A novel approach to understand a mechanism of proteostatic decline with aging

摘要 前列腺癌的质量控制机制随着年龄的增长而失效，导致受损和 功能失调的蛋白质。蛋白质分解和代之以合成新蛋白质(统称为 作为蛋白质周转)是随着时间的推移减缓受损蛋白质积累的主要机制，以及 因此是一种关键的蛋白抑制机制。由于蛋白质组学机制与农业病原体的生物学高度相关。 有针对性地增加健康寿命的ING和干预措施，更准确地评估PRO- 静止期将为了解它们的机制提供重要的见解。这个项目的总体目标是用一本小说 克服研究蛋白质随衰老周转的障碍并揭示其机制的途径 细胞特有的蛋白质恒定过程。在过去的十年里，我们开发了稳定同位素方法- 了解蛋白质代谢中蛋白质周转的机制基础。这份提案确定了 在设计蛋白质周转率和蛋白质周转率的研究时经常无法解释的三种生理机制 老化：1)单个蛋白质的含量和半衰期不同的数量级，2)增殖能力 一种细胞类型会影响蛋白质周转率的测量，以及3)蛋白质可以变得抵抗分解 (例如聚集或交叉桥联)，这改变了动态蛋白质池的大小。科技广告-- 改进使我们能够克服这些以前的限制，并明确地解决蛋白质周转与农业- ING和治疗(雷帕霉素和卡路里限制(CR))可以延长健康寿命。拟议中的项目 将使用新描述的核标签和翻译核糖体亲和纯化(NuTRAP)小鼠， 靶向蛋白质组学和用于检测细胞类型特异性个体蛋白的新型氧化氢(D2O)标记 周转和复制。这项研究利用NuTRAP x Cre小鼠的细胞类型特异性检测了三个 脑中的细胞类型和骨骼肌中的三种细胞类型。增生性和非增殖性混合疾病的检查 每个组织中的增殖性细胞类型将有助于确定蛋白质周转的变化如何有助于降低 蛋白稳定与年龄的关系，以及延缓衰老的治疗(雷帕霉素和CR)是否保持蛋白稳定 通过改善蛋白质周转。假设是：1)随着年龄的增长，蛋白质的异质性变化 蛋白质、细胞和组织的周转导致蛋白质稳定性的丧失和动态蛋白质池大小的减少，以及 2)在脑和骨骼肌中，雷帕霉素和CR会增加而不是减少，关键的周转与衰老有关 蛋白质，减少增殖细胞类型的复制，并维持更大的动态蛋白质池。通过使用 旨在具体解决传统上下落不明的机制的方法，以及 预测生物信息学的方法，预计该项目的完成将克服重大 阻碍了我们对蛋白质随着年龄的增长而恶化的理解，并提供了对蛋白质- 静态调节。这些结果将促进针对肌肉和大脑的干预措施的发展 抑制蛋白质的过程来减缓衰老过程。

SUMMARY Proteostatic quality control mechanisms fail with advancing age, resulting in the accumulation of damaged and dysfunctional proteins. Protein breakdown and replacement with synthesis of new proteins (collectively referred to as protein turnover) is the primary mechanism to mitigate accumulation of damaged proteins over time, and is thus a critical proteostatic mechanism. As proteostatic mechanisms are highly relevant to the biology of ag- ing and interventions targeted to increase healthspan, a more accurate assessment of the components of pro- teostasis will provide important insights into their mechanisms. The overall goal of this project is to use a novel approach to overcome barriers to progress in understanding protein turnover with aging and to reveal mecha- nisms of cell-specific proteostatic processes. For the last decade we have developed stable isotope approach- es to understand the mechanistic underpinnings of protein turnover in proteostasis. This proposal identifies three physiological mechanisms that are often unaccounted for when designing studies of protein turnover and aging: 1) content and half-lives of individual proteins vary by orders of magnitude, 2) the proliferative capacity of a cell type impacts protein turnover measurements, and 3) that proteins can become resistant to breakdown (e.g. aggregation or cross bridging), which changes the size of the dynamic protein pool. Technological ad- vancements allow us to overcome these previous limitations and definitively address protein turnover with ag- ing and treatments (rapamycin and caloric restriction (CR)) that increase health- lifespan. The proposed project will use the newly described Nuclear tagging and Translating Ribosome Affinity Purification (NuTRAP) mouse, targeted proteomics, and novel deuterium oxide (D2O) labeling to examine cell-type-specific individual protein turnover and replication. This study leverages the cell-type specificity of NuTRAP x Cre mice to examine three cell types in brain and three cell types in skeletal muscle. Examination of a mix of proliferative and non- proliferative cell types within each tissue will help determine how changes in protein turnover contribute to de- clining proteostasis with age, and if treatments that slow aging (rapamycin and CR) maintain proteostasis through improved protein turnover. The hypotheses are that: 1) with aging, heterogeneous changes in protein turnover by protein, cell, and tissue cause a loss of proteostasis and decreased dynamic protein pool size, and 2) in brain and skeletal muscle, rapamycin and CR will increase, not decrease, turnover of key aging-related proteins, decrease replication of proliferative cell types, and maintain a larger dynamic protein pool. By using approaches designed to specifically address mechanisms that are traditionally unaccounted for, as well as a predictive bioinformatics approach, it is expected that the completion of this project will overcome a significant barrier in our understanding of proteostatic deterioration with age, and provide mechanistic insight into proteo- static regulation. These outcomes will facilitate the development of interventions in muscle and brain that target proteostatic processes to slow the aging process.