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人类心脏发育与疾病候选基因LBH的相互作用蛋白的鉴定

发布时间:2019-05-17 14:25
【摘要】: 心脏病已经成为威胁人类健康和生命最为严重的疾病之一,近几年,心血管疾病的死亡人数占到疾病死亡总人数的三分之一。为攻克这一顽症,首先必须从分子水平上系统地研究控制心脏发育的基因及其相互调控关系以及导致该疾病的分子机理。 小鼠Lbh(Limb-bud-and-heart)基因在心脏和肌肉组织特异表达,在对Lbh转基因小鼠模型的研究中发现该小鼠心脏表型异常,类似人类先天性心脏疾病的症状。人类LBH基因与小鼠的Lbh基因在蛋白质水平的相似性高达89%,由此推测它可能与人类心脏疾病发生有关。对人LBH基因的深入研究对于探讨人心脏发育的基因调控机制,对于心脏疾病的诊断和治疗具有重大的理论和实践意义。 生物体内的许多简单的生理活动都是在众多蛋白质的协调相互作用下进行的,每个蛋白质行使其功能都要通过和其他与其相互作用的蛋白质来协调作用完成。研究一个蛋白质功能的工作包括确定它与哪些蛋白质发生作用,而鉴定这些与其发生作用的蛋白质也可以为研究这个蛋白质本身的功能提供很多的线索。酵母双杂交技术简便,灵敏,高效,而且能够大规模筛选活细胞内的蛋白质相互作用。它在基因功能的研究中得到了广泛的应用。目前,对于LBH基因还没有做任何深入的研究,LBH的功能尚不清楚,利用酵母双杂交技术筛选鉴定相互作用蛋白,从而研究该基因的可能功能作用,是一个简单而有效的方法。 为了研究LBH在心脏中的作用机制,本研究利用酵母双杂交方法在成人心脏文库中筛选鉴定到7个与LBH发生相互作用的蛋白质,其中αB-crystallin(CRYAB)是一小分子热休克蛋白,在心肌细胞表达非常丰富;作者用免疫共沉淀和Pull-Down验证了LBH与αB-crystallin确实发生相互作用。利用免疫荧光研究LBH和αB-crystallin在COS-7细胞的定位情况发现,,LBH主要定位在细胞核,细胞质中也有一定量的表达,αB-crystallin主要在细胞质中表达,二者共同转入细胞时,αB-crystallin发生了入核现象,说明细胞核中的二者相互作用,荧光报告基因分析发现LBH和αB-crystallin的共同表达协同抑制p53、p21转录活性;另外,Tcap是一种肌节蛋白,对心脏发育和心脏功能都有重要作用,并且其突变均心肌病密切相关。作者用免疫共沉淀和Pull-Down验证了LBH与Tcap确实发生相互作用。利用免疫荧光研究LBH和Tcap在COS-7细胞的定位情况发现,LBH主要定位在细胞核,细胞质中也有一定量的表达,Tcap在核质均有表达,当二者共同转入细胞时,二者在细胞核质共定位表达情况一致。综合目前的研究结果来看,LBH很可能通过与αB-crystallin、Tcap的相互作用参与心脏功能并且与心脏疾病相关联。
[Abstract]:Heart disease has become one of the most serious diseases threatening human health and life. In recent years, the number of deaths from cardiovascular diseases accounts for 1/3 of the total number of deaths. In order to overcome this persistent disease, it is necessary to systematically study the genes that control cardiac development and their mutual regulation at the molecular level, as well as the molecular mechanism leading to the disease. Mouse Lbh (Limb-bud-and-heart) gene was specifically expressed in heart and muscle tissue. In the study of Lbh transgenic mouse model, it was found that the mouse heart phenotype was abnormal, which was similar to the symptoms of human congenital heart disease. The similarity between human LBH gene and mouse Lbh gene at protein level is as high as 89%, which suggests that it may be related to the occurrence of human heart disease. The further study of human LBH gene is of great theoretical and practical significance for exploring the gene regulation mechanism of human heart development and for the diagnosis and treatment of heart diseases. Many simple physiological activities in organisms are carried out under the coordinated interaction of many proteins, and each protein exercises its function by coordinating with other proteins that interact with it. The study of the function of a protein includes determining which proteins it interacts with, and identifying the proteins that interact with it can also provide a lot of clues to the study of the function of the protein itself. Yeast two-hybrid technique is simple, sensitive and efficient, and can screen protein interactions in living cells on a large scale. It has been widely used in the study of gene function. At present, no further research has been done on LBH gene, and the function of LBH is not clear. It is a simple and effective method to screen and identify the interacting protein by yeast two-hybrid technique, so as to study the possible functional effect of the gene. In order to study the mechanism of LBH in heart, seven proteins interacting with LBH were screened and identified by yeast two-hybrid method in adult heart library, in which 伪 B-crystallin (CRYAB) was a small molecule heat shock protein. The expression was very rich in cardiomyocytes. Immunoprecipitation and Pull-Down were used to verify the interaction between LBH and 伪 B-crystallin. The localization of LBH and 伪 B-crystallin in COS-7 cells was studied by immunofluorescence. It was found that LBH was mainly located in the nucleus and a certain amount of expression was also found in the cytoplasm. 伪 B-crystallin was mainly expressed in the cytoplasm. 伪 B-crystallin entered the nucleus, indicating the interaction between the two in the nucleus. Fluorescence reporter gene analysis showed that the co-expression of LBH and 伪 B-crystallin coordinated the inhibition of p53 and the transcriptional activity of p21. In addition, Tcap is a sarcoprotein, which plays an important role in cardiac development and cardiac function, and its mutation is closely related to cardiomyopathy. Immunoprecipitation and Pull-Down were used to verify the interaction between LBH and Tcap. The localization of LBH and Tcap in COS-7 cells was studied by immunofluorescence. It was found that LBH was mainly located in the nucleus, and there was also a certain amount of expression in cytoplasm, and Tcap was expressed in both nucleoplasms. when both of them were transferred into the cells, The co-localization and expression of the two in nucleus and cytoplasm were the same. Based on the results of current research, LBH is likely to be involved in cardiac function and associated with heart disease through the interaction with 伪 B 鈮

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