Dissecting the integrated mechanisms of protein turnover to prevent proteostatic decline with aging

摘要 随着年龄的增长，前列腺癌的机制失效，导致损伤和功能障碍的积累。 蛋白质。蛋白质的分解和被新合成的蛋白质替代(蛋白质周转)是主要的 减少受损蛋白质随时间积累的机制。有几个相互矛盾的发现 与蛋白质周转、衰老和延缓衰老的治疗有关，这给该领域留下了根本的 关于蛋白质周转作为蛋白质抑制机制的矛盾。目前的项目寻求 了解调节蛋白平衡维持的基本机制，以便我们能够 克服障碍，瞄准这一老龄化支柱，减缓与年龄相关的下降。我们的研究表明： 1)衰老并不普遍降低蛋白质的周转率，事实上它增加了许多蛋白质的周转率， 2)老化减少了周转的蛋白质数量(为此，我们引入了术语动态池大小)，3) 延长寿命的治疗方法含有几乎相同数量的增加和减少蛋白质的蛋白质 合成，这与延长寿命的治疗方法减缓蛋白质合成的概念相矛盾，以及4)一些 延长寿命的治疗方法会减少细胞增殖，而细胞增殖本身就会降低非 决定性的态度。到目前为止，这些研究仅限于组织样本，这限制了 了解组织中的单个细胞类型如何影响整个组织的蛋白质平衡。要克服 对于这些未知因素，拟议中的研究使用了允许特定细胞分离蛋白质和细胞核的小鼠模型 在骨骼肌和大脑中。该项目将使用基于发现的蛋白质组学和靶向蛋白质组学 氧化氢(D2O)标记以检测特定细胞类型的个体蛋白质周转和细胞复制。 通过蛋白质维持的丧失(老化)和蛋白质维持的增加(减慢的治疗 ，我们将解决以下具体目标：确定易感细胞类型的特定蛋白 蛋白质稳定性随着年龄的增长而下降，以确定抑制mTOR的治疗如何改善蛋白质稳定性，并 确定不直接抑制mTOR的治疗如何改善蛋白稳定性。假设是这样的： 随着年龄的增长，蛋白质稳定性的丧失是由于个体蛋白质周转率和细胞类型的特定变化造成的。 减少动态蛋白质池大小，抑制mTOR通过以下方式改善蛋白质静态维持 减少细胞增殖，同时增加易感蛋白质的周转率和动态池大小 蛋白抑制剂下降，而不直接抑制mTOR的延长寿命的治疗改善了 通过不包括减慢细胞增殖的机制来维持蛋白质的稳定。在目标方面的进展 与年龄相关的蛋白降解酶的恶化受到来自蛋白质的矛盾和矛盾的结果的阻碍 离职率研究。我们预计，我们的方法将缩小易受蛋白质抑制剂影响的蛋白质的范围。 下降，更重要的是，将在靶向这些蛋白质的策略方面取得重大进展 随着年龄的增长保持蛋白质平衡。

SUMMARY Proteostatic mechanisms fail with advancing age, resulting in the accumulation of damaged and dysfunctional proteins. Protein breakdown and replacement with newly synthesized proteins (protein turnover) is the primary mechanism to mitigate accumulation of damaged proteins over time. There are several conflicting findings related to protein turnover, aging, and treatments that slow aging, which leave the field with fundamental contradictions regarding protein turnover as a proteostatic mechanism. The current project seeks to understand the fundamental mechanisms that regulate proteostatic maintenance so that we can overcome a barrier to targeting this pillar of aging to slow age-related decline. Our studies indicate that: 1) aging does not universally decrease protein turnover, and in fact it increases the turnover of many proteins, 2) aging deceases the number of proteins that turnover (for which we introduce the term dynamic pool size), 3) treatments that extend lifespan have nearly an equal number of proteins that increase and decrease protein synthesis, which contradicts the notion that treatments that extend lifespan slow protein synthesis, and 4) some treatments that increase lifespan decrease cell proliferation, which by itself decreases protein turnover in a non- determinant manner. To date, these studies have been limited to tissue samples, which has restricted the understanding of how individual cell types within a tissue influence overall tissue proteostasis. To overcome these unknowns, the proposed studies use mouse models that allow cell-specific isolation of proteins and nuclei in skeletal muscle and brain. The project will use both discovery-based and targeted proteomics with novel deuterium oxide (D2O) labeling to examine cell-type-specific individual protein turnover and cell replication. Through loss of proteostatic maintenance (aging) and gain of proteostatic maintenance (treatments that slow aging), we will address the following specific aims: to determine cell-type specific proteins susceptible to proteostatic decline with aging, to determine how a treatment that inhibits mTOR improves proteostasis, and to determine how a treatment that does not directly inhibit mTOR improves proteostasis. The hypotheses are that: the loss of proteostasis with aging results from cell-type specific changes in individual protein turnover rates and decreases in the dynamic protein pool size, that inhibiting mTOR improves proteostatic maintenance by decreasing cell proliferation while increasing turnover and dynamic pool size of proteins that are susceptible to proteostatic decline, and that a lifespan-extending treatment that does not directly inhibit mTOR improves proteostatic maintenance by mechanisms that do not include slowed cell proliferation. Progress toward targeting age-related proteostatic deterioration has been hindered by contradictory and paradoxical results from protein turnover studies. We expect that our approaches will narrow the scope of proteins susceptible to proteostatic decline, and more importantly, will make significant advancements toward strategies to target these proteins to maintain proteostasis with age.