Small GTPases and Lung Endothelial Apoptosis

内皮细胞凋亡是调节血管生成的重要手段，在血管生成中可能发挥作用。 涉及肺泡毛细血管隔膜的疾病的发病机制。小GTP酶具有C末端 CAAX基序并进行翻译后加工，最终导致C-末端的羧基甲基化。 异戊二烯半胱氨酸羧基甲基转移酶（ICMT）。根据初步结果，我们 假设ICMT抑制降低Ras和RhoA GTP酶的羧基甲基化， 粘着斑复合物（FAC）的破坏，半胱天冬酶活化，FAC蛋白组分的蛋白水解， 和凋亡。初步结果表明，ICMT抑制改变了GRP 94的表达和电荷， 一种在未折叠蛋白质反应（UPR）中很重要的伴侣蛋白。由于故障的 UPR和内质网（ER）应激反应引起细胞凋亡，我们假设UPR和ER应激反应的减少， ICMT活性和导致的Ras或RhoA活性降低改变了GRP 94的功能，导致细胞凋亡， 普遍定期审议机制失灵。1.我们将确定ICMT抑制对FAC形成的影响， 失巢。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 GTd 3以及使用培养的肺血管内皮细胞的细胞凋亡，B.我们将 通过比较Ras和RhoA GTP酶在ICMT抑制引起的内皮失巢凋亡中的作用， Ras或RhoA GT3及其下游信号分子的过表达对FAC的影响 由ICMT抑制引起的破坏和凋亡，c.我们将确定ICMT抑制对 肺血管内皮细胞凋亡。2.我们将确定ICMT抑制对 GRP 94和ER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GRP 94表达，翻译后加工， 亚细胞定位B.我们将通过评估以下因素来确定小GT酶活性降低的作用： Ras和RhoA过表达对ICMT诱导的GRP 94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.我们将确定下列因素的影响： Ras和/或RhoA过表达对UPR/ER应激反应的影响。e.我们将确定以下因素的影响： GRP 94过表达对半胱天冬酶活化、FAC破坏、FAC组分的蛋白水解和失巢凋亡的影响 通过抑制ICMT诱导。本申请中提出的工作将确定 GT3甲基化和GRP 94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（如肺气肿）的认识。在 此外，对细胞凋亡的理解可能为依赖于细胞凋亡的肺部疾病的治疗提供线索。 异常内皮增生，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.