Calcium Influx Factor

描述（由申请人提供）：π的实验室的长期目标是定义分子组件和存储的机制——运营Ca2 +条目(跌倒),这是众所周知的,被激活在消耗细胞内的钙离子在兴奋和non-excitable细胞这一创新提案的重点是钙流入的因素(CIF),一个出了名的难以捉摸,仍然不确定信使,产生损耗的商店和被发现在所有活细胞检测到目前为止(从酵母到人类)。我们在CIF提取方面有独特的知识和丰富的经验。我们已经发现了CIF（一种特定的质膜结合Ca2+不依赖磷脂酶A2）的生理靶点，并证明了它在SOCE中的关键作用，这被其他研究者证实。最近，我们发现STIM1是内质网中CIF产生的触发因子。在我们之前的研究中，主要关注CIF的产生机制、作用及其在SOCE通路中的作用，PI的实验室积累了独特的知识和专业知识，可能最终允许分子鉴定这种极其重要但仍然难以捉摸的信使。我们建立了CIF的高级多步骤纯化和生物检测的新方法，可以获得足够数量的最高纯度的CIF，这是仪器分析所必需的。我们新提案的目标是CIF的分子鉴定。我们知道这是一个雄心勃勃的目标，但我们相信，我们终于拥有了成功实现这一目标的一切条件：1)关于CIF生产的分子触发和CIF的生理目标的知识，2)CIF提取物的丰富经验，3)新的丰富的CIF来源，4)所有用于其精细纯化的工具和新方法，5)新的有效和敏感的生物测定方法和方法，用于测试样品和CIF活性的候选分子，6)最先进的设施，并与世界一流的质谱专家建立了合作，核磁共振和SOCE机制。我们独特的专业知识和广泛的初步数据充分支持了CIF纯化和鉴定其分子结构的可行性。本提案的具体目的是确定CIF。我们将：使用先进的质谱和核磁共振技术确定CIF的化学和结构成分。1.2. 合成CIF分子及其无活性类似物。1.3. 验证CIF在生物测定系统中的身份，并将其与内源性CIF活性相匹配。1.4. 确定CIF生产的生化途径。公共卫生相关性：本提案的目标是确定钙内流因子（CIF），这是一种新的第二信使，目前已测试的所有细胞类型（从酵母到人类）都普遍产生，并参与激活储存操作的Ca2+进入途径，这是决定健康和疾病中Ca2+稳态的主要机制之一。我们独特的专业知识、广泛的初步数据和PI实验室已经开发的先进方法充分支持了CIF识别的可行性。

DESCRIPTION (provided by applicant): The long term goal of the PI's laboratory is to define the molecular components and mechanism of the store- operated Ca2+ entry (SOCE), which is known to be activated upon depletion of intracellular Ca2+ stores in excitable and non-excitable cells The focus of this innovative proposal is on calcium influx factor (CIF), a notoriously elusive and still un- identified messenger, that is produced upon depletion of the stores and was found in all living cells tested so far (from yeast to humans). We have unique knowledge and extensive experience in working with CIF extracts. We had discovered the physiological target for CIF (a specific plasma membrane bound Ca2+ independent phospholipase A2), and demonstrated its crucial role in SOCE, which was confirmed by other investigators. Most recently, we identified STIM1 as a trigger for CIF production in the endoplasmic reticulum. During our previous studies that focused on the mechanism of CIF production, action and its role in SOCE pathway, PI's laboratory had accumulated unique knowledge and expertise that may finally allow molecular identification of this extremely important but still elusive messenger. We had established new methods for advanced multi-step purification and biological testing of CIF, and can obtain sufficient quantities of CIF of the highest purity that is essential for its instrumental analysis. The goal of our new proposal is molecular identification of CIF. We understand that this is an ambitious goal, but we believe that we finally have everything for its successful achievement: 1) the knowledge about the molecular trigger of CIF production, and about physiological target of CIF, 2) extensive experience in working with CIF extracts, 3) new abundant source of CIF, 4) all the tools and novel approaches for its fine purification, 5) new effective and sensitive bioassay approaches and methods for testing samples and candidate molecules for CIF activity, 6) state-of-the-art facilities and established collaborations with the world class experts in mass spectrometry, NMR and SOCE mechanism. The feasibility of CIF purification and identification of its molecular structure is fully supported by our unique expertise, and extensive preliminary data. Specific Aim of this proposal is to identify CIF. We will: 1.1. Determine chemical and structural components of CIF using advanced mass spectrometry and NMR techniques. 1.2. Synthesize CIF molecule and its inactive analogs. 1.3. Verify CIF identity in bioassay systems, and match it with endogenous CIF activity. 1.4. Identify the biochemical pathway for CIF production. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to identify calcium influx factor (CIF), a novel second messenger that is ubiquitously produced by all the cell types tested so far (from yeast to human), and is involved in activation of the store-operated Ca2+ entry pathway, one of the major mechanisms that determine Ca2+ homeostasis in health and disease. The feasibility of CIF identification is fully supported by our unique expertise, extensive preliminary data and advanced approaches that are already developed in PI's lab.