues of the two binding domains have not been determined. [13. Since the interaction between proteins is achieved through the formation of special spatial structures of peptide segments within molecules (e.g. "ligand binding pockets", which are responsible for the recognition and binding between receptors and ligands). Usually only a few key functional residues are required to determine the efficiency of their interactions. The functional fragments of CUB7-8 which can bind to albumin were selected as the research object, and the 3D model of CUB8 functional domain was established by homologous modeling method. The candidate amino acid residues of CUB8 domain and albumin were predicted by computer assisted molecular docking method, and the wild-type and mutants CUB7-8 protein fragments were expressed by prokaryotic expression. The affinity of wild-type and mutant CUB7-8 protein fragments to albumin was determined by biosensor technique to determine the key amino acid residues of cubilin binding to albumin. To lay a foundation for further study of physiological function and pathophysiological significance of cubilin. 1. NCBI and PDB databases were used to search for the amino acid sequence of CUB8 and the template protein with high similarity to the fragment. Insight II workstation was used for homologous modeling of CUB8 protein fragment, and then the CUB8 protein fragment and albumin were docked. To search for candidate binding amino acid residues of albumin and CUB8.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R341

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