Interspecies differences in proteome turnover kinetics are correlated with lifespans and energetic demands

Kyle Swovick, Denis Firsanov, Kevin A. Welle, Jennifer R. Hryhorenko, John P. Wise, Craig George, Todd L. Sformo, Andrei Seluanov, Vera Gorbunova, Sina Ghaemmaghami

Posted on: 18 May 2020

Preprint posted on 27 April 2020

Article now published in Molecular & Cellular Proteomics at http://dx.doi.org/10.1074/mcp.RA120.002301

Live long and proteostasis: slower protein turnover correlates with a longer lifespan and reduced energy consumption.

Selected by Teresa Rayon

Categories: biochemistry, cell biology, evolutionary biology

Protein homeostasis (proteostasis) is essential for an organism to maintain its normal cellular function, as cells need to continually degrade and replace damaged and old proteins. Proteostasis ensures timely disposal of misfolded proteins and the correct balance between protein production and degradation (i.e protein turnover) As we age, the capacity of cells to maintain proteostasis declines progressively. In addition, several models for lifespan extension in mammalian systems have shown reduced protein turnover rates (Basisty et al., 2018). But is enhanced protein quality and reduced protein turnover the underlying mechanism behind longevity? In this preprint, Swovick et al. measure protein turnover kinetics across twelve different mammals with lifespans ranging from 4 years in mouse to 200 years in the bowhead whale to answer this question.

In a tour de force, the authors measure degradation rates of single proteins genome-wide by using quantitative proteomics (pulsed Stable Isotope Labeling in Cell Culture -pSILAC-) of quiescent fibroblasts. They find that longer-lived organisms generally have slower global protein turnover rates. This interesting correlation drives the authors to ask why long-lived organisms have slower rates of protein turnover. They perform an in-depth comparison between the mouse and the evolutionarily related and similar sized naked mole-rat, which has cancer resistance and a long lifespan and therefore is broadly used as a model in the aging field. Through a number of experiments to measure energy consumption and response to proteotoxic stress, they find that increased protein stability reduces ATP demands and reduces the production of reactive oxygen species (ROS) over the course of a long lifespan.

**Figure** 1. Cross-species comparisons of lifespan, gestation period and degradation rates. (A) Phylogenetic tree and maximal lifespans and gestational period of the species analysed in this study. (B) Experimental design to measure protein stability by pSILAC. Blue and red colors indicate unlabeled and isotopically labeled spectra/cells, respectively. (C) Correlation between species’ median degradation rates (kdeg) and maximal lifespans. r value indicates Pearson correlation coefficient. Adapted from Figures 1 and 2 of this preprint.

Why I chose the paper:

I have been wondering for a while why developmental pace differs across species. The gestational period is highly species-specific, lasting 420 days in bowhead whales, 280 days in humans, 70 days in naked mole rats and 20 days in mouse, and correlates with the animals’ lifespan. In our own recent work, we find that interspecies differences in protein stability correlate with developmental pace (Rayon et al., 2019). Interestingly, the preprint highlighted here studies the relationship between lifespan -total length of time from birth to death- and protein turnover, and it describes a correlation between protein stability and lifespan in the fibroblasts of 12 different mammalian species. Only recently, advances in quantitative proteomics have enabled the genome-wide measurement of protein turnover rates for individual proteins. I find the use of this state-of-the-art technique to compare between quiescent fibroblasts from multiple species really elegant. This is an impressively large cross-species comparison that includes amazing mammalian species such as the bowhead whale and the naked mole-rat. Moreover, their findings go beyond a correlation of protein stability and lifespan to show that highly abundant proteins in long-lived organisms are significantly more stable. Since protein turnover is highly expensive energetically (Ramsey et al., 2000), this underscores the fact that the cost of proteostasis might be a target for longevity. In agreement with this, they also demonstrate slower rates of ATP production and reduced levels of respiration and glycolytic proteins in long-lived naked mole-rat fibroblasts. Finally, their comparison between mouse and naked mole-rat allows them to show a lower accumulation of ROS in the latter, suggesting that longevity might be related to a reduction in energy demand and ROS production.

Aging is an intrinsic decline in physiological function, that is thought to be due to direct damage, accumulation of cellular waste, errors, and imperfect repairs. Thus, it could be argued that aging starts during development, and it might suggest that proteostasis is a common cellular clock that tracks the course of time during development and adulthood.

Why I think this work moves the field forward

The quest for the elixir of youth is linked to the idea that most of us want to live long, or age slowly, if at all. Faster protein turnover is generally associated with youth, and the progressive loss of proteostasis is a hallmark of aging. Constitutive protein turnover accounts for as much as 25% of the total energy expenditure in the body (Ramsey et al., 2000), and the findings by Swovick et al. support that slower protein turnover rates reduce ATP demand and lower the production of ROS over the course of a long lifespan. Altogether, this work establishes how the energetic costs in protein turnover might determine longevity in mammals. In the future, understanding how long-lived animals ease aging by studying the relationship between metabolism and proteostasis may shed some light towards finding the elixir of youth to extend the lifespan in humans.

Questions to authors

Fibroblasts senesce and lose amplification potential over passages in culture, and it is thought that the age of the donors’ fibroblasts determines the efficiency of reprogramming. Did the authors take into account the age of the donor species for the isolated fibroblasts and the number of passages of the fibroblasts? I wonder if there would be identifiable differences between old-donor versus young-donor fibroblasts across species.
Unlike most mammals, naked mole rats do not regulate their body temperature. Do the authors think that the energetic cost and the mechanisms to lower ROS production to maintain a slower protein turnover in other long-lived species would be conserved? Do they think temperature can have an effect on proteostasis?
In this work the authors measure degradation rates of long-lived proteins. Do the authors think that they would find differences if they measured the stability of short-lived proteins?
The authors test the ability of naked mole rat and mouse fibroblasts to tolerate proteotoxic stress. Have the authors considered looking separately at the unfolded protein response or the heat shock response?
The authors measured protein turnover in quiescent cells, what do the authors think would be the effect of cell proliferation rate?

References

Basisty, N., Meyer, J. G. and Schilling, B. (2018). Protein Turnover in Aging and Longevity. PROTEOMICS 18, 1700108.

Ramsey, J. J., Harper, M. E. and Weindruch, R. (2000). Restriction of energy intake, energy expenditure, and aging. Free Radical Biology and Medicine 29, 946–968.

Rayon, T., Stamataki, D., Perez-carrasco, R., Garcia-perez, L., Barrington, C., Melchionda, M., Exelby, K., Tybulewicz, V., Fisher, E. M. C. and Briscoe, J. (2019). Species-specific developmental timing is associated with global differences in protein stability in mouse and human. bioRxiv.

Tags: aging, proteomics, proteostasis

doi: https://doi.org/10.1242/prelights.20619

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Author's response

Kyle Swovick shared

I really appreciate the great questions you asked and I’ve included the answers to each one below.

Q1. This is a great point and is perhaps the biggest caveat in our study. It has been documented that as mammals age, protein turnover rates generally decrease. Therefore, the age of individual organisms at the time of fibroblast isolation may have an effect on measured turnover rates. To alleviate this, cells isolated from species raised in house (mouse, naked mole rat, blind mole rat) were harvested at times so that they were developmentally age matched, and the human fibroblast was from a young male that was also age matched. Unfortunately, the other 8 species were all harvested from individuals from the wild, so we were unable to determine their age. Our assumption is that interspecies variabilities in turnover rates are significantly greater than any age-dependent variabilities in turnover rates within a species. I would love to conduct an experiment where we measure protein turnover rates for all these species at different ages. However, this may be difficult for species like whales!

Q2. It has been shown that temperature can have dramatic effects on degradation rates of individual proteins within cells. The extent to which this affects entire proteomes is yet unclear. But it would be very interesting to determine if there are differences between species regarding how their rates of protein degradation change in response to temperature shifts. It is important to note that in this study turnover rates were analyzed at the same temperature (37°) for all species. But at any given temperature, it is possible that different organisms may differentially activate protein degradation pathways depending on their inherent temperature regulatory mechanisms.

Q3. Actually, in this study we got fairly deep coverage in our proteomic data, and were able to measure degradation rates of some relatively short-lived proteins as well as long-lived proteins. But it is true that short-lived proteins generally have lower abundances in the cell and are underrepresented in proteomic datasets. Rapid protein turnover is energetically expensive, so I would venture to guess that for many transient proteins with very short half-lives, their rapid turnover is functionally critical. For example, many signaling proteins have short half-lives because they need to be rapidly regulated and their steady-state levels need to be quickly adjusted in response to changes in their synthesis rates. For this subset of proteins, slowing down their turnover rates may not be viable in long-lived organisms.

Q4. In this study, we used the amino acid analogue AZC to induce protein misfolding and proteotoxic stress. AZC results in a broad proteotoxic stress response in cells and has been shown to induce ER stress, UPR as well as components of the heat shock response. Analyzing each of these responses individually in long-lived organisms is definitely important and something we plan to do in the future.

Q5. We chose to conduct our analyses in quiescent non-dividing cells mostly due to an important practical consideration. When we conduct isotopic labelling experiments in dividing cells, the observed rate of fractional labelling for a protein is the summation of its rate of degradation and its rate of cellular dilution due to cell division. In cases where the rate of protein degradation is significantly slower than the rate of cell division, the measured rate of fractional labelling is dominated by the rate of cell division. Therefore, it is very difficult to accurately measure degradation rates of long-lived proteins in rapidly dividing cells using this methodology. Measuring degradation rates in quiescent cells alleviates this complication. Interestingly, all the different cells we used from different species have varying proliferation rates. Analyzing these cells in a quiescent state took this variability out of the equation. That being said, we and others have shown that protein degradation pathways such as autophagy are upregulated in quiescent fibroblasts. Because of this, the interspecies trends in protein degradation kinetics we observe in quiescent cells may be different, and perhaps less prominent, in dividing cells.

Thank you for showing interest in our preprint!

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Interspecies differences in proteome turnover kinetics are correlated with lifespans and energetic demands

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