Protein stability and resistance to oxidative stress are determinants of longevity in the longest-living rodent, the naked mole-rat

Abstract

The widely accepted oxidative stress theory of aging postulates that aging results from accumulation of oxidative damage. Surprisingly, data from the longest-living rodent known, naked mole-rats [MRs; mass 35 g; maximum lifespan (MLSP) > 28.3 years], when compared with mice (MLSP 3.5 years) exhibit higher levels of lipid peroxidation, protein carbonylation, and DNA oxidative damage even at a young age. We hypothesize that age-related changes in protein structural stability, oxidation, and degradation are abrogated over the lifespan of the MR. We performed a comprehensive study of oxidation states of protein cysteines [both reversible (sulfenic, disulfide) and indirectly irreversible (sulfinic/sulfonic acids)] in liver from young and old C57BL/6 mice (6 and 28 months) and MRs (2 and >24 years). Furthermore, we compared interspecific differences in urea-induced protein unfolding and ubiquitination and proteasomal activity. Compared with data from young mice, young MRs have 1.6 times as much free protein thiol groups and similar amounts of reversible oxidative damage to cysteine. In addition, they show less urea-induced protein unfolding, less protein ubiquitination, and higher proteasome activity. Mice show a significant age-related increase in cysteine oxidation and higher levels of ubiquitination. In contrast, none of these parameters were significantly altered over 2 decades in MRs. Clearly MRs have markedly attenuated age-related accrual of oxidation damage to thiol groups and age-associated up-regulation of homeostatic proteolytic activity. These pivotal mechanistic interspecies differences may contribute to the divergent aging profiles and strongly implicate maintenance of protein stability and integrity in successful aging.

Fig. 1.

Age-related changes in cysteine oxidation in cytosolic liver homogenates from young (open bars) and old (solid bars) mice and MRs. Data are the means of 8 (mice) and 10 (MRs) ± SEM. The * denotes values that are significantly (P ≤ 0.05) different from young mice as analyzed by nonparametric ANOVA. (A) Disulfide bond content increases 1.3-fold in mice with age, but is unchanged (#, P = 0.07) with age in MRs. (B) Sulfenic acid levels increase 1.5-fold with age in mice (P = 0.04), but are unchanged with age in MRs. (C) Total cysteine content in MRs is 1.6-fold higher than in mice (P = 0.05). Whereas total cysteine declines significantly with age in mice, no age-related changes are evident in MRs. (D) Irreversible oxidation increases 3.4-fold more with age in mice than in MRs. Levels of irreversible cysteine oxidation are obtained from the differences (Δ) between the values of total cysteine in young and old mice and MRs.

Fig. 2.

Proteins from both young and old MRs are extremely resistant to protein unfolding, whereas those from mice are markedly more susceptible. Protein unfolding is measured by incorporation of the BisANS fluorescence probe when hydrophobic pockets are exposed and is expressed as fluorescent units/mg protein as a percentage of control (100%). Data are mean ± SEM (n = 6). The # (P = 0.0038) and * (P = 0.048) denote those values that are significantly different from untreated samples when analyzed by nonparametric ANOVA. (A) Basal protein unfolding is unchanged with age (P = 0.094) in mice and MRs. (B) Young mice have markedly higher levels of protein unfolding after urea treatment than do young MRs with maximal BisANS incorporation at 1 M urea. (C) BisANS incorporation in response to1 M urea is abrogated with age, although old mice show less resistance to unfolding than do old MRs. (D) GAPDH activity declines less with urea treatment in MRs than it does in mice regardless of age, indicating that MRs are better able to maintain protein structure and function than mice. A representative Western blot of GAPDH expression is shown. Letters a–e represent significant differences among comparative datasets (a = 0.03; b = 0.05; c = 0.05; d = 0.05; e = 0.05), while f (representing age-related differences in untreated GAPDH activity in MRs) is not significant (P = 0.24).

Fig. 3.

Ubiquitinated proteins increase with age in mice, but are similar in samples from 2-year-old and 26-year-old MRs. (A) A representative Western blot of ubiquitinated proteins is shown. (B) Levels of ubiquitinated protein as measured by Western blot analysis in cytosolic protein homogenates from the livers of young (open bars) and old (solid bars) mice and MRs are shown. Data (mean ± SEM; n = 3) were analyzed by nonparametric ANOVA. The * denotes values that are significantly (P ≤ 0.05) different from young mice. (C) The levels of proteasome (20S) are similar in both mouse and MRs. The total amount of 20S proteasome subunit is measured by Western blot analysis. (D) Proteasome activity is greater in old MRs than in old mice (*, P = 0.052). Chymotrypsin-like proteasome activity is measured in cytosolic protein homogenates from livers of young (open bars) and old (solid bars) mice and MRs by using the 20S proteasome fluormetric (AMC) assay kit. Data (mean ± SEM; n = 3) were analyzed by nonparametric ANOVA.