Regulation of Membrane Dynamics by Sec7 Domain Arf Nucle
Sec7 域 Arf 核对膜动力学的调节
基本信息
- 批准号:7334112
- 负责人:
- 金额:--
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:
- 资助国家:美国
- 起止时间:至
- 项目状态:未结题
- 来源:
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项目摘要
This unit studies the Sec7 domain guanine nucleotide exchange factors (GEFs) for the Arf family of small GTPases. We are interested in the roles of these proteins in membrane dynamics and protein trafficking. The Arfs and the Arf GEFs are important regulators of both organelle structure and protein transport throughout the cell. Eukaryotic cells are characterized by their internal membrane structure, which is essential for the correct spatial organization of the many biochemical reactions that take place within cells. The nucleus is separated from the surrounding cytoplasm by the nuclear envelope, a double membrane structure that is continuous with the endoplasmic reticulum (ER). Transmembrane domain proteins, proteins destined for secretion, and soluble resident proteins of organelles such as the lysosome/vacuole are translocated across the ER membrane into the lumen of this organelle. From here they are transported to their final destination via the Golgi apparatus. The ER spreads throughout cells, and gives rise to multiple Golgi elements that in mammalian cells are transported via microtubules to a region adjacent to the nucleus, thus forming the Golgi apparatus. A major function of the Golgi apparatus is the post-translational modification of proteins traveling through it, and sorting of these proteins to their correct destination in the cell. We are focusing our attention on a subfamily of Arf GEFs involved in transport through the Golgi apparatus, both in budding yeast and in mammalian cells. A central question in cell biology is how the elaborate and dynamic structures of membrane systems are maintained in the face of constant trafficking into and out of each organelle. In particular, the way organelle structure is generated and maintained, and how structure is correlated with the underlying molecular events of protein sorting and membrane remodeling are pressing questions. Evidence that the Arf GEFs play a key role in membrane dynamics and organelle structure came from studies of the cellular effects of brefeldin A (BFA), a drug that has profound effects on organelles of the secretory and endocytic pathways in a wide range of cell types. BFA causes the complete and rapid disassembly of the Golgi apparatus and its fusion with the ER, as well as fusion of the trans-Golgi network with endosomes. These experiments were the first to show the incredibly dynamic nature of the Golgi apparatus, whose elaborate structure would seem to suggest a more stable state. We demonstrated that three Arf GEFs in yeast are the major targets of the drug in the yeast secretory pathway. We also demonstrated the mechanism of action of this drug. BFA binds to a normally very short-lived reaction intermediate in the exchange reaction, an Arf-GDP-Sec7 domain complex, and forms an abortive quaternary complex that prevents the reaction from proceeding to completion. This unusual mechanism of action provides a paradigm for development of novel drugs. Instead of the usual search for drugs competing for a given substrate, screens could be designed for drugs that block reactions through stabilization of reaction intermediates. We are using a combination of techniques, including yeast genetics, molecular biology, imaging of yeast and mammalian cells and biochemistry, to elucidate the roles of the Arf GEFs in protein transport and organelle structure. An important step towards understanding the mechanisms of membrane trafficking will be to define the roles of the Arf GEFs at the molecular level, through identification of interacting partners, elucidation of membrane localization mechanisms and analysis of Arf GEF mutants in vivo. We have identified a number of interesting partners of the Arf GEFs in both budding yeast (Saccharomyces cerevisiae) and in mammalian cells, and are currently characterizing these novel partners and their roles in protein trafficking and membrane dynamics.
Enteroviruses are members of the Picornaviridae family of positive strand non-enveloped RNA viruses, and include a number of important human pathogens such as poliovirus, coxsackievirus and echovirus. Despite the fact that enteroviruses (and other non-enveloped viruses) are not enclosed by a membrane, their replication never-the-less depends completely on host cell membranes, although the molecular details of this requirement are not understand. Enteroviruses are implicated in a wide range of human diseases including poliomyelitis, meningoencephalitis, encephalitis, pancreatitis and myocarditis. It is estimated that throughout the world, one billion human infections per year are caused by enteroviruses. All enteroviruses have a similar RNA genome of approximately 7.5 kb that encodes four capsid proteins and a number of nonstructural proteins. These non-capsid proteins are involved in viral replication and in virus-induced host cell membrane reorganization. One of the most dramatic effects of enterovirus infection is the massive reorganization of intracellular membrane systems, which ultimately involves all internal cell membrane systems except mitochondria and the nuclear envelope. The membrane structures produced upon viral infection are absolutely required for viral replication, which takes place on the membrane surface. Membrane reorganization occurs within two to three hours after viral infection. Subsequently, at three to four hours post-infection, enteroviruses completely inhibit intracellular protein transport. This effect can be produced by expression of only the viral 2B (or its precursor 2BC) protein in mammalian cells, or the viral 3A protein alone. The ability of the 2B(C) protein to inhibit transport is linked to its ability to rearrange membranes of the secretory pathway, but the 3A protein blocks transport by a mechanism that is not dependent on membrane reorganization. The transport inhibition and replication functions of the 3A protein can be genetically separated by mutations in 3A that affect one or the other function independently. The capacity of enteroviruses to block trafficking is important for viral infectivity, probably by suppressing both innate and adaptive immune responses and by suppressing the apoptotic pathway induced by viral infection. Recent studies have demonstrated a role for the Arf GTPases in poliovirus and coxsackievirus infection. Arf1, Arf3 and Arf5 (but not Arf6) are activated and recruited to membranes in an in vitro poliovirus replication assay. In addition, Arf1 is relocated from the Golgi apparatus to virus-induced membrane structures between two and three hours post-infection. A role for the Arf GEFs in viral infectivity was initially suggested by the fact that replication of poliovirus is completely inhibited by brefeldin A (BFA). In collaboration with the groups of Dr. Frank van Kuppeveld and Dr. Ellie Ehrenfeld, we have obtained direct proof that the BFA-sensitive Arf GEFs have important functions in at least two stages of viral infection, and have begun to elucidate their precise roles in these processes.
本单元研究小 GTP 酶 Arf 家族的 Sec7 结构域鸟嘌呤核苷酸交换因子 (GEF)。我们对这些蛋白质在膜动力学和蛋白质运输中的作用感兴趣。 Arf 和 Arf GEF 是整个细胞内细胞器结构和蛋白质运输的重要调节因子。真核细胞的特征在于其内部膜结构,这对于细胞内发生的许多生化反应的正确空间组织至关重要。细胞核通过核膜与周围的细胞质分开,核膜是一种与内质网 (ER) 连续的双层膜结构。跨膜结构域蛋白、用于分泌的蛋白以及细胞器(例如溶酶体/液泡)的可溶性驻留蛋白穿过内质网膜易位到该细胞器的内腔中。从这里,它们通过高尔基体被运送到最终目的地。内质网遍布整个细胞,并产生多种高尔基体元件,在哺乳动物细胞中,这些高尔基体元件通过微管转运到邻近细胞核的区域,从而形成高尔基体。高尔基体的主要功能是对通过它的蛋白质进行翻译后修饰,并将这些蛋白质分类到细胞中的正确目的地。我们将注意力集中在参与芽殖酵母和哺乳动物细胞中高尔基体运输的 Arf GEF 亚家族。细胞生物学的一个核心问题是,面对每个细胞器的不断进出,如何维持膜系统复杂和动态的结构。特别是,细胞器结构的生成和维持方式,以及结构如何与蛋白质分选和膜重塑的潜在分子事件相关,都是紧迫的问题。 Arf GEF 在膜动力学和细胞器结构中发挥关键作用的证据来自对布雷菲德菌素 A (BFA) 细胞效应的研究,布雷菲德菌素 A (BFA) 是一种对多种细胞类型的分泌和内吞途径的细胞器具有深远影响的药物。 BFA 导致高尔基体完全快速分解并与内质网融合,以及跨高尔基体网络与内体的融合。这些实验首次展示了高尔基体令人难以置信的动态性质,其复杂的结构似乎表明了一种更稳定的状态。我们证明酵母中的三个 Arf GEF 是酵母分泌途径中药物的主要靶标。我们还展示了这种药物的作用机制。 BFA 与交换反应中通常寿命非常短的反应中间体(Arf-GDP-Sec7 结构域复合物)结合,并形成无效的四元复合物,阻止反应进行至完成。这种不寻常的作用机制为新药的开发提供了范例。可以设计筛选通过稳定反应中间体来阻断反应的药物,而不是通常寻找与给定底物竞争的药物。我们正在结合使用酵母遗传学、分子生物学、酵母和哺乳动物细胞成像以及生物化学等技术来阐明 Arf GEF 在蛋白质运输和细胞器结构中的作用。了解膜运输机制的一个重要步骤是通过鉴定相互作用伙伴、阐明膜定位机制和分析体内 Arf GEF 突变体,在分子水平上定义 Arf GEF 的作用。我们已经在芽殖酵母(酿酒酵母)和哺乳动物细胞中鉴定出许多有趣的 Arf GEF 伙伴,目前正在表征这些新伙伴及其在蛋白质运输和膜动力学中的作用。
肠道病毒是正链无包膜RNA病毒小核糖核酸病毒科的成员,包括许多重要的人类病原体,例如脊髓灰质炎病毒、柯萨奇病毒和埃可病毒。尽管肠道病毒(和其他无包膜病毒)没有被膜包围,但它们的复制仍然完全依赖于宿主细胞膜,尽管这一要求的分子细节尚不清楚。肠道病毒与多种人类疾病有关,包括脊髓灰质炎、脑膜脑炎、脑炎、胰腺炎和心肌炎。据估计,全世界每年有十亿人感染由肠道病毒引起。所有肠道病毒都具有相似的约 7.5 kb 的 RNA 基因组,编码四种衣壳蛋白和许多非结构蛋白。这些非衣壳蛋白参与病毒复制和病毒诱导的宿主细胞膜重组。肠道病毒感染最显着的影响之一是细胞内膜系统的大规模重组,最终涉及除线粒体和核膜外的所有内部细胞膜系统。病毒感染时产生的膜结构对于病毒复制是绝对必需的,病毒复制发生在膜表面。病毒感染后两到三个小时内发生膜重组。随后,在感染后三到四个小时,肠道病毒完全抑制细胞内蛋白质转运。这种效应可以通过在哺乳动物细胞中仅表达病毒2B(或其前体2BC)蛋白或单独表达病毒3A蛋白来产生。 2B(C) 蛋白抑制转运的能力与其重新排列分泌途径膜的能力有关,但 3A 蛋白通过不依赖于膜重组的机制阻断转运。 3A 蛋白的转运抑制和复制功能可以通过 3A 中的突变在遗传上分开,这些突变独立影响一种或另一种功能。肠道病毒阻止贩运的能力对于病毒感染性很重要,可能是通过抑制先天性和适应性免疫反应以及抑制病毒感染诱导的细胞凋亡途径来实现的。最近的研究证明了 Arf GTP 酶在脊髓灰质炎病毒和柯萨奇病毒感染中的作用。在体外脊髓灰质炎病毒复制测定中,Arf1、Arf3 和 Arf5(但不是 Arf6)被激活并募集到细胞膜上。此外,在感染后两到三小时内,Arf1 从高尔基体转移到病毒诱导的膜结构中。布雷菲德菌素 A (BFA) 完全抑制脊髓灰质炎病毒的复制,这一事实最初表明了 Arf GEF 在病毒感染性中的作用。通过与 Frank van Kuppeveld 博士和 Ellie Ehrenfeld 博士的团队合作,我们获得了直接证据,证明 BFA 敏感的 Arf GEF 在病毒感染的至少两个阶段具有重要功能,并已开始阐明它们在这些过程中的确切作用。
项目成果
期刊论文数量(12)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
Brefeldin A revealing the fundamental principles governing membrane dynamics and protein transport.
Brefeldin A 揭示了控制膜动力学和蛋白质运输的基本原理。
- DOI:10.1007/0-306-46824-7_6
- 发表时间:2000
- 期刊:
- 影响因子:0
- 作者:Jackson,CL
- 通讯作者:Jackson,CL
Functional analysis of ADP-ribosylation factor (ARF) guanine nucleotide exchange factors Gea1p and Gea2p in yeast.
酵母中 ADP-核糖基化因子 (ARF) 鸟嘌呤核苷酸交换因子 Gea1p 和 Gea2p 的功能分析。
- DOI:10.1016/s0076-6879(01)29090-9
- 发表时间:2001
- 期刊:
- 影响因子:0
- 作者:Peyroche,A;Jackson,CL
- 通讯作者:Jackson,CL
Three dimensional configuration of the secretory pathway and segregation of secretion granules in the yeast Saccharomyces cerevisiae.
酿酒酵母分泌途径的三维结构和分泌颗粒的分离。
- DOI:10.1242/jcs.114.12.2231
- 发表时间:2001
- 期刊:
- 影响因子:4
- 作者:Rambourg,A;Jackson,CL;Clermont,Y
- 通讯作者:Clermont,Y
The ARF exchange factors Gea1p and Gea2p regulate Golgi structure and function in yeast.
ARF 交换因子 Gea1p 和 Gea2p 调节酵母中的高尔基体结构和功能。
- DOI:10.1242/jcs.114.12.2241
- 发表时间:2001
- 期刊:
- 影响因子:4
- 作者:Peyroche,A;Courbeyrette,R;Rambourg,A;Jackson,CL
- 通讯作者:Jackson,CL
N-terminal acetylation targets GTPases to membranes.
N 末端乙酰化将 GTP 酶靶向膜。
- DOI:10.1038/ncb0504-379
- 发表时间:2004
- 期刊:
- 影响因子:21.3
- 作者:Jackson,CatherineL
- 通讯作者:Jackson,CatherineL
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Catherine L Jackson其他文献
Catherine L Jackson的其他文献
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{{ truncateString('Catherine L Jackson', 18)}}的其他基金
Sec7 Domain Arf Exchange Factors In Membrane Traffic
膜流量中的 Sec7 域 Arf 交换因素
- 批准号:
6541339 - 财政年份:
- 资助金额:
-- - 项目类别:
Regulation of Membrane Dynamics by Sec7 Domain Arf Nucle
Sec7 域 Arf 核对膜动力学的调节
- 批准号:
6672659 - 财政年份:
- 资助金额:
-- - 项目类别:
Regulation of Membrane Dynamics by Sec7 Domain Arf Nuc*
Sec7 域 Arf Nuc* 对膜动力学的调节
- 批准号:
6993549 - 财政年份:
- 资助金额:
-- - 项目类别:
Regulation of Membrane Dynamics by Sec7 Domain Arf Nucle
Sec7 域 Arf 核对膜动力学的调节
- 批准号:
7209898 - 财政年份:
- 资助金额:
-- - 项目类别:
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