Superior proteome stability in the longest lived animal

Abstract

Bivalve mollusks have several unique traits, including some species with exceptionally long lives, others with very short lives, and the ability to determine the age of any individual from growth rings in the shell. Exceptionally long-lived species are seldom studied yet have the potential to be particularly informative with respect to senescence-resistance mechanisms. To this end, we employed a range of marine bivalve mollusk species, with lifespans ranging from under a decade to over 500 years, in a comparative study to investigate the hypothesis that long life requires superior proteome stability. This experimental system provides a unique opportunity to study closely related organisms with vastly disparate longevities, including the longest lived animal, Arctica islandica.Specifically, we investigated relative ability to protect protein structure and function, both basally and under various stressors in our range of species. We found a consistent relationship between species longevity, resistance to protein unfolding, and maintenance of endogenous enzyme (creatine kinase) activity. Remarkably, our longest-lived species, Arctica islandica (maximum longevity >500 years), had no increase in global proteome unfolding in response to several stressors. Additionally, the global proteome of shorter-lived species exhibited less resistance to temperature-induced protein aggregation than longer-lived species. A reporter assay, in which the same protein's aggregation properties was assessed in lysates from each study species, suggests that some endogenous feature in the cells of long-lived species, perhaps small molecular chaperones, was at least partially responsible for their enhanced proteome stability. This study reinforces the relationship between proteostasis and longevity through assessment of unfolding, function, and aggregation in species ranging in longevity from less than a decade to more than five centuries.

Fig. 1

Phylogeny and maximum longevities of the species. Arctica = Arctica islandica, longevity documented in (Butler et al. 2013); Callista = Callista chione, longevity documented in (Powell and Cummins 1985); Mercenaria = Mercenaria mercenaria, longevity documented in (Ungvari et al. 2011); Ruditapes = Ruditapes philippinarum, longevity documented in (Ponurovskii 2008). Phylogeny from (Giribet ; Taylor et al. 2007). In parentheses, maximum reported longevity in years

Fig. 2

a Protein unfolding: BisANS incorporation indicative of global proteome surface hydrophobicity after 1 M urea or 20 μM TBHP stress as compared to basal levels. In both results, there are highly statistically significant differences among species. Bars not sharing a common letter are significantly different (p < .05) as assessed by Tukey's HSD after one-way analysis of variance. Species effects under the urea stress: F(3,8) = 8.73, p = .007 and TBHP: F(3,8) = 14.34, p = .001. b Enzyme activity: reduction in endogenous creatine kinase activity in response to stress with 1 M urea. Although the overall ANOVA is only marginally significant, the order of activity loss is, as expected, inversely related to species longevity. F(3,15) = 2.81, p = .075. Rud = Ruditapes, Cal = Callista, Mer = Mercenaria, Arc = Arctica. Numbers in parentheses are maximum species longevity in years

Fig. 3

a Serial temperature-induced aggregation: we partitioned pure soluble cytosolic lysates into soluble and insoluble fractions after temperature stress. The surviving soluble fraction is reused for each higher temperature, with insoluble temperature-induced aggregates removed each time by 100,000 g ultracentrifugation for 1 h. Note that fewer aggregates are seen in the longest-lived bivalve at each temperature, and most aggregates are seen in the mouse at each temperature. These differences were assessed by two-way analysis of variance, finding a main effect of species, F(3,24) = 7.04, p = .001, and of stress, F(2,24) = 20.40, p < .001. There was no significant interaction between species and stress levels, F(6,24) = 2.00, p = .105. b Denatured aggregation: we partitioned pure soluble cytosolic lysates into soluble and insoluble fractions after boiling for 15 min and 14,000 g centrifugation for 5 min. The results were assessed by one-way analysis of variance, F(4,15) = 353.99, p < .0001. Individual differences between species were assessed post hoc by Tukey's HSD, and bars not sharing a common letter are statistically different p < .05

Fig. 4

FITC-tagged BSA aggregation: using the same reporter protein in the lysates of our various species allowed us to investigate the potential role of molecular chaperones and co-chaperones in the protein stability properties of our species. Data represents BSA aggregates in the pellet after 2-h temperature stress at 42 or 52 °C and 1-h ultracentrifugation at 100,000 g. The two shorter-lived species clustered together and aggregated more BSA than the two longer-lived species, which also clustered together. Results were assessed by two-way analysis of variance, indicating a significant main effect of species, F(3,15) = 3.90, p = .030, but no difference between 42 and 52 °C, F(1,15) = .788, p = .389. There was also no interaction effect, F(3,15) = .068, p = .976