Regulation of eNOS by Subcellular Targeting

说明(申请人提供)：心血管疾病是男性和女性的主要死亡原因，不分种族，高达25%的人口患有某种形式的慢性心血管疾病。内皮细胞通过内皮型一氧化氮合酶(ENOS)产生一氧化氮(NO)的能力降低是许多心血管疾病的标志，但调节eNOS活性的机制尚不完全清楚。除了转录调控外，eNOS还受翻译后机制的调控，包括钙、磷酸化、蛋白质-蛋白质相互作用和亚细胞定位。在培养的内皮细胞和血管内，eNOS定位于质膜和核周高尔基复合体。ENOS在这两个区域的相对比例是不同的，最近发现的一种eNOS结合蛋白NOSIP调节这些区域中eNOS的数量。然而，内皮型一氧化氮合酶在高尔基体或质膜上的个体功能尚不清楚。在我们的初步结果中，我们已经表明，通过将eNOS特异性地定位于高尔基体或质膜，细胞内的位置可以直接影响NO的释放量和导致eNOS激活的机制。质膜eNOS具有较高的基础活性，对钙敏感，并在丝氨酸1179上发生结构性磷酸化。相反，高尔基体eNOS表现出较低水平的钙依赖活性，并被Akt介导的磷酸化高度激活。基于这些发现，这一建议的中心假设是，利用Akt依赖通路的激动剂优先激活高尔基体eNOS，钙依赖激动剂优先激活质膜eNOS。为了验证这一假说，提出了4个具体目标：第一个目标将确定利用Akt或钙依赖机制的激动剂是否优先激活高尔基体或质膜eNOS。第二个目标将确定高尔基体和质膜eNOS活性差异的机制(钙和磷酸化)。第三个目的将确定NOSIP蛋白是否浓缩高尔基体中的eNOS，从而调节特定激动剂的活性。第四个目标将确定eNOS亚cetlutar靶向在分离血管中对血管功能的重要性。

DESCRIPTION (provided by applicant): Cardiovascular disease is the leading cause of death for men and women regardless of ethnicity and up to 25% of the population live with some form of chronic cardiovascular disease. Reduced capacity of the endothelium to produce nitric oxide (NO), via the enzyme endothelial nitric oxide synthase (eNOS), is a hallmark of many cardiovascular diseases yet the mechanisms regulating eNOS activity are not fully understood. In addition to transcriptional control, eNOS is regulated by post-translational mechanisms including calcium, phosphorylation, protein-protein interactions and subcellular localization. Within endothelial cells, both cultured and in blood vessels, eNOS is localized to the plasma membrane and the perinuclear Golgi complex. The relative proportion of eNOS in both locations is variable and a recently discovered eNOS binding protein, NOSIP, regulates the amount of eNOS within these domains. However, the individual function of eNOS at the Golgi or plasma membrane is not yet known. In preliminary results we have shown that by targeting eNOS exclusively to the Golgi or plasma membrane, intracellular location can directly influence both the amount of NO released and the mechanisms leading to eNOS activation. Plasma membrane eNOS has elevated basal activity, is calcium-sensitive and is constitutively phosphorylated on serine 1179. In contrast, Golgi eNOS displays lower levels of calcium-dependent activity and is highly activated by Akt mediated phosphorylation. Based on these findings, the central hypothesis of this proposal is that agonists utilizing Akt- dependent pathways preferentially activate Golgi eNOS and that calcium-dependent agonists preferentially activate plasma membrane eNOS. To test this hypothesis, 4 specific aims are proposed: The first aim will determine whether agonists utilizing Akt or calcium-dependent mechanisms preferentially activate Golgi or plasma membrane eNOS. The second aim will determine the mechanisms (calcium and phosphorylation) underlying the divergent activities of Golgi and plasma membrane eNOS. The third aim will determine whether the protein NOSIP concentrates eNOS in the Golgi and thus modulates the activity of specific agonists. The fourth aim will determine the importance of eNOS subcetlutar targeting to vascular function in isolated blood vessels.