Calcium Influx Factor

描述（申请人提供）：PI实验室的长期目标是确定钙库操纵的钙内流（SOCE）的分子组成和机制，已知SOCE在可兴奋和不可兴奋细胞中的细胞内钙库耗尽时被激活。这项创新建议的重点是钙内流因子（CIF），一种众所周知的难以捉摸且尚未鉴定的信使，它是在储存耗尽时产生的，并且在迄今为止测试的所有活细胞（从酵母到人类）中发现。我们拥有独特的知识和丰富的经验与CIF提取物。我们发现了CIF的生理靶点（一种特异性的质膜结合的钙离子非依赖性磷脂酶A2），并证明了其在SOCE中的关键作用，这一点已被其他研究者所证实。最近，我们确定了STIM1作为内质网中CIF生产的触发器。在我们以前的研究中，主要集中在CIF的产生，作用及其在SOCE途径中的作用的机制，PI的实验室积累了独特的知识和专业知识，最终可能允许分子鉴定这个极其重要但仍然难以捉摸的信使。我们已经建立了先进的多步纯化和生物学测试的CIF的新方法，并可以获得足够数量的最高纯度的CIF，这是必不可少的仪器分析。我们的新建议的目标是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：1）关于CIF产生的分子触发物和CIF的生理靶点的知识，2）使用CIF提取物的丰富经验，3）CIF的新的丰富来源，4）用于其精细纯化的所有工具和新方法，5）用于测试样品和候选分子的CIF活性的新的有效和灵敏的生物测定方法和方法，6）最先进的设施，并与质谱、NMR和SOCE机制方面的世界级专家建立了合作关系。我们独特的专业知识和广泛的初步数据完全支持CIF纯化和分子结构鉴定的可行性。本提案的具体目的是确定CIF。我们将：1.1.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.在生物测定系统中验证CIF身份，并将其与内源性CIF活性匹配。1.4.确定CIF产生的生化途径。公共卫生相关性：该提案的目标是确定钙内流因子（CIF），这是一种新的第二信使，目前为止测试的所有细胞类型（从酵母到人类）普遍产生，并参与激活钙库操作的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。CIF鉴定的可行性得到了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的充分支持。

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.