Endoplasmic reticulum chaperone protein GRP78 protects cells from apoptosis induced by topoisomerase inhibitors - Role of ATP binding site in suppression of caspase-7 activation
Endoplasmic reticulum chaperone protein GRP78 protects cells from apoptosis induced by topoisomerase inhibitors - Role of ATP binding site in suppression of caspase-7 activation
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DOI：
10.1074/jbc.m212328200
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发表时间：
2003-06-06
影响因子：
4.8
通讯作者：
Lee, AS
中科院分区：
文献类型：
--
作者：
Reddy, RK;Mao, CH;Lee, AS
大量的相关研究已经证实，未折叠蛋白反应（UPR）的激活改变了细胞对化疗药物的敏感性。虽然葡萄糖调节蛋白（GRP）的诱导通常被用作UPR的指标，但GRP在赋予对DNA损伤剂的抗性中的直接作用尚未得到证实。我们在这里报告，没有使用内质网（ER）应激诱导剂，特定的过度表达GRP 78的结果在减少细胞凋亡和更高的集落存活时，挑战与拓扑异构酶II抑制剂，依托泊苷和阿霉素，拓扑异构酶I抑制剂，喜树碱。在研究GRP 78对依托泊苷诱导的细胞死亡的保护作用的机制时，我们发现与UPR相反，GRP 78过表达不会导致G（1）阻滞或拓扑异构酶II的耗尽。Caspase-7是一种与ER相关的执行caspase，被依托泊苷激活。我们发现GRP 78的特异性表达在体内和体外都能阻断依托泊苷对caspase-7的激活，并且这种作用可以通过在无细胞系统中加入dATP来逆转。最近，据报道，异位表达的GRP 78和半胱天冬酶-7和-12形成复合物，从而将ER应激耦合到细胞死亡程序。然而，GRP 78（一种推测为ER腔蛋白）如何调节细胞凋亡的胞质效应物的机制尚不清楚。在这里，我们提供的证据表明，GRP 78的亚群可以作为ER跨膜蛋白存在，以及与caspase-7共定位，如荧光显微镜所证实的。免疫共沉淀研究进一步揭示了内源性GRP 78与半胱氨酸天冬氨酸蛋白酶原-7而不是半胱氨酸天冬氨酸蛋白酶原-3组成型相关。最后，GRP 78突变体的ATP结合结构域删除不能结合procaspase-7，并失去其对依托泊苷诱导的细胞凋亡的保护作用。
A large number of correlative studies have established that the activation of the unfolded protein response (UPR) alters the cell's sensitivity to chemotherapeutic agents. Although the induction of the glucose-regulated proteins (GRPs) is commonly used as an indicator for the UPR, the direct role of the GRPs in conferring resistance to DNA damaging agents has not been proven. We report here that without the use of endoplasmic reticulum (ER) stress inducers, specific overexpression of GRP78 results in reduced apoptosis and higher colony survival when challenged with topoisomerase II inhibitors, etoposide and doxorubicin, and topoisomerase I inhibitor, camptothecin. While investigating the mechanism for the GRP78 protective effect against etoposide-induced cell death, we discovered that in contrast to the UPR, GRP78 overexpression does not result in G(1) arrest or depletion of topoisomerase II. Caspase-7, an executor caspase that is associated with the ER, is activated by etoposide. We show here that specific expression of GRP78 blocks caspase-7 activation by etoposide both in vivo and in vitro, and this effect can be reversed by addition of dATP in a cell-free system. Recently, it was reported that ectopically expressed GRP78 and caspases-7 and -12 form a complex, thus coupling ER stress to the cell death program. However, the mechanism of how GRP78, a presumably ER lumen protein, can regulate cytosolic effectors of apoptosis is not known. Here we provide evidence that a subpopulation of GRP78 can exist as an ER transmembrane protein, as well as co-localize with caspase-7, as confirmed by fluorescence microscopy. Co-immunoprecipitation studies further reveal endogenous GRP78 constitutively associates with procaspase-7 but not with procaspase-3. Lastly, a GRP78 mutant deleted of its ATP binding domain fails to bind procaspase-7 and loses its protective effect against etoposide-induced apoptosis.