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，可能在肺泡毛细血管间隔病变的发病机制中发挥作用。小的gtpase具有c端CAAX基序，并进行翻译后加工，最终通过异戊酰半胱氨酸羧甲基转移酶（ICMT）将c端半胱氨酸羧甲基化。基于初步结果，我们假设ICMT的抑制降低了Ras和RhoA gtpase的羧基甲基化，并导致Focal Adhesion Complexes （FAC）的破坏、caspase的激活、FAC蛋白组分的蛋白水解和细胞凋亡。初步结果表明，ICMT抑制改变了GRP94的表达和电荷，GRP94是未折叠蛋白反应（UPR）中重要的伴侣蛋白。由于UPR和内质网（ER）应激反应的功能障碍导致细胞凋亡，我们假设ICMT活性的降低和Ras或RhoA活性的降低改变了GRP94的功能，导致UPR功能障碍导致细胞凋亡。1. 我们将确定ICMT对FAC形成和纤维化的抑制作用。a.我们将利用培养的肺血管内皮细胞确定ICMT抑制剂对Ras和RhoA GTPase的甲基化、定位和激活以及对细胞凋亡的影响。b.我们将通过比较Ras或RhoA GTPase过表达和下游信号分子对ICMT抑制引起的FAC破坏和细胞凋亡的影响，确定Ras和RhoA GTPase在ICMT抑制引起的内皮细胞损伤中的作用。c.我们将在体内确定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。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。b.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caspase激活、FAC破坏、FAC组分蛋白水解以及ICMT抑制诱导的anokiis的影响。本研究旨在明确GTPase甲基化的机制以及GRP94在内皮细胞凋亡调控中的作用。这项工作将提高对肺气肿等以内皮细胞凋亡为特征的肺部疾病的认识。此外，了解细胞凋亡可能为治疗依赖内皮细胞异常增殖的肺部疾病（如肺动脉高压和肺癌）提供线索。

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.