Multiple oxidative stress-response members of the Adapt78 family.
Multiple oxidative stress-response members of the Adapt78 family.
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Adapt78 家族的多个氧化应激反应成员。
DOI：
10.1016/j.freeradbiomed.2004.05.014
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发表时间：
2004
影响因子：
7.4
通讯作者：
Crawford,DanaR
中科院分区：
文献类型：
--
作者：
Michtalik,HenryJ;Narayan,AnanthV;Bhatt,Nishant;Lin,HY;Mulligan,MichaelT;Zhang,ShenLi;Crawford,DanaR
Adapt78是一个氧化和钙应激反应基因。其蛋白质产物是细胞内钙信号蛋白钙调磷酸酶的有效天然抑制剂。关于Adapt78蛋白的大部分已知信息都是基于细胞转染研究。为了理解天然内源性Adapt78，我们使用了针对细胞Adapt78的抗体，最近确定内源性Adapt78蛋白，如其mRNA，是氧化和钙应激反应。在这里，我们报告了该41 kDa蛋白家族的第二种内源性形式的鉴定。人HeLa细胞的亚细胞分级显示，在以前的转染研究的结果相反，大多数内源性Adapt78，其特征在于29和41 kDa的电泳双联体，驻留在细胞的胞质溶胶。Adapt78的41 kDa形式是丰富的，并发现表现出许多共同的特征与以前报道的氧化应激反应的29 kDa形式，包括低磷酸化和过度磷酸化的变体，氧化应激后的低磷酸化形式的快速损失，对各种激酶和磷酸酶抑制剂的反应，和本地化。然而，它也表现出一些独特的特征，最明显的是缺乏钙诱导。最后，与41 kDa Adapt78形式相比，29 kDa形式在氧化剂暴露后表现出短得多的半衰期和强稳定性。这些数据揭示了一种新的氧化应激反应性41 kDa Adapt78物种的存在，进一步深入了解Adapt78蛋白家族及其分布，并挑战了以前使用转染方案获得的结论。
Adapt78 is an oxidative and calcium stress-response gene. Its protein product is a potent natural inhibitor of the intracellular calcium signaling protein calcineurin. Much of what is known about Adapt78 protein is based on cell-transfection studies. Toward understanding natural endogenous Adapt78, we used an antibody raised against cellular Adapt78 and recently determined that endogenous Adapt78 protein, like its mRNA, is oxidative and calcium stress responsive. Here we report the identification of a second endogenous form of this protein family of 41 kDa. Subcellular fractionation of human HeLa cells revealed that in contrast to results of previous transfection studies, most endogenous Adapt78, characterized as 29 and 41 kDa electrophoretic doublets, resides in the cellular cytosol. The 41 kDa form of Adapt78 was abundant and found to exhibit many characteristics in common with the previously reported oxidative stress-responsive 29 kDa form, including hypo- and hyperphosphorylation variants, rapid loss of the hypophosphorylated form following oxidative stress, response to various kinase and phosphatase inhibitors, and localization. However, it also exhibited some unique characteristics, most notably the lack of calcium inducibility. Finally, the 29 kDa form exhibited a much shorter half-life and strong stabilization following oxidant exposure compared with the 41 kDa Adapt78 form. These data reveal the presence of a novel oxidative stress-responsive 41 kDa Adapt78 species, lend further insight into the Adapt78 family of proteins and their distribution, and challenge previous conclusions obtained using transfection protocols.