The measurement of protein synthesis for assessing proteostasis in studies of slowed aging

Abstract

Slowing the aging process can reduce the risk for multiple chronic diseases simultaneously. It is increasingly recognized that maintaining protein homeostasis (or proteostasis) is important for slowing the aging process. Since proteostasis is a dynamic process, monitoring it is not a simple task and requires use of appropriate methods. This review will introduce methods to assess protein and DNA synthesis using deuterium oxide (D2O), and how protein and DNA synthesis outcomes provide insight into proteostatic mechanisms. Finally, we provide a discussion on how these assessments of protein and DNA synthesis are "mechanistic" investigations and provide an appropriate framework for the further development of slowed aging treatments.

Keywords: Deuterium oxide; Long-lived model; Mitochondria; Proliferation; Stable isotope.

Figure 1. Using D₂O for assessing synthesis of DNA and protein

Following an intraperitoneal injection, D₂O (²H₂O) is administered via the animal's drinking water where it rapidly equilibrates with the body water pool (A). Incorporation of deuterium from body water into labile hydrogens of precursor building blocks and subsequent incorporation of those precursor species into stable C-H bonds allows monitoring of rates of newly synthesized proteins and DNA up until a period in which all are 100% new (B). The synthesis of new protein and DNA control protein concentration in vivo (C).

Figure 2. Representative protein synthesis, DNA synthesis, and protein synthesis:DNA synthesis in three models of slowed aging

(A) Synthetic rates of DNA and protein in three fractions of skeletal muscle (MIXED = myofibrillar, CYTO = cytoplasmic, MITO = Mitochondrial) in lifelong calorically restricted mice (CR) and ad libitum fed controls (AL). Although there is a trend for greater mitochondrial protein synthesis in CR, protein synthesis rates are largely similar between CR and AL (data reproduced from (Miller et al., 2013; 2012)). (B) When the data are expressed as a synthesis ratio, it is clear that there is increase protein synthesis dedicated to proteostasis in CR animals as compared to AL. (C) Synthetic rates of DNA and three fractions of heart in chronically fed rapamycin treated (RAP) and age matched controls (CON). There are no differences between RAP and CON for protein synthetic rates, although DNA synthesis is lower in RAP compared to CON. (D) When the data are expressed as a synthesis ratio, there is increased protein synthesis dedicated to maintaining existing protein structures in RAP compared to CON (data reproduced from (Drake et al., 2013)). (E) Synthetic rates of DNA and three fractions of skeletal muscle in crowded litter mice (CL) and normal litter sized controls (CON). As opposed to CR and RAP models, there is greater protein synthesis in CL compared to CON and no difference in DNA synthesis (data reproduced from (Drake et al., 2014)). (F) Despite qualitative differences in synthesis rates in the CL model compared to CR and RAP, the ratio of protein and DNA synthesis rates still imply maintained proteostasis. Data are means ± SEM, n = 4-12/group. *p<0.05 compared to CON for the same fraction.

Figure 3

(A) Individual protein components of ATP Synthase (· ·), Ribosome (· ·), and urea cycle (◇) are synthesized significantly more slowly (٭, p<0.05) during CR. (B) Comparison of DNA:Protein synthesis rate ratios suggests that synthesis of urea cycle proteins is maintained at higher comparative rate in CR compared to AL controls.