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

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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.