Small GTPases and Lung Endothelial Apoptosis

内皮细胞凋亡是调节血管生成的重要手段，可能在血管生成中发挥作用。 肺泡毛细血管隔膜疾病的发病机制。小分子GTP酶具有C-末端 CAAX基序，并经过翻译后加工，最终导致C-末端的羧甲基化 半胱氨酸由异丙基半胱氨酸羧甲基转移酶(ICMT)催化。根据初步结果，我们 假设ICMT的抑制减少了RAS和RhoA GTP酶的羧甲基化，并导致 焦点黏附复合体(FAC)的破坏，caspase的激活，FAC蛋白质组分的蛋白质分解， 和细胞凋亡。初步结果表明，ICMT抑制改变了GRP94的表达和电荷， 一种伴侣蛋白，在未折叠蛋白反应(UPR)中起重要作用。由于出现故障， UPR和内质网(ER)应激反应导致细胞凋亡，我们假设减少 ICMT活性和由此导致的RAS或RhoA活性降低改变了GRP94的功能，导致细胞凋亡 与普遍定期审议的故障有关。1.我们将确定ICMT抑制对FAC形成和FAC形成的影响 阿尼基斯。A.我们将确定ICMT抑制剂对甲基化、定位和激活的影响 RAS和RhoA GTP酶以及对培养的肺血管内皮细胞的凋亡的影响，b.我们将 通过比较确定Ras和RhoA GTP酶在ICMT抑制引起的内皮细胞失巢中的作用 Ras或RhoA GTPase及其下游信号分子过表达对FAC的影响 ICMT抑制引起的干扰和凋亡，c.我们将确定ICMT抑制对 活体肺血管内皮细胞凋亡。2.检测ICMT抑制对大鼠脑内神经元胞浆内皮细胞的影响 GRP94和内质网应激反应在ICMT抑制引起的内皮细胞凋亡中的作用，a。 我们将确定ICMT抑制对GRP94表达、翻译后处理和 亚细胞定位。B.我们将通过评估确定小分子GTP酶活性降低的作用 RAS和RhoA过表达对ICMT诱导的GRP94变化的影响C.我们将确定 ICMT抑制对UPR/ER应激反应标志物的影响D.我们将确定 RAS和/或RhoA在UPR/ER应激反应中的过度表达。E.我们将确定以下因素的影响 GRP94在caspase激活、Fac破坏、Fac组分的蛋白分解和失巢凋亡中的过表达 ICMT抑制所致。本申请中提出的工作将确定 GTP酶甲基化及GRP94在内皮细胞凋亡调控中的作用。这项工作将 提高对以内皮细胞凋亡为特征的肺部疾病的认识，如肺气肿。在……里面 此外，对细胞凋亡的了解可能会为依赖于 血管内皮细胞异常增殖，如肺动脉高压和癌症。

Endothelial cell apoptosis is an important means of regulating angiogenesis and may play a role in the pathogenesis of diseases involving the alveolar capillary septum. Small GTPases possess a C-terminal CAAX motif and undergo post-translational processing, culminating in carboxyl methylation of C-terminal cysteine by isoprenylcysteine carboxyl methyltransferase(ICMT). Based on Preliminary Results, we hypothesize that inhibition of ICMT decreases carboxyl methylation of Ras and RhoA GTPases, and causes disruption of Focal Adhesion Complexes (FAC), caspase activation, proteolysis of FAC protein components, and apoptosis. Preliminary Results indicate that ICMT inhibition changes expression and charge of GRP94, a chaperone protein that is important in the Unfolded Protein Response (UPR). Since malfunction of the UPR and endoplasmic reticulum (ER) stress response causes apoptosis, we hypothesize that decreased ICMT activity and resulting decreased Ras or RhoA activity alter GRP94 function, resulting in apoptosis due to malfunction of the UPR. 1. We will determine the effects of ICMT inhibition on FAC formation and anoikis. a. We will determine the effects of inhibitors of ICMT on methylation, localization, and activation of Ras and RhoA GTPase and on apoptosis using cultured pulmonary vascular endothelial cells, b. We will determine the role of Ras and RhoA GTPases in endothelial anoikis caused by ICMT inhibition by comparing the effects of over-expression of Ras or RhoA GTPase and downstream signaling molecules on FAC disruption and apoptosis caused by ICMT inhibition, c. We will determine the effects of ICMT inhibition on pulmonary vascular endothelial apoptosis in vivo. 2. We will determine the effect of ICMT inhibition on GRP94 and the role of the ER Stress Response in endothelial cell apoptosis caused by ICMT inhibition, a. We will determine the effects of ICMT inhibition on GRP94 expression, post-translational processing, and sub-cellular localization. b.We will determine the role of decreased small GTPase activity by assessing effects of Ras and RhoA over-expression on ICMT-induced changes in GRP94. c.We will determine the effects of ICMT inhibition on markers of the UPR/ER Stress Response. d.We will determine the effects of Ras and/or RhoA over-expression on the UPR/ER Stress Response. e.We will determine the effects of GRP94 over-expression on caspase activation, FAC disruption, proteolysis of FAC components, and anoikis induced by inhibition of ICMT. The work proposed in this application will ascertain the mechanism of GTPase methylation and the role of GRP94 in regulation of endothelial cell apoptosis. This work will improve understanding of lung diseases characterized by endothelial apoptosis, such as emphysema. In addition, understanding of apoptosis may provide clues to treatment of lung diseases dependent upon abnormal endothelial proliferation, such as Pulmonary Arterial Hypertension and cancer.