III型CD38的信号调控分子机制

项目名称： III型CD38的信号调控分子机制

项目编号： No.31501134

项目类型： 青年科学基金项目

立项/批准年度： 2016

项目学科： 生物科学

项目作者： 刘隽

作者单位： 北京大学

项目金额： 21万元

中文摘要： 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。

中文关键词： CD38；膜蛋白；互作蛋白；CIB1；磷酸化

英文摘要： 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.

英文关键词： CD38;membrane protein;interaction protein ;CIB1;phosphorylation