Function and assembly of toxin-stabilized domains

毒素稳定结构域的功能和组装

• 批准号: 8532431
• 负责人: Anne K Kenworthy
• 金额: $ 32.3万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-15 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8532431
• 关键词: Actins; Address; Affinity; Bacterial Toxins; Binding; Binding Sites; Biological Assay; Cell membrane; Cell physiology; Cells; Cellular biology; Ceramides; Cholera; Cholera Toxin; Cholera Toxin Protomer B; Clathrin; Complex; Conflict (Psychology); Crosslinker; Cytoskeleton; Data; Diffuse; Disease; Endocytosis; Event; Ganglioside GM1; Glycolipids; Label; Life; Link; Lipid Binding; Lipids; Liquid substance; Membrane; Membrane Microdomains; Membrane Protein Traffic; Modeling; Pathway interactions; Play; Process; Property; Proteins; Reagent; Role; Signal Transduction; Site; Sorting - Cell Movement; Structure; Study models; Testing; Toxin; Vesicle; Work; base; glycolipid receptor; insight; interest; mutant; nanoscale; novel; pathogen; public health relevance; receptor; research study; tool; unilamellar vesicle; uptake

DESCRIPTION (provided by applicant): How lipids and proteins cooperate to form functional membrane domains in cells remains a major unanswered question in cell biology. The lipid raft hypothesis has become a widely studied model for understanding this process. Rafts have been implicated in normal cellular functions ranging from membrane trafficking to cell signaling. They have also have been linked to a number of diseases and are thought to represent a major site for pathogen entry into cells. Current models propose that raft domains normally exist as nanoscale compositional fluctuations at steady state in cells, but can be stabilized to form functional rafts. However, how stabilized rafts assemble and function remains unclear. In this application, we propose to address these fundamental questions using the non-toxic membrane binding B-subunit of cholera toxin (CTxB) as a model. CTxB is a homopentamer that contains 5 binding sites for its glycolipid receptor, ganglioside GM1, itself a raft-associated molecule. Give its ability to cluster multiple GM1 molecules simultaneously, CTxB is widely regarded to serve as a raft crosslinker that builds stabilized raft domains. Moreover, the entry of cholera toxin int cells is thought to depend importantly on its targeting to raft-dependent, clathrin-independent endocytic pathways. However, the mechanisms by which these CTxB- stabilized domains form, how this process is regulated in cells, and how stabilized rafts are preferentially sorted into clathrin-independent endocytic carriers remain poorly understood. Here, we propose to investigate these questions though the following aims: 1) to test the hypothesis that CTxB must cluster raft-associated glycolipids to assemble stabilized raft domains; 2) to determine how toxin-stabilized rafts interact with the actin cytoskeleton; and 3) to determine if stabilized raft play an active role in targeting CTxB for uptake by clathrin independent endocytosis by mechanically deforming membranes. Successful completion of these aims will provide mechanistic insights into proteins and lipids cooperate to form stabilized rafts, how raft assembly is regulated by the cytoskeleton, and the role of stabilized rafts in endocytosis.

描述（由申请人提供）：脂质和蛋白质如何合作在细胞中形成功能性膜结构域仍然是细胞生物学中的主要未解决问题。脂质筏假说已成为一个广泛研究的模型，用于理解这一过程。筏已经与正常细胞功能有关，从膜运输到细胞信号。它们也与多种疾病有关，被认为代表了病原体进入细胞的主要部位。当前的模型表明，筏结构域通常是在细胞中稳态处作为纳米级组成波动而存在的，但可以稳定以形成功能性筏。但是，稳定的筏的组装和功能尚不清楚。在此应用程序中，我们建议使用霍乱毒素（CTXB）作为模型的非毒性膜结合B-亚基的解决这些基本问题。 CTXB是一种同质体，其中包含5个用于其糖脂受体的结合位点，神经脂GM1本身是与筏相关的分子。 CTXB赋予其同时聚集多个GM1分子的能力，被广泛认为是木筏交叉链接器，该交联链构建稳定的筏域。此外，认为霍乱毒素INT细胞的进入重要地取决于其靶向筏依赖性，非甲状腺素独立的内吞途径。然而，这些CTXB稳定结构域形成的机制，在细胞中如何调节该过程的方式以及如何优先将稳定的筏分类为非网状蛋白独立的内吞载体仍然较少了解。在这里，我们建议通过以下目的调查这些问题：1）检验CTXB必须簇簇相关的糖脂才能组装稳定的筏域的假设； 2）确定毒素稳定的筏如何与肌动蛋白细胞骨架相互作用； 3）确定稳定的筏在靶向CTXB中是否起着积极作用，通过机械变形膜来靶向CTXB摄取CTXB以摄取独立的内吞作用。这些目标的成功完成将提供对蛋白质和脂质配合以形成稳定筏的机械见解，如何通过细胞骨架调节木筏组装，以及稳定筏在内吞作用中的作用。