Regulation of eNOS by the cell division cycle homolog, cdc37

描述（由申请人提供）：内皮型一氧化氮合酶（eNOS）通过在血管内皮中产生一氧化氮（NO）在心血管生物学中起关键作用。已知NO调节血管舒张、血小板聚集、血小板和白细胞粘附于内皮、内皮素-1生成和血管平滑肌增殖，从而在血压调节和动脉粥样硬化的更广泛问题中发挥作用。此外，eNOS解偶联（其中NO产生和超氧化物产生的平衡向有利于超氧化物的方向转移）与包括高血压和糖尿病在内的各种病理状况中发生的内皮功能障碍相关。eNOS功能的精确调节对于维持血管内环境的稳定至关重要。eNOS受共翻译和翻译后机制、磷酸化和蛋白质-蛋白质相互作用的调节。了解这些调节机制之间的关系是一个重要的研究领域。在我们的初步数据中，我们已经确定了一种新的eNOS相互作用蛋白，它可能在介导eNOS蛋白复合物的形成以及随后的eNOS磷酸化和激活中发挥重要作用。使用免疫共沉淀和体外结合实验，我们已经确定，细胞分裂周期37同源物（cdc 37）直接与eNOS相互作用，并在没有其他蛋白质的情况下断言eNOS活性的抑制性影响。Cdc 37是热休克蛋白90（Hs 90）和Akt（两种关键eNOS调节蛋白）之间的蛋白复合物的关键组分。然而，以前的研究没有检查cdc 37和eNOS之间的潜在相互作用。基于这些观察和我们的初步工作，我们将使用纯化的蛋白质在体外结合和激酶实验，以及共免疫沉淀和腺病毒介导的基因转移在内皮细胞培养和分离的血管，以探讨这些相互作用。本研究的总体目标是通过检测cdc 37在协调eNOS与Hsp 90和Akt的相互作用以及随后的磷酸化和活化中的作用来研究cdc 37对eNOS功能的影响。本研究的具体目标和假设是：目的1 -为了检验可以鉴定cdc 37和eNOS中控制两种蛋白质之间直接相互作用的特定结构域的假设，目的2 -为了检验cdc 37介导eNOS与Hsp 90和Akt的复合物形成随后改变eNOS磷酸化的假设，目的3 -检验cdc 37在体外和体内均修饰eNOS功能的假设。收集的数据将为血管调节机制提供新的见解，并帮助我们了解与高血压，糖尿病和动脉粥样硬化等疾病相关的血管功能障碍的潜在机制。

DESCRIPTION (provided by applicant): Endothelial nitric oxide synthase (eNOS) via the production of nitric oxide (NO) in the vascular endothelium plays a key role in cardiovascular biology. NO is known to regulate vasodilation, platelet aggregation, platelet and leukocyte adhesion to the endothelium, endothelin-1 generation, and vascular smooth muscle proliferation thereby playing a role in the broader issues of blood pressure regulation and atherosclerosis. In addition, eNOS uncoupling in which the balance of NO production and superoxide production is shifted in favor of superoxide is associated with the endothelial dysfunction that occurs in various pathological conditions including hypertension and diabetes. Precise regulation of eNOS function is of vital importance to maintain vascular homeostasis. eNOS is regulated by co- and posttranslational mechanisms, phosphorylation, and protein-protein interactions. Understanding the relationship between these regulatory mechanisms is an important area of investigation. In our preliminary data, we have identified a novel eNOS interacting protein which may play an important role in mediating the eNOS protein complex formation as well as subsequent eNOS phosphorylation and activation. Using co-immunoprecipitation and in vitro binding experiments, we have determined that the cell division cycle 37 homolog (cdc37) interacts directly with eNOS and in the absence of other proteins asserts an inhibitory influence on eNOS activity. Cdc37 is critical component of the protein complex between heat shock protein 90 (Hs90) and Akt, two key eNOS regulatory proteins. However, no previous studies have examined the potential interaction between cdc37 and eNOS. Based on these observations and our preliminary work, we will use purified proteins in in vitro binding and kinase experiments as well as coimmunoprecipitation and adenovirus mediated gene transfer in endothelial cell cultures and isolated vessels to explore these interactions. The overall goals of this project are to investigate the effects of cdcd37 on eNOS function by examining cdc37's role in coordinating the interaction of eNOS with Hsp90 and Akt as well as subsequent phosphorylation and activation. The specific aims and hypotheses of this study are: Aim 1 - To test the hypothesis that specific domains in cdc37 and eNOS can be identified which govern the direct interaction between the two proteins, Aim 2 - To test the hypothesis that cdc37 mediates the eNOS complex formation with Hsp90 and Akt subsequently altering eNOS phosphorylation, and Aim 3 - To test the hypothesis that cdc37 modifies eNOS function both in vitro and in vivo. The data gathered will provide new insight into the mechanisms by which blood vessels are regulated and help us understand the underlying mechanisms of vascular dysfunction associated with diseases such as hypertension, diabetes, and atherosclerosis.