STRUCTURE AND FUNCTION OF CYSTEINE STRING PROTEINS

描述：（申请人摘要） 半胱氨酸串蛋白是一类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、 最近的证据强烈支持这样的假设，即CSP是一个独特的 发生在对接的突触囊泡和 突触前钙通道。这一点的功能后果是， 突触前钙通道的开放能力 响应于膜去极化。然而，分子和生物物理 这种相互作用的机制仍然未知。因为CSP与 Hsp 70分子伴侣的特异性亚型，我们假设， csp-Ca通道连接涉及Hsp 70的特异性同种型。具体目标1 直接用生物化学和生理学的方法来检验这一假设 接近。其次，我们假设csp参与的事件是 突触囊泡与质膜融合所必需的。的 这一建议的理由是考虑到不寻常的结构， 的CSP。这些蛋白质的半胱氨酸串结构域含有多达11个 连续的半胱氨酸残基。据我们所知，所有这些半胱氨酸 残基被脂肪酰化。具有这种疏水结构域的蛋白质 可以在膜界面处呈现一种构象， 交叉连接相邻的膜（提出了这种相互作用的模型）。 具体目标2通过以下方式测试了这种膜交联模型的有效性： 研究csps对脂质体聚集、融合或溶解的影响 在不同的经验条件下。最后，具体目标3考虑了几个 相关的结构研究的csp是相关的其推定 功能协调发展的计划中的研究包括以下努力：（一）明确解决 天然CSP的脂肪酰化程度;（ii）检查二级 膜结合CSP的结构;和（iii）使用蛋白质 棕榈酰硫酯酶来探测膜束缚和棕榈酰的作用 CSP中的残基在神经末梢起作用。总之，这些调查 将推进我们的知识csp在几个方面是密切相关的， 在突触上起作用。由于csp广泛分布于神经末梢， 在其他分泌细胞中，这项工作对我们的研究具有根本的重要性。 了解正常和病理性膜运输事件 情节

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.