Regulation of eNOS by S-Nitrosylation in Diet Associated Erectile Dysfunction

项目摘要 富含来自动物源的糖和脂肪以及廉价植物油的饮食被称为 “西方饮食”。西方饮食的消费现在在美国很流行， 在过去的15年里，西方饮食的消费与病因有关 勃起功能障碍（艾德），一种与生活质量下降密切相关的疾病。一氧化氮是一种主要的 一氧化氮合酶是阴茎勃起的介质。在过度氧化的条件下， 应激时，内皮型一氧化氮合酶（eNOS）可能变得解偶联，从而产生超氧化物 而不是一氧化氮，它会进一步传播氧化应激。认为eNOS偶联状态是 通过辅因子和底物的充分存在来调节。然而，最近的数据表明，eNOS 解偶联和功能障碍可以通过S-亚硝基化来促进，S-亚硝基化可以依赖于氧化的 细胞氧化还原状态转变。此外，雷帕霉素的哺乳动物靶标（mTOR）是一种细胞信号传导途径， 这种分子可能会诱导氧化应激反应，以应对过量的脂肪和糖的消耗。主 这一建议的假设是，在阴茎氧化还原环境中的氧化转变，由mTOR介导， 依赖营养信号对西方饮食的反应促进了由eNOS引起的勃起功能障碍 通过脱亚硝基化系统受损诱导的过度S-亚硝基化引起的调节异常。具体目标 该建议的目的是：1）确定西方饮食诱导的艾德是否是由氧化还原状态的氧化转变介导的 2）确定氧化还原状态的mTOR依赖性转变是否施加改变 3）确定S-亚硝基化的调节是否控制饮食依赖性勃起功能， 方式拟议的研究计划包括使用西式啮齿动物饮食， 药理学、分子学、蛋白质组学和生理学研究。这些研究利用西方饮食， 与氧化还原状态的药理学转变、mTOR的抑制和蛋白质的遗传操作的组合 调节脱硝作用的蛋白质建议的分子研究包括蛋白质结构、功能和 翻译后修饰影响eNOS、mTOR信号通路和调节细胞凋亡的蛋白质。 细胞S-亚硝基化建议的蛋白质组学研究包括评估阴茎的S-亚硝基化状态 蛋白质组，以及阴茎eNOS的单个巯基的S-亚硝基化。分子和蛋白质组学研究 将通过响应于这些扰动的生理勃起功能测量来丰富。的 提出的研究包括使用遗传小鼠模型，其中S-亚硝基化的调节是 操纵，和大鼠模型，其中S-亚硝基化的调节是专门在阴茎操纵。它 预计这项提案的发现将使人们更好地了解导致 西医饮食与艾德有关，并为加强治疗提供依据。

PROJECT SUMMARY A diet abundant in sugar and fats derived from animal sources and cheap vegetable oils has been termed the "Western diet". Consumption of the Western diet is now prevalent in the United States and has been spreading throughout the world over the past 15 years. Consumption of a Western style diet is implicated in the etiology of erectile dysfunction (ED), a condition strongly associated with reduced quality of life. Nitric oxide is a primary mediator of penile erection, which is produced by nitric oxide synthase. Under conditions of excessive oxidative stress, endothelial nitric oxide synthase (eNOS) may become uncoupled, whereby superoxide is produced rather than nitric oxide, which propagates further oxidative stress. It is thought that eNOS coupling status is regulated by adequate presence of co-factors and substrates. However, recent data indicate that eNOS uncoupling and dysfunctionality may be promoted by S-nitrosylation, which may be dependent on an oxidative shift in cellular redox state. Additionally, the mammalian target of rapamycin (mTOR) is a cellular signaling molecule that may induce oxidative stress in response to excessive fat and sugar consumption. The primary hypothesis of this proposal is that an oxidative shift in the penile redox environment, mediated by mTOR- dependent nutrient signaling in response to the Western diet promotes erectile dysfunction due to eNOS dysregulation via excessive S-nitrosylation induced by impairment of the denitrosylation system. Specific aims for the proposal are: 1) to determine if Western diet-induced ED is mediated by an oxidative shift in redox state dependent upon mTOR signaling; 2) to determine if the mTOR dependent shift in redox state exerts alteration of S-nitrosylation; 3) to determine if regulation of S-nitrosylation controls erectile function in a diet-dependent manner. The proposed research plan includes the use of a Western style rodent diet in combination with pharmacologic, molecular, proteomic, and physiological studies. These studies utilize the Western diet in combination with pharmacologic shifts in redox state, inhibition of mTOR, and genetic manipulation of proteins that regulate denitrosylation. Molecular studies proposed include assessment of protein structure, function, and post-translational modifications influencing eNOS, the mTOR signaling pathway, and the proteins that regulate cellular S-nitrosylation. Proteomic studies proposed include assessment of S-nitrosylation status of the penile proteome, as well as S-nitrosylation of individual thiol groups of penile eNOS. Molecular and proteomic studies will be enriched by physiologic erectile function measurements in response to these perturbations. The proposed studies include the use of a genetic mouse model where the regulation of S-nitrosylation is manipulated, and a rat model where the regulation of S-nitrosylation is manipulated specifically in the penis. It is anticipated that the findings of this proposal will provide greater understanding of molecular events leading to Western diet associated ED, and provide a basis for enhanced treatment.