Understanding the molecular regulation of autophagy during aging

了解衰老过程中自噬的分子调控

• 批准号: 9374136
• 负责人: Rajat Singh
• 金额: $ 25.05万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE, INC
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9374136
• 关键词: Acetylation; Age; Age of Onset; Aging; Arginine; Autophagocytosis; Autophagosome; Binding; Biochemical; Cells; Cis-Acting Sequence; Development; Disease; Elements; FRAP1 gene; Failure; Fatty acid glycerol esters; Fibronectins; Genes; Genetic; Genetic Translation; Goals; Half-Life; Human; In Vitro; Individual; Knock-out; Light; Lipids; Liver; Longevity; Malignant Neoplasms; Mediating; Membrane Lipids; Messenger RNA; Metabolic Diseases; Metabolic syndrome; Metabolism; Molecular; Mus; Mutagenesis; Mutant Strains Mice; Nerve Degeneration; Organelles; PIK3CG gene; Pathology; Pathway interactions; Phosphotransferases; Play; Post-Translational Protein Processing; Proteins; Proteomics; Quality Control; RNA; RNA Binding; Regulation; Risk; Role; Sequence Analysis; Starvation; Stress; Testing; Transcript; Transgenic Organisms; age related; aged; cis acting element; disorder prevention; feeding; in vivo; insight; mRNA Stability; mutant; novel; novel strategies; overexpression; prevent; protein aggregate; reconstitution; restoration; sarcopenia

Abstract The remarkable increase in human longevity over the last century has, unfortunately, increased the risk of developing a number of age-related conditions, which underscores the need to develop strategies to extend human healthspan. Cells recycle their damaged organelles and protein aggregates via autophagy whereby cytoplasmic components are sequestered within autophagosomes (APh) and targeted for lysosomal degradation. Briefly, class III PI3K, ULK1 kinase, and a conjugation cascade involving core autophagy-related (ATG) proteins, ATG7, ATG5, ATG12, ATG16L1 and ATG8/light chain 3 (LC3) regulate autophagosome (APh) formation by lipidating cytosolic LC3-I into APh-bound LC3-II. Aging associates with autophagy failure, which is a critical factor in the development of age-associated conditions. How autophagy decreases with age remains unclear. While sustained mTOR activation, altered membrane lipid composition, and acetylation of core ATG proteins each contribute to autophagy failure; none of these mechanisms can completely explain why ATG proteins decrease with age. We have found that LC3, a structural component of APh, binds to Atg16l1 mRNA. LC3 has recently been shown to bind and enhance fibronectin mRNA translation – thus revealing a novel role of LC3 in RNA metabolism. Our preliminary analyses revealed that starvation, which activates autophagy, leads to increased LC3-Atg16l1 mRNA binding. Livers knocked out for Atg7 (that do not form LC3-II), and livers from aged mice, each display loss of LC3-Atg16l1 mRNA binding, suggesting that LC3-II, and not LC3- I, binds to Atg16l1 mRNA. In addition, overexpression of LC3 enhances ATG16L1 protein half-life. Furthermore, Atg16l1 mRNA sequence analysis revealed presence of several AU-rich elements (ARE), which are cis-acting sequences known to facilitate binding of RNA to specific proteins. These observations have led to our hypothesis that LC3 binds to Atg16l1 mRNA in a feed-forward regulatory loop that stabilizes Atg16l1 mRNA, and thereby maintains ATG16L1 protein levels and autophagy. We hypothesize further that disruption of this regulatory loop contributes to autophagy failure. The overall goal of this proposal is to: 1) examine whether age-associated decrease in LC3-Atg16l1 mRNA binding leads to decreased Atg16l1 mRNA levels and autophagy failure, 2) determine the mechanism of LC3- mediated Atg16l1 mRNA stability by using novel LC3 mutants in vitro, and a novel RRRLC3QQQ mutant mouse wherein a triple arginine (RRR) motif in LC3, which we propose allows LC3-Atg16l1 mRNA binding, has been inactivated, and 3) test whether genetic reconstitution of LC3-II in aged livers can restore Atg16l1 mRNA levels, autophagy, and lipohomeostasis. Our studies will provide new mechanistic insight into how autophagy decreases with age, and provide a novel strategy to prevent the decline of this important quality control pathway with age. Significance: Aged individuals are at increased risk for developing a number of diseases, neurodegeneration, metabolic syndrome, and cancers, to mention a few. Studies of the loss of autophagy in young mice or genetic overexpression of specific Atg genes in mice have shown the critical role of autophagy in disease prevention. How autophagy decreases with age remains unclear. Our studies will reveal a new mechanism of regulating autophagy by enhancing the stability of an Atg mRNA, Atg16l1, by an ATG protein, LC3. Understanding how autophagy decreases with age is critical for the development of new strategies to extend human healthspan – the number of years one remains healthy and active.

摘要 不幸的是，在过去的世纪里，人类寿命的显著延长增加了这种风险。 发展一些与年龄有关的条件，这强调需要制定战略， 延长人类的健康寿命。细胞通过以下途径回收它们受损的细胞器和蛋白质聚集体： 自噬，其中细胞质组分被隔离在自噬体内（APh）， 靶向溶酶体降解。简而言之，III类PI 3 K、ULK 1激酶和缀合级联 涉及核心自噬相关（ATG）蛋白、ATG 7、ATG 5、ATG 12、ATG 16 L1和ATG 8/轻链 3（LC 3）通过将胞质LC 3-I脂化为APh结合的LC 3-II来调节自噬体（APh）的形成。 衰老与自噬失败有关，自噬失败是年龄相关性肿瘤发生的关键因素。 条件自噬如何随着年龄的增长而减少仍不清楚。在持续mTOR激活的同时， 改变的膜脂质组成和核心ATG蛋白的乙酰化都有助于自噬 这些机制都不能完全解释为什么ATG蛋白随着年龄的增长而减少。我们 已经发现LC 3，APh的结构组分，与Atg 16 l1 mRNA结合。LC 3最近 显示结合并增强纤连蛋白mRNA翻译-从而揭示LC 3在RNA中的新作用 新陈代谢.我们的初步分析表明，饥饿，激活自噬，导致 增加LC 3-Atg 16 l1 mRNA结合。敲除Atg 7的肝脏（不形成LC 3-II），以及 来自老年小鼠的LC 3-Atg 16 l1 mRNA结合均丧失，表明LC 3-II而非LC 3- I结合Atg 16 l1 mRNA。此外，LC 3的过表达增强了ATG 16 L1蛋白的半衰期。 此外，Atg 16 l1 mRNA序列分析显示存在几种富含AU的元件（ARE）， 其是已知促进RNA与特定蛋白质结合的顺式作用序列。这些 观察导致我们的假设，LC 3结合Atg 16 l1 mRNA的前馈调节 稳定Atg 16 l1 mRNA的环，从而维持ATG 16 L1蛋白水平和自噬。我们 进一步假设该调节环的破坏有助于自噬失败。整体 本研究的目的是：1）研究是否与年龄相关的LC 3-Atg 16 l1 mRNA结合减少 导致Atg 16 l1 mRNA水平降低和自噬失败，2）确定LC 3- 通过使用新的LC 3突变体在体外介导的Atg 16 l1 mRNA稳定性，以及新的RRRLC 3QQQ突变体， 小鼠，其中三重精氨酸（RRR）基序在LC 3中，我们建议允许LC 3-Atg 16 l1 mRNA 结合，已被灭活，和3）测试是否LC 3-II的遗传重建，在老年肝脏， 恢复Atg 16 l1 mRNA水平、自噬和脂质稳态。我们的研究将提供新的 自噬如何随着年龄的增长而减少，并提供了一种新的策略，以防止自噬。 这一重要的质量控制途径随着年龄的增长而下降。 意义：老年人患多种疾病的风险增加， 神经变性、代谢综合征和癌症，仅举几例。研究损失 年轻小鼠中的自噬或小鼠中特定Atg基因的遗传过表达表明， 自噬在疾病预防中的重要作用。自噬如何随着年龄的增长而减少仍不清楚。 我们的研究将揭示一种通过增强Atg的稳定性来调控自噬的新机制 mRNA，Atg 16 l1，通过ATG蛋白，LC 3。了解自噬如何随着年龄的增长而减少至关重要 为了制定新的战略，以延长人类的健康寿命- 健康和活跃。