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）：脂质和蛋白质如何协同形成细胞中的功能性膜结构域仍然是细胞生物学中尚未解答的主要问题。脂筏假说已成为理解这一过程的广泛研究模型。筏与从膜运输到细胞信号传导等正常细胞功能有关。它们还与许多疾病有关，并被认为是病原体进入细胞的主要部位。目前的模型表明，筏域通常以纳米级成分波动的形式存在于细胞中，但可以稳定形成功能筏。然而，稳定筏如何组装和发挥作用仍不清楚。在此应用中，我们建议使用霍乱毒素的无毒膜结合 B 亚基 (CTxB) 作为模型来解决这些基本问题。 CTxB 是一种同五聚体，含有 5 个糖脂受体神经节苷脂 GM1 的结合位点，神经节苷脂 GM1 本身是一种筏相关分子。 CTxB 具有同时聚集多个 GM1 分子的能力，因此被广泛认为可用作构建稳定筏结构域的筏交联剂。此外，霍乱毒素进入细胞被认为重要地取决于其对筏依赖性、网格蛋白独立的内吞途径的靶向。然而，这些 CTxB 稳定结构域形成的机制、该过程在细胞中如何调节以及稳定的筏如何优先分选到不依赖于网格蛋白的内吞载体中仍然知之甚少。在这里，我们建议通过以下目标来研究这些问题：1）检验 CTxB 必须聚集筏相关糖脂以组装稳定的筏结构域的假设； 2) 确定毒素稳定筏如何与肌动蛋白细胞骨架相互作用； 3) 确定稳定筏是否在通过机械变形膜通过网格蛋白独立内吞作用靶向 CTxB 的摄取中发挥积极作用。成功完成这些目标将为蛋白质和脂质合作形成稳定筏、筏组装如何受细胞骨架调节以及稳定筏在内吞作用中的作用提供机制见解。