Cytosolic interaction of type III human CD38 with CIB1 modulates cellular cyclic ADP-ribose levels
Cytosolic interaction of type III human CD38 with CIB1 modulates cellular cyclic ADP-ribose levels
复制标题
III 型人 CD38 与 CIB1 的胞质相互作用调节细胞环状 ADP-核糖水平
DOI：
10.1073/pnas.1703718114
复制
发表时间：
2017-08-01
影响因子：
11.1
通讯作者：
Lee, Hon Cheung
中科院分区：
文献类型：
--
作者：
Liu, Jun;Zhao, Yong Juan;Lee, Hon Cheung
意义本研究探讨了环ADP-核糖(CADPR)介导的钙信号转导中的一个拓扑悖论。信使是由CD38合成的，CD38被认为是一种LI型胞外酶。相反，我们证明了它自然存在于两个相反的膜方向，并且类型III，其催化结构域面向胞浆，在产生细胞cADPR方面是活跃的。它的拓扑结构是通过一种同时针对蛋白质鉴定的双重表位的技术来解决的，该技术明确地识别了细胞内的III型CD38。研究还表明，其cADPR合成活性受胞内与钙离子和整合素结合蛋白1(CIB1)相互作用的调节。结果表明，膜蛋白的表达并不一定像人们普遍认为的那样，只有一个独特的膜方向由序列基序设定。CD38催化合成钙信使环状ADP-核糖(CADPR)。它通常被认为是催化结构域向外的II型蛋白。它如何催化细胞内针对内质钙储存的cADPR的合成还没有解决。我们提出CD38也可以以相反的III型取向存在，其催化结构域面向胞浆。在这里，我们开发了一种方法，使用特定的纳米体来同时针对III型CD38催化区域上的两个不同的表位进行免疫，并确定它是自然存在于人类多发性骨髓瘤细胞中的。由于III型CD38在拓扑上是胞浆调节的，我们使用酵母双杂交筛选来确定胞浆钙离子和整合素结合蛋白1(CIB1)作为它的相互作用伙伴。免疫沉淀、酶联免疫吸附试验和双分子荧光互补的结果证实，CIB1在体内和体外都能与CD38的催化结构域特异结合。突变研究证实，CIB1的N端是相互作用的结构域。用shRNA敲除CIB1，用Cas9/Guide RNA敲除CIB1，细胞内cADPR水平与CIB1水平呈正相关。结果表明，III型CD38具有产生细胞内cADPR的功能，并且该活性是通过与胞内CIB1的相互作用而特异性地调节的。
Significance This study addresses a topological paradox in Ca2+ signaling mediated by cyclic ADP-ribose (cADPR). The messenger is synthesized by CD38, thought to be a type ll ectoenzyme. Instead, we show that it exists naturally in two opposite membrane orientations and that the type III, with its catalytic domain facing the cytosol, is active in producing cellular cADPR. Its topology was resolved by a technique simultaneously targeting dual epitopes for protein identification, which identifies the intracellular type III CD38 unambiguously. It is also shown that its cADPR-synthesizing activity is regulated by cytosolic interactions with Ca2+ and integrin-binding protein 1 (CIB1). The results indicate that membrane proteins are not necessarily expressed with only one unique membrane orientation set by sequence motifs as generally believed. CD38 catalyzes the synthesis of the Ca2+ messenger, cyclic ADP-ribose (cADPR). It is generally considered to be a type II protein with the catalytic domain facing outside. How it can catalyze the synthesis of intracellular cADPR that targets the endoplasmic Ca2+ stores has not been resolved. We have proposed that CD38 can also exist in an opposite type III orientation with its catalytic domain facing the cytosol. Here, we developed a method using specific nanobodies to immunotarget two different epitopes simultaneously on the catalytic domain of the type III CD38 and firmly established that it is naturally occurring in human multiple myeloma cells. Because type III CD38 is topologically amenable to cytosolic regulation, we used yeast-two-hybrid screening to identify cytosolic Ca2+ and integrin-binding protein 1 (CIB1), as its interacting partner. The results from immunoprecipitation, ELISA, and bimolecular fluorescence complementation confirmed that CIB1 binds specifically to the catalytic domain of CD38, in vivo and in vitro. Mutational studies established that the N terminus of CIB1 is the interacting domain. Using shRNA to knock down and Cas9/guide RNA to knock out CIB1, a direct correlation between the cellular cADPR and CIB1 levels was demonstrated. The results indicate that the type III CD38 is functionally active in producing cellular cADPR and that the activity is specifically modulated through interaction with cytosolic CIB1.