III型CD38的信号调控分子机制

CD38 is the dominant enzyme in mammalian tissues responsible for synthesizing two Ca2+ messenger molecules, cyclic ADP-ribose (cADPR) and Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP). It is thought to be a type II transmembrane protein with its carboxyl-terminal catalytic domain facing outside; thus, the mechanism by which CD38 metabolizes intracellular Ca2+ messengers has been controversial. We have recently provided direct evidence that two opposite membrane orientations of CD38, type II and type III (which has its catalytic domain facing cytoplasm), exist naturally in human cells. In this proposal, we aim to research the regulation of the type III CD38 signaling mechanism. Firstly, we used the yeast two hybrid assay to screen the possible interaction proteins of the catalytic domain of CD38 in the cytosol, and the calcium- and integrin-binding protein 1 (CIB1) was identified. To ascertain that CIB1 is not a false positive and can indeed interact with CD38, we will employ both in vivo and in vitro approaches, including immunoprecipitaion and the solid-phase binding assay. We will use recombinant approaches to produce large quantities of the CD38/CIB1 complex and solve its crystal structure. And then, we need to analyze the effect of CIB1 on the enzymatic activity of CD38. Last but not least, we will ascertain if the interaction of CIB1 with CD38 can lead to phosphorylation of CD38 by protein kinases, resulting in a change of its enzymatic activity. The results will provide insights and a theoretical basis for understanding the type III CD38 signaling pathway in cells.

CD38是哺乳动物组织中负责合成钙离子信使cADPR和NAADP的主要酶，它被认为是一类II型跨膜蛋白，即C端催化结构域位于细胞外。因此，CD38如何催化合成细胞内的信使分子存在争议。最近，我们提供直接的证据支持CD38可能存在两种相反的膜定向方式，即II型和III型（C端催化结构域位于细胞内）。本项目拟对III型CD38的调控机制进行探究。首先，我们利用酵母双杂交筛选CD38催化结构域在细胞内的互作蛋白，由此鉴定到一个阳性克隆为钙离子与整合素结合蛋白1（CIB1）。为了验证CIB1与CD38的相互作用，我们拟采用免疫沉淀、酶联免疫吸附等技术分别从体内和体外进行实验，并将尝试利用晶体学解析CD38/CIB1的复合物结构。最后，我们将考察CIB1与CD38 的结合是否通过影响CD38的磷酸化水平，从而影响CD38酶活性。这些结果将为揭示III型CD38的信号通路提供重要的理论基础。

CD38是哺乳动物细胞中负责合成具有钙动员活性的第二信使环腺苷二磷酸核糖（cADPR）和烟酸腺嘌呤二核苷酸磷酸 (NAADP)的主要酶，发挥重要的生物学功能。目前CD38在制药领域的重要性得到体现，包括：多发性骨髓瘤等癌细胞上CD38异常高表达使它成为良好药靶，人们利用它来识别并特异杀伤相应肿瘤细胞；其次，肿瘤浸润T细胞发生免疫抑制过程中CD38表达的激活，对CD38功能机制的深刻理解将有助于通过靶向该分子开发针对多种肿瘤的免疫疗法。通常CD38被认为是一类Ⅱ型跨膜蛋白，其C端催化结构域位于细胞外，由此产生了CD38的拓扑学悖论，即CD38如何催化合成位于细胞内的底物。为了阐明上述问题，我们提出"Ⅲ型CD38"的假说并提供了直接证据。本项目开发了能够特异识别内源性Ⅲ型CD38的工具，成功在不同的多发性骨髓瘤细胞株中验证到Ⅲ型CD38的存在；利用酵母双杂交技术寻找到CD38在细胞内的底物蛋白-CIB1;通过免疫共沉淀、酶联反应和双分子荧光互补技术验证了CIB1与CD38的催化结构域结合；并采用shRNA进行CIB1的敲低以及利用Cas9/gRNA的技术敲除CIB1，证明了CIB1含量与细胞内cADPR水平具有直接正相关的关系。这些结果将为夯实“Ⅲ型CD38”假说以及揭示Ⅲ型CD38在肿瘤发生过程中的作用机制提供重要的理论基础，为今后抗肿瘤研究提供新的分子作用靶点，也对CD38进行药物开发指明方向。