Identification of Calcium Influx Factor (suppl. to RO1)

描述（由申请人提供）：我们的长期目标是确定Ca2+容性内流的关键因素，并建立存储操作的Ca2+传导（SOC）通道激活的分子机制。尽管商店运作的Ca2+内流通路非常重要，但其关键元件之一Ca2+内流因子（CIF）的身份仍然是一个谜。CIF是由内质网在Ca2+储存耗尽后产生的，可以从细胞中提取。最近，我们已经证明CIF激活了不同细胞类型的单SOC通道和Ca2+内流，但这个神秘信使的分子身份仍然未知。我们最近的研究为这个长达十年的谜团带来了一个非常重要的突破——我们发现CIF的生物学靶点不是SOC通道本身，而是一种Ca2+不依赖的磷脂酶A2 (iPLA2)，我们发现它是导致SOC通道激活的新膜划定级联反应的关键决定因素。我们发现CIF取代iPLA2中的抑制性CaM导致其活化，并且其溶血磷脂（LysoPL）产物反过来激活SOC通道。这一最新发现使得开展一项新的研究成为可能，这项研究将最终确定CIF的分子特性。这是一个独立的新项目，大大扩展了我原来的R01拨款（专门用于CIF下游信号级联的详细研究）。我们以前没有提出过CIF鉴定，但这是我们正在进行的成功研究的直接和合乎逻辑的延伸。为了实现这一新的目标，需要额外的资金。这就是为什么我们建议将CIF鉴定作为我现有RO1基金的补充。这个集中补充的目标是确定CIF的分子特性。我们知道这是一个雄心勃勃的目标，但我们相信我们最终拥有了成功鉴定CIF的所有关键因素：1)关于CIF作用靶点和机制的新知识，2)丰富的CIF工作经验，3)新的丰富的CIF来源，4)CIF纯化的所有工具和新方法，5)新的有效和敏感的生物测定系统和方法，用于测试样品和CIF活性的候选分子。本补充建议的目的是确定ClF的分子特性。我们将使用血小板作为CIF的来源，顺序高效液相色谱步骤进行最精细的纯化，质谱结合碎片化技术确定其化学成分，并使用几种新的生物测定方法来检测其活性和测试候选分子。所有方法都已建立，并已在我的实验室中成功应用。方法是直接的，这个建议的可行性充分支持广泛的初步数据。我们拥有所有的知识和工具，可以在3年内确定到岸价。本补充建议的具体目标是：目标1。确定CIF的分子特性。我们将：从血小板提取物中完成CIF的精制纯化。1.2. 测试物理/化学/酶处理对CIF稳定性/有效性的影响。1.3. 确定CIF分子的确切结构成分并确定其分子特性。1.4. 使用我们先进的生物测定系统测试所有候选分子的cif样活性。我们将测试它们是否从iPLA2中取代CaM，并激活SOC通道和容性Ca2+内流。

DESCRIPTION (provided by applicant): Our long-term goal is to define the key elements in capacitative Ca2+influx, and to establish the molecular mechanism of activation of store-operated Ca2+-conducting (SOC) channels. In spite of the tremendous importance of the store-operated Ca 2+ influx pathway, the identity of one of its crucial elements, Ca2+ influx factor (CIF), remains a mystery. CIF is produced by the endoplasmic reticulum following depletion of Ca2+stores, and can be extracted from the cells. Recently we have shown that CIF activates single SOC channels and Ca2+ influx in different cell types, but the molecular identity of this mysterious messenger remains unknown. Our most recent studies brought a very important break-through in this decade-long mystery - we found that a biological target for CIF is not SOC channel itself, but it is a Ca2+-independent phospholipase A2 (iPLA2) that we found to be a crucial determinant of the new membrane-delimited cascade of reactions that leads to activation of SOC channels. We found that CIF displaces inhibitory CaM from iPLA2 leading to its activation, and its lysophospholipid (LysoPL) products in turn activate SOC channels. This recent discovery made it possible to launch a new study that will finally determine the molecular identity of CIF. This is a separate new project that significantly extends my original R01 grant (which is devoted to the detailed studies of the signaling cascade downstream from CIF). We have not proposed CIF identification before, but it is a direct and logical extension of our successful ongoing studies. For achieving this new goal, additional funds are required. That is the reason why we are proposing CIF identification as a supplement to my existing funded RO1 grant. The goal of this focused supplement is to determine the molecular identity of CIF. We understand that this is an ambitious goal, but we believe that we finally have everything crucial for our success in CIF identification: 1) the new knowledge on the target and mechanism of CIF action, 2) extensive prior experience in working with CIF, 3) new abundant source of CIF, 4) all the tools and novel approaches for CIF purification, 5) new effective and sensitive bioassay systems and methods for testing samples and candidate molecules for CIF activity. The goal of this supplemental proposal is to determine the molecular identity of ClF. We will use platelets as a source of CIF, sequential HPLC steps for its finest purification, mass spectrometry combined with fragmentation techniques for the determination of its chemical composition, and several new bioassays to detect its activity and test candidate molecules. All the methods are established and have been successfully used in my lab. The approaches are straightforward, and feasibility of this proposal is fully supported by extensive preliminary data. We have all the knowledge and tools to make CIF determination possible in 3-year time frame. Specific aims of this supplemental proposal are: Aim 1. To determine the molecular identity of CIF. We will: 1.1. Complete refined purification of CIF from platelet extracts. 1.2. Test the effect of physical/chemical/enzymatic treatments on CIF stability/effectiveness. 1.3. Determine the exact structural components of CIF molecule and define its molecular identity. 1.4. Test all the candidate molecules for CIF-like activity using our advanced bioassay systems. We will test if they displace CaM from iPLA2, and activate SOC channels and capacitative Ca2+ influx.