Functional architecture of intracellular Ca2+ signals

项目摘要 由细胞内Ca 2+通道激活引起的细胞内Ca 2+增加调节许多生理学过程。 事件细胞内Ca 2+信号通路的功能障碍涉及许多疾病状态。欣赏 细胞功能是如何被Ca 2+信号控制的，以及病理学的线索是如何破坏它们的功能的，我们必须 了解细胞内离子通道的“功能结构”如何控制Ca 2+信号动力学。 在这个提议中，我们研究了第二信使介导的Ca 2+释放的机制基础， 烟酸腺嘌呤二核苷酸磷酸（NAADP）。NAADP有效地从内体释放Ca 2+， 通过激活称为双孔通道（TPC）的阳离子通道家族来激活溶酶体。这些信号在本地 调节内溶酶体货物的运输，并可通过Ca 2 +- 通过内质网（ER）上邻近的Ca ~（2+）通道诱导Ca ~（2+）释放。但 NAADP诱导的Ca 2+释放的分子基础尚不清楚，因为在细胞表面没有发现NAADP结合位点。 TPC蛋白相反，NAADP通过一种未鉴定的NAADP结合蛋白（NAADP-BP）起作用， TPC复合物介导TPC活化。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，已确定难以捉摸的NAADP-BP作为木星 微管相关同源物2（JPT-2），和（ii）模拟NAADP的优化的新型TPC活化配体 行动上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： (1)NAADP如何与JPT 2结合，以及JPT 2如何与TPC结合。我们将定义核苷酸 JPT 2的结合特性，以及JPT 2在赋予TPC NAADP敏感性中的作用 (2)JPT 2对NAADP诱发的Ca 2+信号的功能影响。我们将探讨其本质和细胞 JPT 2的生物学，解决其在内源性NAADP诱发的Ca 2+信号中的作用，并靶向TPC， 与内质网Ca 2+通道偶联的特殊微区（膜接触位点）。 (3)NAADP敏感性如何在生理上调节。JPT 2的鉴定提供了一种分子 研究细胞NAADP敏感性如何通过JPT 2的调节进行生理控制 稳定我们显示JPT 2和TPC与端锚聚合酶（一种聚ADP核糖基转移酶）相互作用。我们 假设JPT 2是端锚聚合酶底物，细胞JPT 2水平受ADP核糖基化控制。

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.