Department of Medicine, Vascular Research Laboratory, Providence Veterans Affairs Medical Center, Alpert Medical School of Brown University, Providence, Rhode Island 02908, USA.
Am J Physiol Lung Cell Mol Physiol. 2010 Apr;298(4):L501-8. doi: 10.1152/ajplung.00416.2009. Epub 2010 Jan 22.
We have previously demonstrated that inhibition of CAAX carboxyl methylation with AGGC caused redistribution and condensation of the ER molecular chaperones, glucose-regulated protein (GRP)-94 and calnexin; an effect that was attenuated by overexpression of dominant active RhoA. We have also shown that AGGC decreased GRP94 protein level; an effect that was dependent on caspase activity. In the present study, we tested the effects of inhibition of posttranslational processing of CAAX proteins on localization and protein levels of molecular chaperones and phosphorylation and protein level of eIF2alpha. We found that both AGGC, which inhibits CAAX carboxyl methylation, and simvastatin, which inhibits CAAX geranylgeranylation, caused relocalization of GRP94, calnexin, and calreticulin, effects that were not seen during endothelial apoptosis induced by TNF-alpha or ultraviolet (UV) irradiation. These results suggest that posttranslational processing of CAAX proteins is important in maintaining localization of molecular chaperones normally found in the ER. We also noted that AGGC, but not simvastatin, TNF-alpha, or UV irradiation, decreased protein levels of most molecular chaperones. Increased eIF2alpha phosphorylation was observed in the early stages of apoptosis, which was independent of the cause of apoptosis. These results suggest that eIF2alpha phosphorylation is a common early response to apoptosis-inducing stimuli. Interestingly, eIF2alpha protein level was decreased in the late stages of apoptosis induced by AGGC, TNF-alpha, and UV irradiation: an effect that was prevented by caspase inhibition. Thus we speculate that caspase(s)-dependent proteolysis of molecular chaperones and eIF2alpha may be novel signaling pathways of apoptosis. We also speculate that increased eIF2alpha phosphorylation is a defensive response against endothelial cell apoptosis.
我们之前已经证明，用 AGGC 抑制 CAAX 羧基甲基化会导致内质网分子伴侣葡萄糖调节蛋白（GRP）-94 和钙连蛋白的重分布和浓缩；这种效应被显性激活 RhoA 的过表达所减弱。我们还表明，AGGC 降低了 GRP94 蛋白水平；这种效应依赖于半胱天冬酶的活性。在本研究中，我们测试了抑制 CAAX 蛋白的翻译后加工对分子伴侣的定位和蛋白水平以及 eIF2alpha 的磷酸化和蛋白水平的影响。我们发现，抑制 CAAX 羧基甲基化的 AGGC 和抑制 CAAX 香叶基香叶基化的辛伐他汀都会导致 GRP94、钙连蛋白和钙网蛋白的重新定位，而在 TNF-α或紫外线（UV）照射诱导的内皮细胞凋亡过程中则没有观察到这些效应。这些结果表明，CAAX 蛋白的翻译后加工对于维持内质网中正常存在的分子伴侣的定位是重要的。我们还注意到，AGGC 而不是辛伐他汀、TNF-α或 UV 照射会降低大多数分子伴侣的蛋白水平。在凋亡的早期阶段观察到 eIF2alpha 的磷酸化增加，这与凋亡的原因无关。这些结果表明，eIF2alpha 磷酸化是凋亡诱导刺激的常见早期反应。有趣的是，在 AGGC、TNF-α和 UV 照射诱导的凋亡晚期，eIF2alpha 蛋白水平下降：这种效应被半胱天冬酶抑制所阻止。因此，我们推测 caspase 依赖性分子伴侣和 eIF2alpha 的蛋白水解可能是凋亡的新信号通路。我们还推测，eIF2alpha 磷酸化可能是内皮细胞凋亡的一种防御反应。
