Rab家族及其在囊泡运输中的作用

Rab蛋白家族是Ras超家族中最大的亚家族,为小GTP结合蛋白。目前发现的Rab家族成 员已达60多种,各成员之间有相似的结构。除少数特异表达于某一细胞或组织,大多数Rab蛋白广泛 存在于组织细胞中,Rab蛋白是囊泡运输重要的调节因子。Rab突变或表达异常引起囊泡运输异常,从 而导致一些疾病产生。     

Rab家族及其在囊泡运输中的作用

Rab蛋白家族是Ras超家族中最大的亚家族，为小GTP结合蛋白。目前发现的Rab家族成员已达60多种，各成员之间有相似的结构。除少数特异表达于某一细胞或组织，大多数Rab蛋白广泛存在于组织细胞中，Rab蛋白是囊泡运输重要的调节因子。Rab突变或表达异常引起囊泡运输异常，从而导致一些疾病产生。     

Rab家族与遗传性疾病的研究进展

Rab家族在人类基因组中含有60多个成员,参与囊泡运输的调节.越来越多的证据表明,如果Rab蛋白发生突变或者表达水平发生异常,会导致囊泡运输的障碍,使被运输的物质不能到达目的区域,最终可能出现生理失调乃至疾病的发生,如遗传性疾病、老年痴呆症、癌症等.该文就Rab异常导致的遗传性疾病进行综述.     

人类Rab7的结构与功能

Rab蛋白家族是一类Ras相关的GTP结合蛋白,在细胞中发挥确保囊泡转运特异性定位的作用。作为Rab家族的一员,Rab7与其他的Rab成员间既有相似性,又有特异性。其相似性表现在：Rab7与其他的Rab成员有着相似的结构、相似的异戊二烯化过程以及相似的GDP/GTP结合功能;特异性表现在：Rab7特异识别晚期胞内体等囊泡,介导晚期胞内体与溶酶体的膜融合以完成溶酶体转运,发挥特有的生物学功能。Rab7蛋白的功能缺陷和多种人类遗传疾病相关。     

Rho GTPase在信号转导和细胞骨架中的作用

Rho GTPases参与多种重要的细胞生命活动,如肌动蛋白细胞骨架的重构、细胞黏附、细胞运动、囊泡运输、转录激活、基因表达和细胞周期的调控等。当Rho GTPase蛋白水平改变,活性状态改变,效应蛋白丰度改变后,出现异常的Rho 信号,从而影响细胞骨架重组使细胞迁移。调节这些生物信号的转导通路非常复杂, 因此,Rho GTPases已成为近年来的研究热点。     

蛋白质从内质网到高尔基器间运输的分子机制

膜性细胞器间的大分子运输是由有被小泡介导的囊泡转运，ＥＲ到Ｇｏ之间的运输的中介物质主要是ＣＯＰ（ＣｏａｔＰｒｏｔｅｉｎ）和ＣＯＰⅡ外被小泡外被的转运囊泡。这类囊泡不同于笼蛋白外泡的转运囊泡，其性质与笼蛋白外被小泡有某些相似之处。本文从研究方法入手，介绍了近年来关于ＥＲ及Ｇｏ间运输过程中的发现和问题。随着分子生物学技术的发展，人们对这一过程的研究进入了分子领域并取得初步成果。     

Rab22a调控细胞内吞和免疫功能

Rab22a是Rab家族的一个小GTP酶，显著改变多个膜转运步骤，Rab22a在细胞的初级内吞及质膜循环中发挥着关键的作用。Rab22a不仅参与初级内体的形成，还参与物质回收及质膜循环，介导多种效应蛋白和信号受体的转运。研究显示Rab22a与细胞免疫有着密不可分的关系，因为抗原呈递在很大程度上依赖于在多种细胞中发生的内吞事件和在分泌途径中转运事件的复杂调控，以及这两种途径之间的交联。另外，Rab22a在调节自噬中也具有特殊的功能。Rab22a作为膜循环及细胞内运输的重要调控因子，参与调控囊泡的形成、运输及融合的过程。     

调节膜转运的Rab/效应物复合体结构基础

G蛋白是一个种类繁多可与GTP结合而且活性受到GTP调控的超家族。其中的小分子量G蛋白一般只有1个亚单位,而且这些小分子量G蛋白与信息传递,细胞生长与分化、蛋白质合成、合成后的加工及转运机理有密切的关系。Rab蛋白家庭在调控真核细胞膜性细胞器蛋白或其他分子的装运、归类、分泌等活动中起着重要作用。Rab蛋白所调节的功能很广泛,包括囊泡的迁移,及其在膜上特异融合部位的锚定。Rab蛋白在调控突触可塑性(例如神经递质的释放)方面起着决定性的作用。Rab蛋白拥有一个共同的三维折叠结构域,在与GTP结合状态下,它能与下游的多种效应蛋白相结合。如果GTP被水解,这个“开关”区     

Rab4蛋白的细胞内定位及其活性改变对DC内源抗原递呈的影响

目的观察Rab4突变体在树突状细胞(DC)内的定位,研究其对DC内源抗原递呈的影响。方法构建Rab4突变体的慢病毒表达质粒,以DNA-磷酸钙共沉淀法制备慢病毒,病毒感染DC-OVA细胞并使用流式细胞术检测感染效率,激光共聚焦显微镜观察DC-OVA细胞中Rab4突变体的表达与定位,最后用识别MHC I类分子-OVA肽复合物的T细胞杂交瘤B3Z细胞检测DC-OVA细胞内源抗原递呈的变化。结果慢病毒可高效感染DC-OVA细胞,感染后Rab4突变体可在DC-OVA中稳定表达,主要分布在细胞膜周围的囊泡结构中。抗原递呈检测结果显示Rab4对DC内源性抗原递呈具有正相关性影响。结论成功观察到不同Rab4突变体在DC内的表达与分布,初步证实Rab4蛋白可通过其活性的改变参与调节DC的内源性抗原递呈。     

DMT1在小肠吸收细胞的摄铁机制

铁是生物体最丰富的微量金属元素之一,小肠是机体铁吸收和铁稳态调节最关键结构,小肠吸收细胞对非血红素铁的吸收摄取主要由二价金属离子转运体(divalent metal transporter1,DMT1)介导的。DMT1对铁的吸收转运主要通过囊泡运输和载体运输实现的。囊泡运输主要包括DMT1形成吸收铁的囊泡、与apo-Tf囊泡融合、分离、分选转运完成的;载体运输则是在肠表面H+电化学梯度的驱动下将铁转入细胞内的。本文着重介绍了最近国内外关于DMT1在小肠非血红素铁吸收转运中的作用机制的最新研究进展。     

Roles for the recycling endosome, Rab8, and Rab11 in hantavirus release from epithelial cells

Hantavirus structural proteins are believed to localize to intracellular membranes often identified as Golgi membranes, in virus-infected cells. After virus budding into the Golgi luminal space, virus-containing vesicles are transported to the plasma membrane via trafficking pathways that are not well defined. Using the New World hantavirus, Andes virus, we have investigated the role of various Rab proteins in the release of hantavirus particles from infected cells. Rabs 8 and 11 were found to colocalize with Andes virus proteins in virus infected cells and when expressed from cDNA, implicating the recycling endosome as an organelle important for hantavirus infection. Small interfering RNA-mediated downregulation of Rab11a alone or Rab11a and Rab11b together resulted in a decrease in infectious virus particle secretion from infected cells. Downregulation of Rab8a did not alter infectious virus release but reduction of both isoforms did. These data implicate the recycling endosome and the Rab proteins associated with vesicular transport to or from this intracellular organelle as an important pathway for hantavirus trafficking to the plasma membrane.     

Role of Rab GTPases in membrane traffic and cell physiology

Intracellular membrane traffic defines a complex network of pathways that connects many of the membrane-bound organelles of eukaryotic cells. Although each pathway is governed by its own set of factors, they all contain Rab GTPases that serve as master regulators. In this review, we discuss how Rabs can regulate virtually all steps of membrane traffic from the formation of the transport vesicle at the donor membrane to its fusion at the target membrane. Some of the many regulatory functions performed by Rabs include interacting with diverse effector proteins that select cargo, promoting vesicle movement, and verifying the correct site of fusion. We describe cascade mechanisms that may define directionality in traffic and ensure that different Rabs do not overlap in the pathways that they regulate. Throughout this review we highlight how Rab dysfunction leads to a variety of disease states ranging from infectious diseases to cancer.     

Expression profiling of Rab GTPases reveals the involvement of Rab20 and Rab32 in acute brain inflammation in mice

Rab GTPases have emerged as central regulators of vesicle trafficking and are essential for cytokine production during the pathogenesis of neuroinflammation. To characterize the roles of different Rab proteins in brain inflammation, we used quantitative PCR (qPCR) to examine the expression profiles of all members of the Rab family in an experimental model of brain inflammation in mice. We found that Rab20 and Rab32 were substantially up-regulated during the acute phase of inflammation. The increased expression of Rab20 was also confirmed by immunostaining of inflamed brains at different timepoints. The concomitant overexpression of Rabs (Rab20 and Rab32) and early response proinflammatory cytokines (TNF-α and IL-1β) suggested that these Rabs may be important for subsequent inflammatory responses in brain. Furthermore, we found that the expression of certain Rabs was dramatically reduced in cultured primary microglia, which was not observed in the in vivo profiling. In N9, a microglial cell line, however, there was no increase in the expression of Rab20 or Rab32, but Rab3c was significantly overexpressed. These results collectively indicate that Rabs may participate in inflammatory response in microglia during brain inflammation. The differential regulation of individual Rabs in different experimental systems is a caveat for the analysis of Rab functions.     

Mechanisms of transcellular transport of wheat germ agglutinin-functionalized polymeric nanoparticles in Caco-2 cells

Transcellular transport is essential for transmucosal and plasma-to-tissue drug delivery by nanoparticles, whereas its fundamental pathways have not been fully clarified. In this study, an in-depth investigation was conducted into the intracellular itinerary and the transcytosis pathway of wheat germ agglutinin-functionalized nanoparticles (WGA-NP) with various polymer architectures in the Caco-2 cell model. GFP-Rabs, Rab4, Rab5, Rab7, Rab11, GTPases served as key regulators of vesicular transport, and their mutants were transfected to Caco-2 cells respectively to determine the cellular itinerary of WGA-NP and the role of Rabs therein. Transcytosis inhibition experiments indicated that transcellular transport of WGA-NP (PEG(3000)-PLA(40000) formulation) happened in a cytoskeleton-dependent manner and majorly by means of clathrin-mediated mechanism. Intracellular transport, especially the endolysosome pathway was found largely contribute to the transcytosis of WGA-NP. WGA-NP with shorter surface PEG length (2000) resulted in higher cellular association and more colocalization with the clathrin-mediated transport pathway, while that with longer surface PEG length (5000) avoided the clathrin-mediated transport pathway but achieved higher transcytosis after 4?h incubation. WGA-NP with PLGA as the core materials obtained elevated lysosome escape and enhanced transcytosis after 2?h incubation. These findings provided important evidence for the role of polymer architectures in modulating cellular transport of functionalized nanocarriers, and would be helpful in improving carrier design to enhance drug delivery.     

Rab 11 regulates constitutive dopamine transporter trafficking and function in N2A neuroblastoma cells

The dopamine transporter (DAT) is a crucial regulator of dopaminergic neurotransmission which undergoes constitutive and substrate-mediated trafficking to and from the membrane. Although, considerable research has been done to elucidate the regulation of substrate-stimulated DAT trafficking, less is known about which trafficking proteins are involved in constitutive DAT trafficking. Rab proteins are GTPases known to regulate the trafficking of proteins to and from specific endocytic compartments. Rabs 8 and 11, in particular, are involved in trafficking proteins from intracellular compartments to the plasma membrane. In this study, we sought to determine whether Rabs 8 and 11 would modulate DAT activity and trafficking in N2A neuroblastoma cells. We used Rab mutations known to confer constitutively active or dominant negative activity of these proteins to investigate the role of Rab activity in constitutive DAT trafficking and function. We found that constitutively active Rab 11 upregulates DAT function and surface expression while neither the constitutively active nor the dominant negative mutant of Rab 8 had any effect on DA uptake. Furthermore, immunofluorescence experiments revealed that dominant negative Rab 11 overexpression results in decreased surface DAT indicating a necessary function of Rab 11 in DAT trafficking to the plasma membrane. These data show for the first time a functional role of Rab proteins in the constitutive recycling of DAT to the plasma membrane.     

Rab proteins in endocytosis and Glut4 trafficking

The intracellular trafficking of numerous proteins requires a tight control to fulfil their physiological functions. It is the case of the adipocyte and muscle glucose transporter Glut4 that is retained intracellularly until insulin induces its recruitment to the plasma membrane. Rabs are evolutionarily conserved small GTPases that control intracellular traffic events from yeast to mammalian cells. In the past few decades, considerable progresses have been made in identifying the route of Glut4, the Rabs involved in controlling it, and more recently the connection between insulin signalling and Glut4 trafficking through Rab activity control.     

Production and characterization of isotype-specific monoclonal antibodies to bovine brain Rab GDI

Small GTPases of the Rab family play a key role in controlling vesicular transport, and the Rab GDP-dissociation inhibitor (GDI) is a regulatory protein for the Rab proteins. Here we report the production and characterization of isotype-specific monoclonal antibodies (MAbs) to Rab GDI. Rab GDI was purified from bovine brain in several steps of column chromatography and was injected into BALB/c mice intraperitoneally. The resulting MAbs specifically recognized a single protein band of 55 kDa, which comigrates with purified bovine Rab GDI. To localize Rab GDI, we processed cells from different sources for indirect immunofluorescence microscopy. Interestingly, the MAb stained cytosol and vesicular structures in brain cells, whereas it predominantly stained cytosol in nonbrain cells. Next, we investigated the cross-reactivities of brain Rab GDI from some mammals. The immunoreactive bands on Western blots appeared to be the same in molecular mass, 55 kDa, in all mammalian species tested including human. In summary, we produced a panel of MAbs that are GDI-alpha/1 form-specific and we believe that the MAbs will be valuable tools in elucidating the function of Rab GDI isoforms.     

Screening for target Rabs of TBC (Tre-2/Bub2/Cdc16) domain-containing proteins based on their Rab-binding activity

It has recently been proposed that the TBC (Tre2/Bub2/Cdc16) domain functions as a GAP (GTPase-activating protein) domain for small GTPase Rab. Because of the large number of Rab proteins in mammals, however, most TBC domains have never been investigated for Rab-GAP activity. In this study we established panels of the GTP-fixed form of 60 different Rabs constructed in pGAD-C1, a yeast two-hybrid bait vector. We also constructed a yeast two-hybrid prey vector (pGBDU-C1) that harbors the cDNA of 40 distinct TBC proteins. Systematic investigation of 2400 combinations of 60 GTP-fixed Rabs and 40 TBC proteins by yeast two-hybrid screening revealed that seven TBC proteins specifically and differentially interact with specific Rabs (e.g. OATL1 interacts with Rab2A; FLJ12085 with Rab5A/B/C; and Evi5-like with Rab10). Measurement of in vitro Rab-GAP activity revealed that OATL1 and Evi5-like actually possess significant Rab2A- and Rab10-GAP activity, respectively, but that FLJ12085 do not display Rab5A-GAP activity at all. These results indicate that specific interaction between TBC protein and Rab would be a useful indicator for screening for the target Rabs of some TBC/Rab-GAP domains, but that there is little correlation between the Rab-binding activity and Rab-GAP activity of other TBC proteins.