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

总结 随着年龄的增长，蛋白质抑制质量控制机制失效，导致受损和 功能失调的蛋白质蛋白质分解和替换与新蛋白质的合成（统称为 作为蛋白质周转）是减轻受损蛋白质随时间积累的主要机制， 因此是关键的蛋白质抑制机制。由于蛋白质抑制机制与ag的生物学高度相关， 以增加健康寿命为目标的教育和干预措施， teostasis将提供重要的见解，他们的机制。这个项目的总体目标是用一本小说 方法，以克服障碍，以了解蛋白质周转与衰老的进展，并揭示机制， 细胞特异性蛋白质稳定过程的机制。在过去的十年里，我们开发了稳定同位素方法- 目的是了解蛋白质稳态中蛋白质周转的机制基础。该提案确定了 在设计蛋白质周转研究时，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按数量级变化，2）增殖能力 影响蛋白质周转测量，3）蛋白质可以抵抗分解 (e.g.聚集或交叉桥接），这改变了动态蛋白库的大小。技术广告- 这些进展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ag- 服用和治疗（雷帕霉素和热量限制（CR）），增加健康寿命。拟建项目 将使用新描述的核标记和翻译核糖体亲和纯化（NuTRAP）小鼠， 靶向蛋白质组学和新的氧化氘（D2 O）标记，以检查细胞类型特异性的单个蛋白质 周转和复制。这项研究利用NuTRAP x Cre小鼠的细胞类型特异性， 大脑中的三种细胞类型和骨骼肌中的三种细胞类型。检查增殖性和非增殖性混合 每个组织内的增殖细胞类型将有助于确定蛋白质周转的变化如何有助于去增殖。 随着年龄的增长，蛋白质稳态发生变化，如果延缓衰老的治疗（雷帕霉素和CR）维持蛋白质稳态， 通过改善蛋白质周转。假设：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态的丧失和动态蛋白池大小的降低，以及 2)在大脑和骨骼肌中，雷帕霉素和CR将增加而不是减少与衰老相关的关键代谢物的周转。 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库。通过使用 专门处理传统上未加说明的机制的办法，以及 预测生物信息学方法，预计该项目的完成将克服一个重大的 障碍，我们的理解蛋白质随着年龄的恶化，并提供机制的见解蛋白质， 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， 蛋白质稳定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.