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

项目总结 一种从动物来源和廉价植物油中提取的富含糖和脂肪的饮食被称为 “西方饮食”。西方饮食现在在美国很盛行，而且还在蔓延 在过去的15年里，在世界各地。西式饮食的摄入与病因有关 勃起功能障碍(ED)，这是一种与生活质量下降密切相关的疾病。一氧化氮是主要的 阴茎勃起的介质，由一氧化氮合酶产生。在过度氧化的条件下 应激时，内皮型一氧化氮合酶(ENOS)可能解偶联，从而产生超氧化物 而不是一氧化氮，后者会进一步传播氧化应激。人们认为eNOS的偶联状态是 由足够的辅因子和底物的存在来调节。然而，最近的数据表明，eNOS S-亚硝化可促进解偶联和功能障碍，这可能依赖于一种氧化 细胞氧化还原状态的转变。此外，雷帕霉素的哺乳动物靶点(MTOR)是一种细胞信号 因摄入过多的脂肪和糖类而引起氧化应激的分子。初级阶段 这一设想的假设是，阴茎氧化还原环境中的氧化转变，由mTOR- 响应西方饮食的依赖营养信号促进eNOS引起的勃起功能障碍 反硝化系统受损所致的S亚硝化过度所致的调节失调。具体目标 1)确定西方饮食诱导的勃起功能障碍是否由氧化还原状态的氧化转变所介导 依赖于mTOR信号；2)确定氧化还原状态中依赖mTOR的转变是否产生改变 S亚硝化的研究；3)确定S亚硝化的调节是否控制饮食依赖的勃起功能 举止。拟议的研究计划包括使用西式啮齿动物饮食与 药理学、分子、蛋白质组学和生理学研究。这些研究利用了西方的饮食 结合氧化还原状态的药理变化、mTOR的抑制和蛋白质的遗传操作 调节脱氮反应的物质。建议的分子研究包括评估蛋白质的结构、功能和 翻译后修饰影响eNOS、mTOR信号通路和调节蛋白 细胞S-亚硝化。建议的蛋白质组学研究包括评估阴茎的S-硝化状态 蛋白质组，以及阴茎eNOS各个硫醇基团的S亚硝化。分子和蛋白质组学研究 将通过生理勃起功能测量来丰富对这些扰动的反应。这个 建议的研究包括使用遗传小鼠模型，在该模型中，S-亚硝化的调节是 以及S-亚硝化的调节在阴茎中特定地被操纵的大鼠模型。它 预计这项提案的发现将有助于更好地理解导致 西式饮食与ED相关，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.