线粒体O-GlcNAc糖基化在衰老和卡路里限制过程中对线粒体代谢调控

Thousands of cytoplasmic and nuclear proteins are modified by a single O-linked β-N-acetylglucosamine (O-GlcNAc) moiety at serine or threonine residues, termed O-GlcNAcylation. In mammals, two enzymes catalyze the addition and removal of O-GlcNAc, namely O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. UDP-GlcNAc, the donor substrate for O-GlcNAcylation, is derived from extracellular glucose through the hexosamine biosynthetic pathway. Because UDP-GlcNAc and protein O-GlcNAc levels in the cell fluctuate with the availability of glucose, free fatty acids, uridine and the amino acid glutamine, O-GlcNAc is proposed as a nutrient sensor and metabolic regulator. It regulates fundamental cellular processes, such as signal transduction, transcription, translation and protein degradation. O-GlcNAcylation is conserved throughout metazoans. Ablation of the ogt allele in Caenorhabditis elegans impairs metabolism and longevity. The ogt knockout results in loss of embryonic stem cell viability and mouse ontogeny. In humans, aberrant O-GlcNAc modification has been implicated in a wide range of age-associated diseases, including diabetes, cancer, cardiovascular and neurodegenerative diseases. Mitochondrial dysfunction is a hallmarker of aging. Recently accumulating evidences show that many mitochondrial proteins are also O-GlcNAcylated. But the effects of mitochondrial O-GlcNAcylation have been known very limited. In light of the emerging links between protein O-GlcNAcylation and age, our proposed study is to elucidate the functions and mechanisms of mitochondrial O-GlcNAc in the aging process. We will 1)to study mitochondrial O-GlcNAcylation in the key metabolic tissues of old mice, analyze the relationship between O-GlcNAcylation and mitochondrial respiratory function and mitochondrial metabolism, and identify the specific targets by MASS spectra; 2) to confirm the mitochondrial O-GlcNAcylation in the mouse hepatocytes model of OGT loss- and gain function, study the effects on mitochondrial bioenergetics and metabolism, and identify the sites of O-GlcNAcylation in the specific targets ; 3) to estabolish the caloric restriction model in liver-specific OGT knockout mice and liver-specific OGT trangenic mice, study whether the genetic manipulation of OGT modulates calorie restriction, analyze the effects on mitochondrial bioenergetics and metabolism. The accomplishment of these studies will define mitochondrial O-GlcNAcylation as a mediator of aging and caloric restriction, and calrify the regulation of O-GlcNAc in mitochondrial function and metabolism. It is improtant for exploring O-GlcNAc as a therapeutic target in age-related diseases. And it will provide critical insights into the molecular basis of aging and calorie restriction, and help identify new longevity pathway,CR-mitochondrial O-GlcNAcylation-antiaging pathway.

O-GlcNAct糖基化是一种细胞内广泛存在的、独特的蛋白翻译后修饰，作为营养敏感器调节信号转导、转录、翻译和蛋白质降解等细胞行为。O-GlcNAc转移酶和O-GlcNAc水解酶介导这种动态可逆性修饰。异常O-GlcNAc糖基化与衰老和衰老相关疾病密切相关。线粒体功能障碍是衰老的生物标识之一，近年发现线粒体也存在O-GlcNAc修饰，但功能尚不清。本项目拟深入研究线粒体蛋白O-GlcNAc糖基化对线粒体生物能量和线粒体代谢的调控，揭示其在衰老线粒体功能障碍中的作用，分析鉴定特异性线粒体靶分子。利用肝脏特异性OGT基因敲除小鼠和OGT转基因小鼠卡路里限制(CR)模型，深入分析CR抗衰老作用中线粒体O-GlcNAc糖基化的功能和机制，探讨营养和激素对线粒体O-GlcNAc的调节。这不仅为全面揭示CR抗衰老作用机制开辟新的思路，更阐明了一条新的CR-O-GlcNAc糖基化-线粒体-长寿调控通路。

O-GlcNAc糖基化是一种细胞内广泛存在的、O-GlcNAc转移酶和O-GlcNAc水解酶介导的动态可逆性修饰。这种独特的蛋白翻译后修饰作为营养敏感器，调节信号转导、转录、翻译和蛋白质降解等细胞行为。本项目旨在阐释异常O-GlcNAc糖基化与衰老和衰老相关疾病的关系。线粒体功能障碍是衰老的生物标识之一，线粒体也存在O-GlcNAc 修饰，但其在线粒体功能调节和衰老过程中的作用等功能尚不清。. 本项目首先深入分析了衰老组织中线粒体O-GlcNAc糖基化改变，发现在老年小鼠多种代谢组织中O-GlcNAc糖基化水平普遍增高，其中肝脏组织中线粒体O-GlcNAc糖基化水平明显上调，某些特异的蛋白质O-GlcNAc糖基化改变，导致肝脏线粒体呼吸功能降低。这些结果说明线粒体O-GlcNAc糖基化造成线粒体功能受损，参与衰老过程。其次我们鉴定出线粒体呼吸链上complex II的组成分子SDHA是O-GlcNAc糖基化的靶分子，糖基化可以抑制SDHA酶活性和complexII的电子链传递功能。此外尿素循环第一步限速酶CPS1也是O-GlcNAc糖基化特异性靶分子，其Ser537位点糖基化抑制了CPS1酶活性，影响了nitrogen代谢和血氨稳定。这些结果阐明线粒体内某些关键蛋白的O-GlcNAc糖基化，如SDHA和CPS1，调节了线粒体氧化呼吸功能和物质代谢，从而在衰老过程中发挥重要的作用。利用CR模型，我们分析了CR后线粒体O-GlcNAc糖基化信号的变化，结果发现CR能够有效降低线粒体O-GlcNAc糖基化水平，CR后SDHA糖基化减少，线粒体呼吸功能增强。代谢组学研究结果显示O-GlcNAc糖基化参与调节CR后线粒体内尿素循环和TCA循环。因此，本项目证实营养感受器O-GlcNAc信号与衰老密切相关，阐释了新的寿命调控通路CR-O-GlcNAc糖基化-线粒体-衰老通路，为开发O-GlcNAc成为延缓衰老的靶分子奠定了重要的理论基础。. 最后，本项目探讨了线粒体O-GlcNAc糖基化在肿瘤中的功能和分子机制，结果发现在结肠癌细胞中OGT通过上调线粒体代谢关键酶IDH2转录和O-GlcNAc糖基化增加IDH2蛋白稳定性，发挥促进肿瘤生长作用。该分子机制填补了线粒体O-GlcNAc糖基化在肿瘤研究领域的空白，更为开发新的抗肿瘤治疗策略提供新思路。