CD38拓扑学决定因素及其调节机制研究

CD38 is a trans-membrane protein ubiquitously expressed in virtually all mammalian tissues. Two decades of work has revealed that it is a novel signaling enzyme responsible for synthesizing and metabolizing cyclic ADP-ribose (cADPR), a novel Ca2+ messenger discovered by us that modulates the channel activity of the ryanodine receptor and mobilizes the endoplasmic Ca2+ stores. Gene knockout studies have established that CD38 regulates a wide range of physiological functions, from insulin secretion, susceptibility to bacterial infection, to social behavior of mice through modulating neuronal oxytocin secretion. We have elucidated the mechanism of how it catalyzes the multiple reactions responsible for metabolizing cADPR to atomic resolution by X-ray crystallography. How CD38 is endogenously regulated, however, remains largely unexplored. One of the main unresolved issue centers on its membrane topology. CD38 has long been thought to have a type II membrane orientation with the catalytic C-domain facing outside. It has never been fully understood how an ecto-enzyme can gain access to its intracellular substrates, NAD, and produce the signaling messenger, cADPR that target intracellular Ca2+ stores. The main focus of this application is to elucidate this topological paradox of CD38 and to provide long sought insights into the endogenous regulatory mechanisms of this novel Ca2+ signaling enzyme. The specific Aims are: 1. Generation of specific antibodies for Type III CD38. 2. Topology of CD38 2.1 intracellular nanobody for type III CD38. 2.2 BiFC using nanobody-VenN and CD38-VenC. 2.3 G4S for staining and immunoprecipetation of type III CD38. 3. Type III CD38 and cell differentiation in HL-60 Cells. 4. Determinants of CD38 topology Positively-charged residues, serines and cysteines 5. Lipid content and CD38 topology.

跨膜蛋白CD38负责合成和代谢具有钙动员活性的第二信使环腺苷二磷酸核糖（cADPR），它参与调节多项生理功能，包括胰岛素分泌、催产素分泌相关社会学行为等。我们已阐明CD38体外代谢cADPR多个反应的催化机制，但细胞内代谢机制尚待研究，其中的关键问题是其膜拓扑学。通常认为它是II型膜蛋白，其催化结构域位于细胞外，而胞外酶如何接触胞内底物、生成作用于胞内受体的cADPR的过程一直未得到真正理解。为了诠释该拓扑学悖论，我们提出了"III型CD38"假说并发表了首个证据。本项目将开发更多用于识别III型CD38的工具，包括识别非糖基化CD38的抗体和胞内表达的CD38抗体，用于III型CD38的成像和检测，以及评价HL-60分化过程中III型CD38的功能。我们将通过研究CD38氨基酸序列中的功能性氨基酸和生物膜脂质成分对于CD38膜定向的影响，来探讨这个钙信号酶的内源调节机制。

跨膜蛋白CD38 负责合成和代谢具有钙动员活性的第二信使环腺苷二磷酸核糖（cADPR），它参与调节多项生理功能，包括胰岛素分泌、催产素分泌相关社会学行为等。我们已阐明CD38 体外代谢cADPR多个反应的催化机制，但细胞内代谢机制尚待研究，其中的关键问题是其膜拓扑学。通常认为它是II型膜蛋白，其催化结构域位于细胞外，而胞外酶如何接触胞内底物、生成作用于胞内受体的cADPR 的过程一直未得到真正理解。为了诠释该拓扑学悖论，我们提出了“III型CD38”假说并发表了首个证据。.本项目中，我们开发了更多用于识别非糖基化CD38的抗体G4S，发现CD38跨膜区两端的正电氨基酸对其膜定向起了决定性作用，另外三个丝氨酸的磷酸化可能使CD38发生天然翻转。还开发了一系列纳米抗体，获得了它们与CD38的共晶结构，基于结构信息，我们设计了DepID方法，用来定量细胞内天然存在的III型CD38。利用酵母双杂交等方法，鉴定到了调控CD38活性的蛋白CIB1以及脂滴形成蛋白，揭示了III型CD38的调控机制。还研究了CD38在另一个第二信使烟酸腺嘌呤二核苷酸磷酸（NAADP）的生物合成中的作用。