Pin1 prolyl isomerase regulates endothelial nitric oxide synthase

描述(申请人提供)：内皮源性一氧化氮(NO)调节心血管内稳态的许多重要方面，血管中NO的产生即使是两倍的变化也可能在正常的血管生理学和血管病理生理学中产生重大后果。NO在血管扩张状态的调节中起着至关重要的作用，因此在血压调节中起着至关重要的作用。内皮细胞释放的NO还调节其他过程，包括血小板聚集、血小板和白细胞与内皮细胞的黏附、血管平滑肌细胞增殖、内皮细胞凋亡、血管生成和参与急性炎症的血管渗漏。由于NO在这些过程中的关键作用，血管中NO产生的异常被认为与某些血管疾病的发病机制有关，如动脉粥样硬化、糖尿病和高血压。内皮型一氧化氮合酶(ENOS)催化精氨酸氧化内皮细胞合成NO。因此，详细了解eNOS酶的结构、功能和调控显然是一个有价值的科学研究目标。ENOS在翻译后受到两种主要机制的调节：蛋白质-蛋白质相互作用和磷酸化。关于这两个不同的eNOS监管过程是如何相互关联的，目前尚不清楚。然而，一段时间以来已知的是，eNOS中存在丝氨酸116处的抑制性磷酸化位点(S116)。在基础条件下和暴露于促炎细胞因子后，该位点在内皮细胞中被磷酸化。我们的初步数据提出了这样的假设，即S116上eNOS的Pro-导向的磷酸化促进了Pin1 Pro异构酶的结合，并且Pin1诱导的构象变化抑制了eNOS的活性。初步数据还显示，这一过程可能在动脉粥样硬化疾病中做出了重要贡献。因此，我们建议通过各种不同的方法，使用纯化的蛋白、培养的内皮细胞、完整的血管节段和包括动物疾病模型在内的整个动物来检验这些假说。这些研究将提供比目前更完整的对内皮细胞内eNOS调控的理解，这是基础科学家和临床医生都非常感兴趣的话题。

DESCRIPTION (provided by applicant): Endothelium-derived nitric oxide (NO) regulates many important aspects of cardiovascular homeostasis and alterations in vascular NO production of as little as two-fold can have major consequences in both normal vascular physiology and vascular pathophysiology. NO has a crucial role in regulation of the state of vasodilatation of blood vessels and hence in regulation of blood pressure. NO released from the endothelium also modulates other processes as well including platelet aggregation, platelet and leukocyte adhesion to the endothelium, vascular smooth muscle cell proliferation, endothelial cell apoptosis, angiogenesis, and vascular leakage involved in acute inflammation. Because of the key role of NO in each of these processes, abnormalities in vascular NO production are thought to contribute to the pathogenesis of certain vascular disorders such as those of atherosclerosis, diabetes, and hypertension. NO is synthesized in endothelial cells by oxidation of arginine catalyzed by the enzyme, endothelial nitric oxide synthase (eNOS). Obtaining a detailed understanding of the structure, function, and regulation of eNOS enzyme is thus clearly a worthy goal of scientific investigation. eNOS is regulated post-translationally by two primary mechanisms: protein-protein interactions and phosphorylation. Nothing is yet known about how these two different eNOS regulation processes are interrelated. What has been known for some time, however, is that there exists in eNOS an inhibitory phosphorylation site at serine 116 (S116). This site is phosphorylated in endothelial cells under basal conditions and after exposure to pro-inflammatory cytokines. Our preliminary data give rise to the hypothesis that proline-directed phosphorylation of eNOS at S116 promotes binding of the Pin1 prolyl isomerase and Pin1-induced conformational changes that suppress eNOS activity. Preliminary data also show that this process may make an important contribution to the disease of atherosclerosis. We therefore propose to test these hypotheses by a variety of different methods using purified proteins, cultured endothelial cells, intact blood vessel segments, and whole animals including animal disease models. These studies will provide a more complete understanding than currently exists of regulation of eNOS in endothelial cells, a topic of great interest to both basic scientists and clinicians.