Loco/RGS14 Signaling Pathway in Longevity

描述（由申请人提供）：虽然已知TOR-S6K、AC-PKA-Ras/Raf/MAPkinase、IGF-1等几种保守的信号通路在多种生物体中调节抗逆性和寿命，但RGS （g蛋白信号调节因子）蛋白如何通过g蛋白信号通路影响衰老过程的机制尚不清楚。我们的初步数据显示，1)RGS蛋白Loco的表达减少，导致果蝇寿命延长，对应激源的抵抗力增强，MnSOD活性提高，脂肪含量增加。2)相比之下，过表达loco基因显著缩短了寿命，降低了抗逆性，降低了脂肪含量，也表明其RGS结构域与寿命调控有关。3)有趣的是，苍蝇Loco的同源物酵母RGS2和大鼠RGS14的表达变化也影响了各自物种的氧化应激抗性和寿命，这表明Loco/RGS14信号通路在多种生物中具有进化保守性，调控了寿命。众所周知，Loco使抑制性G¿i¿GTP蛋白失活，从而降低腺苷酸环化酶（AC）的活性，RGS14与活化的H-Ras和Raf-1激酶相互作用，从而抑制ERK磷酸化。本研究的具体假设是，Loco/RGS14蛋白作为AC-cAMP-PKA通路的激活剂和/或作为Ras¿GTP-Raf-MEK-ERK信号通路中分离活性Ras和Raf的分子支架调节抗逆性和寿命。与此一致的是，我们的初步数据显示，下调Loco显著降低cAMP含量，增加p-ERK水平，提高抗氧化应激能力。此外，使用loco突变体和过表达进行的蛋白质组学iTRAQ和2D-gel分析显示，loco信号可增加Rpd3蛋白（HDAC1，组蛋白去乙酰化酶1）的磷酸化水平。据报道，Rpd3蛋白的表达减少可以延长果蝇的寿命。然而，Rpd3/HDAC1活性如何调控长寿机制尚不清楚。基于我们的初步数据，具体目的是检验1)Loco蛋白是否通过Ras/Raf/MAPkinase信号通路之间的直接相互作用来调节Ras/Raf/MAPkinase信号通路，2)Loco/RGS14信号通路是否调节果蝇和哺乳动物中Rpd3/HDAC1蛋白的磷酸化水平，3)HDAC1蛋白磷酸化水平是否通过调节HDAC1蛋白的亚细胞定位和/或活性来决定抗逆性和寿命。我们的方法将发现Loco/RGS14蛋白如何通过新的和/或已知的长寿信号通路调节寿命。我们相信，从我们的研究中获得的信息将促进我们对真核细胞衰老过程机制的一般理解，并帮助我们找到治疗或延缓衰老相关疾病的方法，如阿尔茨海默病。

DESCRIPTION (provided by applicant): Although several conserved signaling pathways such as TOR-S6K, AC-PKA-Ras/Raf/MAPkinase, and IGF-1 are known to regulate stress resistance and longevity in various organisms, the mechanism of how RGS (regulator of G-protein signaling) protein affects aging process through G-protein signaling pathway is not understood yet. Our preliminary data exhibited that 1) reduced expression of a RGS protein, Loco, resulted in a longer lifespan of flies with stronger resistances to stressors, higher MnSOD activity, and increased fat contents. 2) In contrast, overexpression of the loco gene shortened lifespan significantly with lower stress resistance and reduced fat contents, also indicating that its RGS domain is related to the regulation of longevity. 3) Interestingly, expressional changes of yeast RGS2 and rat RGS14, homologues of the fly Loco, also affected oxidative stress resistance and longevity in the respective species, suggesting that Loco/RGS14 signaling pathway is evolutionarily conserved in various organisms for the regulation of longevity. It is known that Loco inactivates inhibitory G¿i¿GTP protein which reduces activity of adenylate cyclase (AC) and RGS14 interacts with activated H-Ras and Raf-1 kinases, which subsequently inhibits ERK phosphorylation. The specific hypothesis for this research is that Loco/RGS14 protein regulates stress resistance and longevity as an activator in AC-cAMP-PKA pathway and/or as a molecular scaffold that sequesters active Ras and Raf from Ras¿GTP-Raf-MEK-ERK signaling pathway. Consistently, our preliminary data showed that down-regulation of Loco significantly diminishes cAMP amounts and increases p-ERK levels with higher resistance to the oxidative stress. In addition, proteomic iTRAQ and 2D-gel analyses using loco mutant and overexpression revealed that Loco signaling increases phosphorylation levels of Rpd3 protein (HDAC1, histone deacetylase 1). Reduced expression of Rpd3 protein is reported to extend lifespan in Drosophila. However, it is not characterized how Rpd3/HDAC1 activity is regulated for longevity mechanism. Based on our preliminary data, the specific aims are to examine I) if Loco protein regulates the Ras/Raf/MAPkinase signaling pathway through direct interaction between them, II) if Loco/RGS14 signaling modulates phosphorylation levels of Rpd3/HDAC1 protein in both of fruitfly and mammal, III) if phosphorylation level of HDAC1 protein determines stress resistance and lifespan with regulating subcellular localization and/or activity of HDAC1 protein. Our approaches will find how the Loco/RGS14 protein regulates longevity with new and/or known longevity signaling pathway(s). We believe that the information obtained from our studies will advance our general understanding of the mechanisms of eukaryotic aging process and help us find ways to cure or delay aging-related diseases such as Alzheimer's disease.