第二信使cADPR的受体鉴定及其探针研究

Cells possess multiple intracellular Ca2+ stores. Mobilization of these stores is a fundamental signaling mechanism responsible for regulating virtually all aspects of cellular activities from fertilization, the beginning of life, to programed cell death, or apoptosis. Cyclic ADP-ribose (cADPR), discovered by us, is a novel molecule for mediating Ca2+ mobilization from intracellular stores. Evidence accumulated over the past two decades establishes that cADPR is a second messenger for Ca2+ mobilization in a wide range of cellular systems spanning three biological kingdoms. The functions that cADPR regulates are equally diverse and range from cell cycle progression in protists, stress responses in plants and sponges, to social behavior in mice through modulation of oxyntocin secretion. We have elucidated the enzymatic synthesis of this messenger and show that cADPR is a cyclic nucleotide derived from NAD and it mobilizes calcium via regulating the calcium channel, ryanodine receptor (RYR), in endoplasmic reticulum (ER).But evidence also showed that RYR is not its receptor. In the first part of this study, we will try to identify the receptor of cADPR via several steps including photolabeling by 8N3-cADPR, purification by fluorescence conversion of cADPR to etheno cADPR or immunoprecipitation by antibody against cADPR and protein profiling by LC/MSMS. In the second part, we will try to develop fluorescent probes of cADPR by combining the techniques such as two-photon microscopy, FRET, cell penetrating carriers,etc, which will provide invaluable tools for monitoring the temporal and spatial changes of cADPR in the cells.

钙离子信号转导调节着生命活动的每个方面，我们发现的钙信使cADPR，广泛调节许多生物学功能。我们已经阐明了它们的酶促合成机制，cADPR由NAD环化而来，调节内质网（ER）上的钙通道兰尼碱受体（RYR）来影响细胞内钙离子的浓度。但cADPR的受体并不是RYR本身，本计划第一部分中我们将利用8N3-cADPR光交联它的相互作用蛋白，并通过荧光标记或免疫沉淀技术，联合LC/MSMS技术鉴定它的身份。第二部分中，我们将利用双光子显微镜、FRET以及细胞穿膜等技术开发cADPR的荧光探针，为研究细胞中cADPR的产生过程提供有效工具。

细胞内钙离子浓度调节着细胞各项生命活动，因此研究胞内释放钙离子的第二信使的作用机制显得很有意义。环腺苷二磷酸核糖（cADPR）是二十多年前由本人发现的一个NAD环形衍生物，它具有调动内质网钙库的活性。这些年许多人都试图寻找它的真正靶蛋白，但是都没有取得实质性的成果。本项目建立了一种新型的联合酶促反应的光交联标记某类核苷小分子结合蛋白的方法，证明可以用于cADPR、NAD、NAADP、NADP等的结合蛋白的鉴定；并成功鉴定到多个cADPR结合蛋白，其功能研究尚在进行中。我们利用cADPR的合成酶CD38或SARM1的碱基交换反应，设计了一类荧光探针，可以指示cADPR合成酶的活性，间接指示cADPR的生成，该工作在投稿过程中。我们完成了CD38的拓扑学机制研究以及SARM1的激活机制，定量了细胞内III型CD38以及鉴定了调节蛋白CIB1及hip（发表于PNAS，2017；JBC, 2019-1），证明II型CD38不参与细胞内cADPR的生物合成（发表于JBC，2019-2），发现另一个信号酶SARM1可以被CD38的抑制剂CZ-48激活，在细胞内合成cADPR（发表于iScience，2019）。