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. ? ?
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