质膜微囊蛋白质Cavin-1的细胞膜与细胞核双重定位的结构基础以及功能

Membrane lipid rafts and caveolae contains a specific set of lipids and structural proteins. Caveolae is a type of membrane lipid microdomains containing caveolins, flotillins and cavins. More importantly, both caveolin-1 and cavin-1 are structural proteins essential for the formation of caveolae. Before the identification of cavin-1 as the caveolae component protein, a nuclear protein important for the release of RNA polymerase I and RNA is identified and named as PTRF (polymerase I and transcript release factor). Late on, cavin-1 and PTRF are found to the same protein. Thus, cavin-1/PTRF is a protein present in caveolae as well as cellular nuclei. In caveolae, it is a structure protein important for the formation of caveolae. In nuclei, it is a protein regulating the release of RNA polymerase I and the transcribed RNA from the transcription complex. To play two different roles in two different cellular organelles, cavin-1/PTRF should contain both the nuclear localization sequence and the membrane lipid microdomain association motif. Currently, two putative nuclear localization sequences are identified in cavin-1/PTRF. However, it is still not clear how cavin-1/PTRF interacts with membrane lipid microdomains. In this application, we propose to investigate the sequence or motif involved in mediating the interaction between cavin-1/PTRF and caveolae. We will also analyze the relation of the nuclear localization sequence and caveolae interaction motif in regulating the balance between the nuclear PTRF and caveolae localized cavin-1. As for the functional study, we will focus on the caveolae related cellular functions of cavin-1/PTRF. Cavin-1/PTRF mutations will be constructed to delete its caveolae association motif, nuclear localization sequence or both. Their cellular localization will be analyzed and their functions will be studied using cavin-1/PTRF MEF cells prepared from cavin-1/PTRF knockout mice. For the functional analysis, we will focus on the cell migration. It is identified that the cell motility of cavin-1/PTRF-/- MEF cell is greatly reduced in comparison with the wild type MEF cell. The exogenous expression of cavin-1/PTRF in cavin-1/PTRF-/- MEF cell can rescue the inhibited cell migration. Using this system, we will analyze the effect of cavin-1/PTRF mutants on cell migraiton of cavin-1/PTRF MEF cell. Based on the results, we would like to understand the function of various domains of cavin-1/PTRF in cell motility control and to understand the relationship of caveolae assocaition and cell motility control by cavin-1/PTRF.

细胞膜脂质微区不但具有特殊的脂质成分，也含有特异性的结构和功能蛋白质。Cavin-1/PTRF是细胞膜脂质微区的重要组成蛋白质，同时也是细胞核中调控RNA聚合酶和RNA释放的蛋白质。它在细胞膜和细胞核中的不同功能源于其具有质膜微囊和细胞核的双重定位。研究表明，cavin-1/PTRF具有两个核定位序列，但其质膜微囊定位的结构序列还有待发现。项目将围绕cavin-1/PTRF与质膜微囊相互作用以及在质膜微囊中的功能，研究cavin-1/PTRF在细胞膜的定位结构序列。通过构建cavin-1/PTRF的突变体，研究其细胞膜定位和细胞核定位的变化以及相互间的关系。围绕cavin-1/PTRF在细胞迁移中的作用，在cavin-1/PTRF-/-细胞中表达不同的cavin-1/PTRF突变体，研究蛋白质结构与细胞迁移的关系以及不同结构域在其中的作用。从而认识其质膜微囊的相互作用对细胞迁移的调控作用。

质膜微囊是细胞膜上的一类功能广泛的细胞膜结构。Cavin-1是质膜微囊中的一个组成蛋白质，它的缺失导致质膜微囊不能形成。我们研究发现它依靠一个leucine-zipper细胞膜定位序列与细胞膜质膜微囊结合。突变或缺失了这个细胞膜定位序列后，cavin-1突变体成为细胞核定位蛋白质。以前文献中报道的与cavin-1是同一个蛋白质的细胞核内RNA转录释放因子（PTRF）应该属于是leucine-zipper突变或缺失的cavin-1突变体，而非cavin-1本身。在质膜微囊的形成过程中，内质网中形成的初始囊泡通过高尔基体加工、分泌，而后整合到细胞质膜形成质膜微囊。这一质膜微囊形成过程的囊泡运输途径也是一条蛋白质分泌途径，分泌型蛋白激酶Fam198a依赖其从内质网直到分泌出细胞。Cavin-1缺失后，Fam198a被阻止在内质网内。因此，Cavin-1是在内质网阶段就参与了囊泡形成以及运输，而非通常认为的是在最后整合到细胞质膜时才参与质膜微囊的形成。Cavin-1还参与肝脏血管/窦内皮细胞上开孔的形成。Cavin-1缺失抑制肝脏血管/窦内皮细胞的分化以及细胞开孔的形成，导致cavin-1缺失小鼠肝脏中血管/窦内皮细胞的通透性下降，血液中的葡萄糖等代谢物不能有效地与肝细胞交换，降低了肝脏作为代谢调控器官的作用与功能，小鼠的糖脂代谢异常。项目的研究结果为澄清cavin-1和PTRF的关系提供了分子结构依据。同时发现了cavin-1在细胞迁移、蛋白质分泌等重要细胞活动中有重要功能。进一步还发现了cavin-1通过影响肝脏血管/窦内皮细胞的通透性而影响肝脏的生理功能和对糖脂代谢的调控。