Loco/RGS14 Signaling Pathway in Longevity

描述(申请人提供)：虽然一些保守的信号通路，如IGF-1，AC-PKA，RAS/Raf/MAPK，TOR-S6K和Sir2已知在各种生物中调节抗逆性和寿命，但RGS(G蛋白信号调节因子)蛋白通过G蛋白信号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，导致果蝇寿命更长，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基因的过度表达显著缩短了植物的寿命，降低了植物的抗逆性和脂肪含量，也表明IS RGS结构域与长寿的调节有关。3)有趣的是，酵母RGS2和大鼠RGS14是苍蝇Loco的同源物，它们的表达变化也影响了各自物种的抗氧化性和寿命，这表明Loco/RGS14信号通路在进化上是保守的，用于调节寿命。众所周知，Fly Loco通过失活抑制的G？I？GTP蛋白来增加腺苷环化酶(AC)的活性，哺乳动物的RGS14与激活的H-Ras和Raf-1激酶相互作用，从而抑制ERK的磷酸化。我们的初步数据证实，LOCO的下调显著减少了cAMP的数量，并增加了p-ERK的水平，从而提高了对氧化应激的抵抗力。此外，我们的蛋白质组学iTRAQ和PHOS-Tag凝胶分析表明，Loco信号的减少降低了Rpd3蛋白(HDAC1，组蛋白脱乙酰酶1)的磷酸化水平，这可能是由Loco途径中的p-ERK调节的。据报道，Rpd3的表达减少可以延长果蝇的寿命。然而，长寿机制中RPD3/HDAC1活性的调节方式尚不清楚。我们还发现，LOCO基因的脂肪身体特异性下调增强了抗应激能力和寿命。根据我们的初步数据，本研究的具体假设是，RGS蛋白Loco/RGS14以组织(脂肪体)特异的方式调节Rpd3/HDAC1蛋白的磷酸化水平，从而调节抗逆性和寿命。其具体目的是检验：i)Loco信号是否通过无偏iTRAQ筛选确定的Rpd3 S419/S421位点的磷酸化来调节Rpd3的活性；以及ii)Loco/RGS14信号中磷酸化-Rpd3/HDAC1水平的变化是否通过组蛋白脱乙酰基酶Rpd3的靶基因决定抗逆性和寿命。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)在衰老过程中如何表观调控染色质重塑复合体(RPD3)。

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.