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

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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.