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容量性Ca 2+内流的关键要素，并建立钙库操纵的Ca 2+传导（SOC）通道激活的分子机制。尽管钙池调控的钙内流途径具有重要意义，但其关键元件之一的钙内流因子（CIF）的身份至今仍是一个谜。CIF由内质网在Ca 2+储存耗尽后产生，并且可以从细胞中提取。最近，我们已经表明，CIF激活单SOC通道和Ca 2+内流在不同的细胞类型，但这个神秘的信使的分子身份仍然未知。我们最近的研究在这个长达十年的谜团中带来了一个非常重要的突破-我们发现CIF的生物靶点不是SOC通道本身，而是一种Ca 2+非依赖性磷脂酶A2（iPLA 2），我们发现它是导致SOC通道激活的新膜界定级联反应的关键决定因素。我们发现CIF从iPLA 2置换抑制性CaM，导致其活化，并且其溶血磷脂（LysoPL）产物反过来活化SOC通道。最近的这一发现使得启动一项新研究成为可能，该研究将最终确定CIF的分子身份。这是一个独立的新项目，大大扩展了我原来的R 01资助（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，但它是我们正在进行的成功研究的直接和逻辑延伸。为了实现这一新目标，需要更多的资金。这就是为什么我们建议CIF识别作为我现有的资助RO 1补助金的补充。这一重点补充的目标是确定CIF的分子身份。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识别CIF的关键因素：1）关于CIF作用的靶点和机制的新知识，2）与CIF一起工作的广泛的先前经验，3）CIF的新的丰富来源，4）用于CIF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系统和方法。本补充提案的目的是确定ClF的分子特性。 我们将使用血小板作为CIF的来源，连续的HPLC步骤，其最好的纯化，质谱结合碎片技术，用于确定其化学成分，以及几种新的生物测定法，以检测其活性和测试候选分子。所有的方法都已建立并成功地应用于我的实验室。这些方法都很简单，这一建议的可行性得到了广泛的初步数据的充分支持。我们拥有所有的知识和工具，可以在3年的时间内确定CIF。本补充提案的具体目标是：目标1。确定CIF的分子鉴别。我们将：1.1.从血小板提取物中完全精制纯化CIF。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分子的确切结构组成并确定其分子身份。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iPLA 2中置换CaM，并激活SOC通道和容量性Ca 2+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.