Integrated mechanisms of protein turnover to prevent proteostasis decline with the aging process

Abstract

Proteostatic quality control mechanisms fail with advancing age, resulting in the accumulation of damaged and dysfunctional proteins. However, our understanding is limited because the prior investigations treated tissues as a homogenous collection of cells. Therefore, a critical unknown is how individual cell types within a tissue influence overall tissue proteostasis. The overall goal of this study will be to elucidate previously unexplored mechanisms of protein turnover for proteostatic maintenance. We will accomplish this goal by examining cell-type specific changes in individual protein turnover with aging or treatments that slow aging through direct inhibition of mTOR (rapamycin) or independent of direct mTOR inhibition (17±-estradiol (17±-E2)). For this, the following specific goals will be used: 1) Determine cell-type specific proteins susceptible to proteostatic decline with aging. 2) Determine how a treatment that inhibits mTOR improves proteostasis. 3) Determine how a treatment that does not directly inhibit mTOR improves proteostasis. We will use novel mouse models (NuTRAP-Cre; i.e HSA-myocyte, Pax7-satellite cell, Neuron-CamkIIa, and astrocyte-Aldh1l1) to isolate DNA, protein and mitochondria in a cell-type-specific manner. Our new approaches use the combination of deuterium oxide labeling and discovery or targeted proteomics. The methodologies used in this project will be single-cell, intact nuclear isolation, mass spectrometry and protein synthesis rate. Our study can contribute to a better understanding of the basic biology of aging and clarify more profoundly the molecular networks behind physiological adaptations promoted by protein turnover. Therefore, the findings open new perspectives and insights for studies based on complex mechanisms that govern cellular longevity. (AU)