Small GTPases and Lung Endothelial Apoptosis

描述(申请人提供)：内皮细胞凋亡是调节血管生成的重要手段，并可能在涉及肺泡毛细血管间隔的疾病的发病机制中发挥作用。小分子GTP酶具有C端的CAAX基序，经过翻译后加工，最终通过异丙基半胱氨酸羧甲基转移酶(ICMT)使C端的半胱氨酸发生羧甲基化。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，并导致局部黏附复合体(FAC)的破坏，caspase激活，FAC蛋白组分的蛋白分解和细胞凋亡。初步结果表明，ICMT抑制改变了GRP94的表达和电荷，GRP94是一种在未折叠蛋白反应(UPR)中起重要作用的伴侣蛋白。由于UPR和内质网(ER)应激反应失灵导致细胞凋亡，我们推测ICMT活性降低和RAS或RhoA活性降低改变了GRP94的功能，从而导致UPR失灵导致细胞凋亡。1.我们将测定ICMT抑制对FAC形成和失巢细胞的影响。B.通过比较Ras或RhoA GTP酶及其下游信号分子的过度表达对ICMT抑制引起的FAC破坏和细胞凋亡的影响，确定Ras和RhoA GTP酶在ICMT抑制引起的内皮细胞失巢中的作用；c.在体内研究ICMT抑制对肺血管内皮细胞凋亡的影响。2.我们将确定ICMT抑制对GRP94的影响以及内质网应激反应在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、翻译后加工和亚细胞定位的影响。我们将通过评估RAS和RhoA过表达对ICMT诱导的GRP94变化的影响来确定小GTPase活性降低的作用。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过表达对UPR/ER应激反应的影响。E.我们将确定GRP94过表达对半胱氨酸天冬氨酸氨基转移酶激活、FAC破坏、FAC组分的蛋白分解以及ICMT抑制诱导的失巢凋亡的影响。本申请中提出的工作将确定GTP酶甲基化的机制和GRP94在调控内皮细胞凋亡中的作用。这项工作将提高对以内皮细胞凋亡为特征的肺部疾病的理解，如肺气肿。此外，对细胞凋亡的了解可能为治疗依赖于内皮细胞异常增殖的肺部疾病提供线索，如肺动脉高压和癌症。

DESCRIPTION (provided by applicant): 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.