STRUCTURE AND FUNCTION OF CYSTEINE STRING PROTEINS

半胱氨酸串蛋白的结构和功能

• 批准号: 6363731
• 负责人: CAMERON B GUNDERSEN
• 金额: $ 20.03万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-03-01 至 2004-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6363731
• 关键词: Torpedo; Xenopus; amidases; calcium channel; cell membrane; circular dichroism; cysteine; enzyme activity; fatty acylation; membrane activity; membrane model; membrane permeability; molecular chaperones; neural transmission; protein isoforms; protein structure function; secretory protein; synapses; voltage /patch clamp

DESCRIPTION: (Applicant's Abstract) Cysteine-string proteins (csps) are a novel family of synaptic vesicle proteins that have been implicated in two important processes at nerve endings. First, recent evidence strongly supports the hypothesis that csps are part of a unique regulatory interaction that takes place between a docked synaptic vesicle and presynaptic calcium (Ca) channels. The functional consequence of this interaction is that the presynaptic Ca channels are rendered competent to open in response to membrane depolarization. However, the molecular and biophysical mechanisms of this interaction remain unknown. Because csps associate with a specific isoform of Hsp70 molecular chaperones, we have hypothesized that the csp-Ca channel link involves a specific isoform of Hsp70. Specific Aim 1 directly tests this hypothesis using both biochemical and physiological approaches. Second, we have postulated that csps participate in events that are necessary for the fusion of synaptic vesicles with the plasma membrane. The rationale for this proposal stems from a consideration of the unusual structure of csps. The cysteine string domain of these proteins contains as many as 11 consecutive cysteine residues. As far as we know, all of these cysteine residues are fatty acylated. A protein with this type of a hydrophobic domain can assume a conformation at membrane interfaces that can effectively cross-link adjacent membranes (a model of this interaction is presented). Specific Aim 2 tests the validity of this model of membrane cross-linking by studying the effect of csps on the aggregation, fusion or lysis of liposomes under different empirical conditions. Finally, Specific Aim 3 considers several inter-related structural studies of csp that are relevant to its presumed functions. Among the planned studies are efforts: (I) to resolve unequivocally the degree of fatty acylation of native csps; (ii) to examine the secondary structure of membrane-associated csps; and (iii) to use a protein palmitoylthioesterase to probe the role of membrane tethering and palmitoyl residues in csp function at nerve endings. In summary, these investigations will advance our knowledge of csps on several fronts that are germane to its function at synapses. Because csps are widely distributed at nerve endings and in other secretory cells, this work is of fundamental importance to our understanding of membrane trafficking events in normal and pathological circumstances.

描述：（申请人的摘要） 半胱氨酸弦蛋白（CSP）是一个新型突触囊泡蛋白的家族 这与神经末梢的两个重要过程有关。第一的， 最近的证据强烈支持CSP是独特的一部分的假设 在停靠的突触囊泡和 突触前钙（CA）通道。功能后果 互动是突触前CA频道具有胜任 响应膜去极化。但是，分子和生物物理 这种相互作用的机制仍然未知。因为CSP与 HSP70分子伴侣的特定同工型，我们假设 CSP-CA通道链路涉及HSP70的特定同工型。具体目标1 直接使用生化和生理学来直接检验该假设 方法。其次，我们假设CSP参与了 突触囊泡与质膜融合所必需的。这 该提议的理由源于对异常结构的考虑 CSP。这些蛋白质的半胱氨酸弦结构域包含多达11 连续半胱氨酸残基。据我们所知，所有这些半胱氨酸 残基是脂肪酰化的。具有这种疏水结构域的蛋白质 可以在膜界面上假设一个构象 交联相邻膜（提出了这种相互作用的模型）。 特定的目标2测试了这种膜交联模型的有效性 研究CSP对脂质体聚集，融合或裂解的影响 在不同的经验条件下。最后，特定目标3考虑了几个 与CSP相关的结构研究与其推测有关 功能。在计划的研究中有努力：（i）明确解决 天然CSP的脂肪酰化程度； （ii）检查次要 膜相关CSP的结构； （iii）使用蛋白质 棕榈酰硫代酯酶探测膜束缚和棕榈酰的作用 CSP功能在神经末端的残基。总之，这些调查 将提高我们对CSP的了解，这些方面的几个方面 在突触中的功能。因为CSP广泛分布在神经末端，并且 在其他分泌细胞中，这项工作对我们来说至关重要 在正常和病理中了解膜贩运事件 情况。