Structural dynamics of the proteins involved in calcium-triggered exocytosis

参与钙触发胞吐作用的蛋白质的结构动力学

• 批准号: 7571054
• 负责人: Justin Worthington Taraska
• 金额: $ 8.4万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-01-15 至 2011-01-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7571054
• 关键词: Address; Affect; Architecture; Award; Behavior; Binding; Binding Proteins; Biochemistry; Biological; Brain; Calcium; Cell membrane; Cells; Color; Complement; Complex; DNA Sequence Rearrangement; Development Plans; Disease; Docking; Endocrine; Enzymes; Exocytosis; Fluorescence; Fluorescence Microscopy; Fluorescence Resonance Energy Transfer; Fluorometry; Grant; Image; Incentives; Investigation; Ion Channel; Laboratories; Life; Ligands; Maps; Measures; Membrane; Membrane Fusion; Membrane Proteins; Mentors; Molecular; Molecular Conformation; Movement; Neuroendocrine Cell; Neurons; Neurotransmitters; Peptides; Phase; Principal Investigator; Protein Dynamics; Proteins; Regulation; Relative (related person); Role; SNAP receptor; Set protein; Signal Transduction; Structure; Synapses; Synaptic Transmission; Synaptic Vesicles; System; Techniques; Testing; Time; Vesicle; Work; career development; cellular imaging; chemical release; in vivo; insight; interest; molecular rearrangement; nanoscale; neurotransmitter release; novel; patch clamp; protein function; protein structure; research study; small molecule; stoichiometry; structural biology; syntaxin; syntaxin 1; target SNARE proteins

Neurons communicate with one another by releasing neurotransmitters, peptides, and small molecules by exocytosis. The fusion of neurotransmitter-containing vesicles with the plasma membrane is driven by a core set of proteins known as SNAREs along with several regulatory factors. While the identity of many of these proteins and their requirement in calcium-triggered exocytosis has been demonstrated, the underlying molecular mechanism by which SNAREs and SNARE-associated proteins function is still unknown. A number of fundamental questions remain: How is the core SNARE fusion machinery activated to drive membrane fusion? What are the orientations, architectures, and stoichiometries of the proteins that constitute the fusion machine? What are the underlying structural rearrangements of the proteins associated with membrane fusion? To address these and related questions, we will employ a combination of novel fluorescence techniques, biochemistry, and live cell imaging. We will focus our efforts at two levels of investigation, studying the structure and dynamics of SNAREs in isolated membrane patches as well as in intact living cells. In this grant two specific aims are proposed: 1) map the orientation of the plasma membrane resident t-SNARE Syntaxin and Syntaxin binding proteins relative to each other and to the plasma membrane with patch-clamp fluorometry; and 2) characterize the stoichiometry, dynamic associations, and functional roles of SNAREs and SNARE-associated enzymes in vivo with total internal reflection fluorescence (TIRF) microscopy of single exocytic vesicles and fluorescently-tagged proteins. These studies will map the precise molecular steps required to activate SNAREs and drive calcium-triggered membrane fusion in neurons and endocrine cells. A detailed understanding of how these enzymes function at the atomic level will provide insights into the control and regulation of synaptic transmission, how its malfunction leads to disease, and how its modulation might be targeted for novel therapies.

神经元通过胞吐作用释放神经递质、肽和小分子来相互交流。含有神经递质的囊泡与质膜的融合是由一组称为SNARE的核心蛋白质沿着几种调节因子驱动的。虽然许多这些蛋白质的身份和它们在钙触发的胞吐作用中的需求已经被证明，但SNARE和SNARE相关蛋白质功能的潜在分子机制仍然未知。一些基本的问题仍然存在：核心的SNARE融合机制是如何被激活来驱动膜融合的？构成融合机器的蛋白质的方向、结构和化学计量是什么？与膜融合相关的蛋白质的潜在结构重排是什么？为了解决这些和相关的问题，我们将采用新的荧光技术，生物化学和活细胞成像的组合。我们将集中精力在两个层次的调查，研究结构和动态的SNARE在孤立的膜补丁，以及在完整的活细胞。本研究提出了两个具体的目标：1）用膜片钳荧光法定位质膜上的t-SNARE Syntaxin和Syntaxin结合蛋白的相对位置以及与质膜的相对位置;和2）表征化学计量，动态缔合，用全内反射荧光（TIRF）研究SNARE及其相关酶在体内的功能作用单个胞吐囊泡和荧光标记蛋白的显微镜检查。这些研究将绘制激活SNARE并驱动神经元和内分泌细胞中钙触发的膜融合所需的精确分子步骤。对这些酶在原子水平上如何发挥作用的详细了解将有助于深入了解突触传递的控制和调节，其功能障碍如何导致疾病，以及其调节如何成为新疗法的目标。