STRUCTURE AND FUNCTION OF CYSTEINE STRING PROTEINS

描述：(申请人摘要) 半胱氨酸串蛋白是一个新的突触囊泡蛋白家族 与神经末梢的两个重要过程有关。第一, 最近的证据有力地支持了这一假设，即CSP是独特的 发生在对接的突触小泡和 突触前钙通道。这样做的功能后果是 相互作用是使突触前钙通道有能力开放 以响应膜的去极化。然而，分子和生物物理 这种相互作用的机制仍不清楚。因为CSP与 Hsp70分子伴侣的特定亚型，我们假设 CSP-Ca通道连接涉及Hsp70的特定亚型。具体目标1 直接使用生化和生理学来检验这一假说 接近了。其次，我们假设CSP参与的活动是 突触小泡与质膜融合所必需的。这个 这项提议的理由源于对这种不寻常的结构的考虑 CSP的数量。这些蛋白质的半胱氨酸串结构域包含多达11个 连续的半胱氨酸残基。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 可以在膜界面采取一种构象，该构象可以有效地 交联型相邻膜(提出了这种相互作用的模型)。 特定目标2通过以下方式测试这种膜交联模型的有效性 CSPs对脂质体聚集、融合或溶解影响的研究 在不同的实验条件下。最后，具体目标3考虑了几个 与其推定相关的CSP相互关联的结构研究 功能。计划中的研究工作包括：(一)明确解决 天然CSP的脂肪酰化程度；(Ii)检查继发性 膜相关CSP的结构；以及(Iii)使用蛋白质 棕榈酰硫酯酶探讨膜系留和棕榈酰化的作用 CSP中的残基作用于神经末梢。总而言之，这些调查 将在与ITS密切相关的几个方面促进我们对CSP的了解 在突触上起作用。因为CSP广泛分布于神经末梢和 在其他分泌细胞中，这项工作对我们的 对正常和病理性膜转运事件的认识 情况。

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.