Lu Qing, Harrington Elizabeth O, Newton Julie, Jankowich Matthew, Rounds Sharon
Providence VA Medical Center, Pulmonary/Critical Care Medicine Section, 830 Chalkstone Avenue, Providence, RI 02908, USA.
Am J Respir Cell Mol Biol. 2007 Jul;37(1):20-30. doi: 10.1165/rcmb.2006-0301SM. Epub 2007 Mar 8.
Isoprenylcysteine-O-carboxyl methyltransferase (ICMT) catalyzes methylation of proteins containing a C-terminal CAAX motif. We have previously shown that chemical inhibition of ICMT caused endothelial cell apoptosis, an effect correlated with decreased Ras and RhoA carboxyl methylation and GTPase activities. In the current study, proteomic analysis of pulmonary artery endothelial cells (PAEC) exposed to the ICMT inhibitor, N-acetyl-geranylgeranyl-cysteine (AGGC), demonstrated a shift in the isoelectric points (pI) of the glucose-regulated protein (GRP) 94. Two-dimensional PAGE and immunoblot analysis further documented that ICMT inhibition caused multiple changes in the pI of GRP94. GRP94 is an endoplasmic reticulum molecular chaperone, a component of the unfolded protein response (UPR), and is involved in apoptosis. Immunofluorescence analyses revealed redistribution and aggregation of GRP94 after 3 h exposure to AGGC. A similar finding was noted with calnexin. In addition, GRP94 protein levels were significantly diminished upon 18 h AGGC exposure or ICMT suppression. The effects of ICMT inhibition on changes in GRP94 subcellular localization and protein content were blunted by overexpression of constitutively active RhoA or a caspase inhibitor. Furthermore, GRP94 depletion augmented endothelial cell apoptosis induced by ICMT inhibition. These results indicate that ICMT inhibition leads to GRP94 relocalization, aggregation, and degradation; effects were dependent upon the activities of RhoA and caspases. We speculate that changes in the pI, subcellular localization, and protein level of GRP94 cause endothelial cell apoptosis, possibly through UPR dysfunction. These studies suggest a novel link between RhoA GTPases and the UPR.
异戊二烯基半胱氨酸 - O - 羧甲基转移酶（ICMT）催化含有C末端CAAX基序的蛋白质的甲基化。我们之前已经表明，ICMT的化学抑制会导致内皮细胞凋亡，这种效应与Ras和RhoA羧基甲基化及GTP酶活性降低相关。在当前研究中，对暴露于ICMT抑制剂N - 乙酰 - 香叶基香叶基半胱氨酸（AGGC）的肺动脉内皮细胞（PAEC）进行蛋白质组学分析，结果显示葡萄糖调节蛋白（GRP）94的等电点（pI）发生了变化。二维聚丙烯酰胺凝胶电泳和免疫印迹分析进一步证明，ICMT抑制导致GRP94的pI发生多种变化。GRP94是一种内质网分子伴侣，是未折叠蛋白反应（UPR）的一个组成部分，并且参与细胞凋亡。免疫荧光分析显示，暴露于AGGC 3小时后GRP94发生了重新分布和聚集。钙联结蛋白也有类似的发现。此外，暴露于AGGC 18小时或抑制ICMT后，GRP94蛋白水平显著降低。组成型活性RhoA的过表达或半胱天冬酶抑制剂可减弱ICMT抑制对GRP94亚细胞定位和蛋白质含量变化的影响。此外，GRP94的缺失增强了ICMT抑制诱导的内皮细胞凋亡。这些结果表明，ICMT抑制导致GRP94重新定位、聚集和降解；这些效应依赖于RhoA和半胱天冬酶的活性。我们推测，GRP94的pI、亚细胞定位和蛋白质水平的变化可能通过UPR功能障碍导致内皮细胞凋亡。这些研究表明RhoA GTP酶与UPR之间存在新的联系。
