Loco/RGS14 Signaling Pathway in Longevity

描述（由申请人提供）：尽管已知几种保守的信号传导途径如IGF-1、AC-PKA、Ras/Raf/MAP激酶、T0 R-S6 K和Sir 2在各种生物体中调节应激抗性和寿命，但RGS（G蛋白信号传导调节剂）蛋白通过G蛋白信号传导途径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，对应激源的抵抗力更强，MnSOD活性更高，脂肪含量增加。2)相反，loco基因的过表达显著缩短了寿命，降低了抗逆性和脂肪含量，也表明RGS结构域与寿命的调节有关。3)有趣的是，酵母RGS 2和大鼠RGS 14的表达变化，苍蝇位置的同源物，也影响氧化应激抗性和寿命在各自的物种，这表明位置/RGS 14信号通路是进化保守的各种生物体的寿命调节。已知果蝇Loco通过使抑制性G i i GTP蛋白失活来增加腺苷酸环化酶（AC）活性，并且哺乳动物RGS 14与活化的H-Ras和Raf-1激酶相互作用，其随后抑制ERK磷酸化。我们的初步数据证实，下调Loco显著减少cAMP量并增加p-ERK水平，对氧化应激具有更高的抵抗力。此外，我们的蛋白质组iTRAQ和Phos-tag凝胶分析显示，Loco信号的减少降低了Rpd 3蛋白（HDAC 1，组蛋白脱乙酰酶1）的磷酸化水平，这可能是由Loco通路中的p-ERK调节的。据报道，Rpd 3的表达减少可延长果蝇的寿命。然而，Rpd 3/HDAC 1活性调节长寿机制的方式尚未确定。我们还发现，脂肪体特异性下调loco基因增强了抗应激能力和寿命。基于我们的初步数据，本研究的具体假设是，RGS蛋白Loco/RGS 14通过以组织（脂肪体）特异性方式调节Rpd 3/HDAC 1蛋白的磷酸化水平来调节应激抗性和寿命。具体目的是检查I）Loco信号传导是否通过从无偏iTRAQ筛选鉴定的Rpd 3 S419/S421位点的磷酸化调节Rpd 3活性，和II）Loco/RGS 14信号传导中磷酸-Rpd 3/HDAC 1水平的变化是否通过组蛋白脱乙酰酶Rpd 3的靶基因决定应激抗性和寿命。我们的研究将探索新的衰老领域，以确定G蛋白信号通路（Loco）如何在衰老过程中表观遗传地调节染色质重塑复合物（Rpd 3）。

DESCRIPTION (provided by applicant): Although several conserved signaling pathways such as IGF-1, AC-PKA, Ras/Raf/MAPkinase, TOR- S6K and Sir2 are known to regulate stress resistance and longevity in various organisms, the mechanism by which the RGS (regulator of G-protein signaling) protein affects the aging process through the 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 content. 2) In contrast, overexpression of the loco gene shortened lifespan significantly with lower stress resistance and reduced fat content, also indicating that is RGS domain is related to the regulation of longevity. 3) Interestingly, the 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 the Loco/RGS14 signaling pathway is evolutionarily conserved in various organisms for the regulation of longevity. It is known that fly Loco increases adenylate cyclase (AC) activity by inactivating the inhibitory G¿i¿GTP protein and that mammalian RGS14 interacts with activated H-Ras and Raf-1 kinases, which subsequently inhibit ERK phosphorylation. Corroboratively, our preliminary data showed that downregulation of Loco significantly diminishes cAMP amounts and increases p-ERK levels with higher resistance to oxidative stress. In addition, our proteomic iTRAQ and Phos-tag gel analyses revealed that reduction of Loco signaling decreases phosphorylation levels of the Rpd3 protein (HDAC1, histone deacetylase 1), which may be regulated by p-ERK in the Loco pathway. Reduced expression of Rpd3 is reported to extend the lifespan in Drosophila. However, the way in which Rpd3/HDAC1 activity is regulated for the longevity mechanism is not characterized. We also found that fat body-specific downregulation of the loco gene enhances stress resistance and longevity. Based on our preliminary data, the specific hypothesis for this research is that the Loco/RGS14, a RGS protein, regulates stress resistance and longevity by modulating the phosphorylation level of Rpd3/HDAC1 protein in a tissue (fat body)- specific manner. The specific aims are to examine I) if Loco signaling modulates Rpd3 activity through phosphorylation of Rpd3 S419/S421 sites identified from unbiased iTRAQ screening, and II) if change of the phospho-Rpd3/HDAC1 level in Loco/RGS14 signaling determines stress resistance and lifespan through the target genes of histone deacetylase Rpd3. Our research will explore new aging field to determine how the G- protein signaling pathway (Loco) epigenetically regulates the chromatin remodeling complex (Rpd3) during aging process.