人类基因RAB24、RAB8B及PDLIM5的功能研究

发布时间：2018-03-13 23:29

本文选题：RAB24　切入点：表达谱分析　出处：《复旦大学》2005年博士论文　论文类型：学位论文

【摘要】：Rab蛋白组成了Ras超家族(即小G蛋白)的一个大家族,主要参与胞内膜泡运输过程,对蛋白的分拣、运输和定位起着非常重要的作用。目前虽然有些Rab蛋白在膜泡运输中的调控机制已经比较明了,但大多数Rab家族成员的具体作用仍不清楚。作为Rab家族的一个非典型成员,Rab24有不少性质与其他成员截然不同。目前有关小鼠的Rab24基因的研究很少,仅有初步的功能探索,还没有明确地阐明它具体参与了什么信号途径,并在这些途径中起了什么作用。而人的RAB24基因虽于2002年在NCBI数据库中释放,至今并无功能方面的研究。我们从本实验室已建立的人全长cDNA文库的序列分析中获得了RAB24基因并对其进行初步的功能研究。现将主要的实验结果罗列如下: (1) 生物信息学分析发现,目前RAB24基因共有四种不同的剪接体,EST分析和RT-PCR实验都表明这四个剪接体在人的多种组织中都是普遍表达的。 (2) 亚细胞定位结果表明,在AD293细胞系中,RAB24定位于细胞质,且在核周位置较为集中,这与以前的报道结果一致;有趣的是,在其它实验室未曾选用的COS7细胞系中,该蛋白意外地定位在细胞核中,仅有少部分定位在核外周。这种定位的不同暗示着Rab24基因在人和猴子中应该具有某些不同的功能。 (3) 突变体RAB24(D1231)在COS7细胞中形成了非常明显的包涵体,包涵体集中分布在核外周,这与已报道的该突变体在其他细胞系中的定位模式似乎正好相反。 (4) 在AD293细胞中,突变体RAB24(G66D)、RAB24(T120N)和RAB24(HH202II)与野生型RAB24的定位模式有所不同,转为明显的全细胞定位,且细胞核中分布较多,这说明RAB24应当存在一种不同于其它RAB蛋白的定位机制。突变体RAB24(Y17A)和RAB24(S67Q)则与野生型RAB24定位模式无明显差异。 (5) 通过酵母双杂交系统的筛选,我们找到了与RAB24相互作用的重要蛋白CYCLOPHILINA、GABARAP。酵母菌结合实验和荧光共定位实验证实了这种相互作用是真实存在的。依次,我们对RAB24可能具有的功能做进一步的探讨。 (6) GTPase活性测定表明,人RAB24与小鼠Rab24一样,活性非常低。而它的两个定点突变体RAB24(Y7A)和RAB24(S67Q)的酶活则有非常显著的增高,这说明这两个位点的存在是造成其酶活性低的重要原因之一。
[Abstract]:Rab proteins form a large family of Ras superfamily (small G protein), mainly involved in the transport of endocytic vesicles and the sorting of proteins. Transport and localization play a very important role. Although the regulatory mechanism of some Rab proteins in membrane vesicle transport has been relatively clear, However, the role of most members of the Rab family is still unclear. As an atypical member of the Rab family, Raban24 has quite a number of properties that are quite different from those of other members. At present, there are few studies on the Rab24 gene in mice, and only a preliminary study of its function. It is not clear what signaling pathways it is involved in and what role it plays in those pathways. The human RAB24 gene was released in the NCBI database on 2002. Up to now, we have obtained the RAB24 gene from the sequence analysis of the human full-length cDNA library established in our laboratory and carried out a preliminary functional study. The main experimental results are listed as follows:. 1. Bioinformatics analysis showed that there were four different splicing bodies in RAB24 gene. The results of EST analysis and RT-PCR experiments showed that the four splicing bodies were universally expressed in various human tissues. The results of subcellular localization showed that RAB24 was located in the cytoplasm of AD293 cell lines and was more concentrated around the nucleus, which was consistent with previous reports. Interestingly, it was found in COS7 cell lines that had not been selected in other laboratories. The protein was accidentally located in the nucleus, with only a small number of the proteins located around the nucleus, suggesting that the Rab24 gene should have some different functions in humans and monkeys. The mutant RAB24D1231) formed a very obvious inclusion body in COS7 cells, and the inclusion bodies were concentrated around the nuclear periphery, which appeared to be the opposite of the reported localization pattern of the mutant in other cell lines. (4) in AD293 cells, the localization patterns of the mutants RAB24G66DNand RAB24H2022) were different from those of wild-type RAB24, and transformed into obvious whole-cell localization, and the distribution of RAB24G66DG-T120Nand RAB24H202II was much more than that of wild-type RAB24. This suggests that RAB24 should have a different localization mechanism from other RAB proteins, while the mutant RAB24AY17A and RAB24S67Q) have no significant difference from wild type RAB24. Through the screening of yeast two-hybrid system, we found the important protein CYCLOOPHILINA GABARAPP interacting with RAB24. The yeast binding experiment and fluorescence co-localization experiment confirmed that the interaction was real. We further explore the possible functions of RAB24. The detection of GTPase activity showed that the activity of human RAB24 was very low as that of mouse Rab24, but the enzyme activity of its two site-directed mutants RAB24OY7A and RAB24S67Q) was significantly increased, which indicated that the existence of these two sites was one of the important reasons for the low enzyme activity.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：Q75

【共引文献】