Functional architecture of intracellular Ca2+ signals

项目总结 激活细胞内钙离子通道导致的细胞内钙离子增加调节许多生理功能 事件。细胞内钙信号通路功能障碍与多种疾病状态有关。欣赏 细胞功能是如何被钙离子信号控制的，以及病理信号如何颠覆它们的功能，我们必须 了解细胞内离子通道的“功能结构”如何控制钙信号动力学。 在这个方案中，我们研究了第二信使介导的钙释放的机制基础。 烟酸腺嘌呤二核苷酸磷酸(NAADP)。NAADP能有效地从内体释放钙离子，并 通过激活一系列被称为双孔通道(TPC)的阳离子通道来激活溶酶体。这些信号在本地 调节内溶酶体货物的转运，并可被钙离子放大为全细胞钙信号 通过内质网(ER)上相邻的钙通道诱导钙释放。然而， NAADP诱导的钙释放的分子基础尚不清楚，因为还没有发现NAADP结合部位 TPC蛋白。相反，NAADP通过一种未知的NAADP结合蛋白(NAADP-BP)发挥作用，该蛋白与 TPC复合体介导TPC激活。在这里，一个多PI团队(Marchant，Patel，Slama)：(I)设计了一个 新的双功能探针使光标记策略成为可能，该策略已将难以捉摸的NAADP-BP确定为木星 微管相关同系物2(JPT-2)，以及(Ii)优化的新型模拟NAADP的TPC激活配体 行动。这些工具提供了最终破译NAADP作用的分子基础的机会。这项建议 结合药物化学、分子分析和高分辨率成像专业知识，以解决以下问题： (1)NAADP如何与JPT2绑定，JPT2如何与TPC关联。我们将定义核苷酸 JPT2的结合特性及其在NAADP对TPC敏感性中的作用 (2)JPT2对NAADP诱导的钙信号的功能影响。我们将探讨它的本质和细胞 JPT2的生物学，解析其在NAADP诱导的内源性钙信号中的作用和TPC的靶向 与内质网钙通道相连的特殊微域(膜接触部位)。 (3)NAADP敏感性的生理调节。JPT2的鉴定提供了一种分子 研究细胞NAADP敏感性如何受JPT2调控的句柄 稳定性。我们发现JPT2和TPC与tankyrase相互作用，tankyrase是一种多聚ADP核糖基转移酶。我们 假设JPT2是Tankyrase底物，细胞JPT2水平受ADP核糖化控制。

PROJECT SUMMARY Increases in cellular Ca2+ resulting from activation of intracellular Ca2+ channels regulate many physiological events. Dysfunction of intracellular Ca2+ signaling pathways is involved in many disease states. To appreciate how cell functions are controlled by Ca2+ signals, and how pathological cues subvert their function, we must understand how the `functional architecture' of intracellular ion channels controls Ca2+ signaling dynamics. In this proposal, we investigate the mechanistic basis of Ca2+ release mediated by the second messenger nicotinic acid adenine dinucleotide phosphate (NAADP). NAADP potently releases Ca2+ from endosomes and lysosomes by activating a family of cation channels called two-pore channels (TPCs). These signals locally regulate trafficking of endolysosomal cargoes and can be amplified into whole cell Ca2+ signals by Ca2+- induced Ca2+ release through neighboring Ca2+ channels on the endoplasmic reticulum (ER). However, the molecular basis of NAADP-evoked Ca2+ release is unclear as no NAADP binding site has been identified on the TPC protein. Rather NAADP acts via an unidentified NAADP binding protein (NAADP-BP) associated with the TPC complex to mediate TPC activation. Here, a multi-PI team (Marchant, Patel, Slama): (i) designed a novel bifunctional probe to enable a photolabeling strategy that has identified the elusive NAADP-BP as Jupiter Microtubule Associated Homolog 2 (JPT-2), and (ii) optimized novel TPC activatory ligands that mimic NAADP action. These tools provide opportunity to finally decipher the molecular basis of NAADP action. This proposal coalesces medicinal chemistry, molecular analyses, and high-resolution imaging expertise to resolve: (1) How NAADP binds to JPT2, and how JPT2 associates with TPCs. We will define the nucleotide binding characteristics of JPT2, and the role of JPT2 in conferring NAADP sensitivity to TPCs (2) The functional impact of JPT2 on NAADP-evoked Ca2+ signals. We will probe the essentiality and cell biology of JPT2, resolving its role in endogenous NAADP-evoked Ca2+ signals and targeting of TPCs to specialized microdomains (membrane contact sites) that couple to endoplasmic reticulum Ca2+ channels. (3) How NAADP sensitivity is regulated physiologically. Identification of JPT2 provides a molecular handle to investigate how cellular NAADP sensitivity is physiologically controlled by regulation of JPT2 stability. We show JPT2 and TPCs interact with tankyrase, a poly-ADP-ribosyltransferase. We hypothesize that JPT2 is a tankyrase substrate, with cellular JPT2 levels controlled by ADP ribosylation.