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Changes of Protein Turnover in Aging Caenorhabditis elegans*

  • Ineke Dhondt
    Footnotes
    Affiliations
    Laboratory for Aging Physiology and Molecular Evolution, Biology Department, Ghent University, K.L. Ledeganckstraat 35, 9000 Ghent Belgium
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  • Vladislav A. Petyuk
    Footnotes
    Affiliations
    Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99352, USA
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  • Sophie Bauer
    Affiliations
    Laboratory for Aging Physiology and Molecular Evolution, Biology Department, Ghent University, K.L. Ledeganckstraat 35, 9000 Ghent Belgium
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  • Heather M. Brewer
    Affiliations
    Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99352, USA
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  • Richard D. Smith
    Affiliations
    Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99352, USA
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  • Geert Depuydt
    Affiliations
    Laboratory for Aging Physiology and Molecular Evolution, Biology Department, Ghent University, K.L. Ledeganckstraat 35, 9000 Ghent Belgium

    Laboratory for Functional Genomics and Proteomics, Department of Biology, KU Leuven, Naamsestraat 59, 3000 Leuven, Belgium
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  • Bart P. Braeckman
    Correspondence
    To whom correspondence should be addressed: Laboratory for Aging Physiology and Molecular Evolution, Biology Department, Ghent University, K.L. Ledeganckstraat 35, 9000 Ghent Belgium. Tel.:+32-9-264-8744; Fax:+32-9-264-8793; E-mail:.
    Affiliations
    Laboratory for Aging Physiology and Molecular Evolution, Biology Department, Ghent University, K.L. Ledeganckstraat 35, 9000 Ghent Belgium
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  • Author Footnotes
    * ID acknowledges a PhD grant from the fund for Scientific Research-Flanders, Belgium (FWO11/ASP/031). Portions of this work were supported by NIH P41GM103493 (to R.D.S). The experimental work described herein was performed in the Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by the DOE and located at Pacific Northwest National Laboratory, which is operated by Battelle Memorial Institute for the DOE under Contract DE-AC05-76RL0 1830.
    This article contains supplemental material.
    ‖ These authors contributed equally to this work.
Open AccessPublished:July 05, 2017DOI:https://doi.org/10.1074/mcp.RA117.000049
      Protein turnover rates severely decline in aging organisms, including C. elegans. However, limited information is available on turnover dynamics at the individual protein level during aging. We followed changes in protein turnover at one-day resolution using a multiple-pulse 15N-labeling and accurate mass spectrometry approach. Forty percent of the proteome shows gradual slowdown in turnover with age, whereas only few proteins show increased turnover. Decrease in protein turnover was consistent for only a minority of functionally related protein subsets, including tubulins and vitellogenins, whereas randomly diverging turnover patterns with age were the norm. Our data suggests increased heterogeneity of protein turnover of the translation machinery, whereas protein turnover of ubiquitin-proteasome and antioxidant systems are well-preserved over time. Hence, we presume that maintenance of quality control mechanisms is a protective strategy in aging worms, although the ultimate proteome collapse is inescapable.
      The downturn of protein homeostasis (proteostasis), including the commonly observed slowdown of protein synthesis and degradation (protein turnover), is a major hallmark of aging (
      • Lopez-Otin C.
      • Blasco M.A.
      • Partridge L.
      • Serrano M.
      • Kroemer G.
      The hallmarks of aging.
      ,
      • Ryazanov A.G.
      • Nefsky B.S.
      Protein turnover plays a key role in aging.
      ). Proteome mismanagement along with age-dependent protein aggregation, oxidation and misallocation, likely leads to the overall functional decline in senescent organisms (
      • Rattan S.I.
      Synthesis, modifications, and turnover of proteins during aging.
      ).
      The nematode Caenorhabditis elegans (C. elegans)
      The abbreviations used are: C. elegans, Caenorhabditis elegans; E. coli, Escherichia coli; FDR, false discovery rate; PTM, Pavlidis Template Matching; ROS, reactive oxygen species; SILAC, stable isotope labeling with amino acids in cell culture; SILeNCe, Stable Isotope Labeling by Nitrogen in Caenorhabditis elegans; TCA, tricarboxylic acid cycle; UPS, ubiquitin-proteasome system.
      1The abbreviations used are: C. elegans, Caenorhabditis elegans; E. coli, Escherichia coli; FDR, false discovery rate; PTM, Pavlidis Template Matching; ROS, reactive oxygen species; SILAC, stable isotope labeling with amino acids in cell culture; SILeNCe, Stable Isotope Labeling by Nitrogen in Caenorhabditis elegans; TCA, tricarboxylic acid cycle; UPS, ubiquitin-proteasome system.
      is one of the best-studied model organisms in aging research because of its relatively short lifespan and the extensive molecular toolbox available for this organism. Microarray experiments (
      • Golden T.R.
      • Melov S.
      Gene expression changes associated with aging in C. elegans.
      ,
      • Lund J.
      • Tedesco P.
      • Duke K.
      • Wang J.
      • Kim S.K.
      • Johnson T.E.
      Transcriptional profile of aging in C. elegans.
      ) and proteomic analyses (
      • Liang V.
      • Ullrich M.
      • Lam H.
      • Chew Y.L.
      • Banister S.
      • Song X.
      • Zaw T.
      • Kassiou M.
      • Gotz J.
      • Nicholas H.R.
      Altered proteostasis in aging and heat shock response in C. elegans revealed by analysis of the global and de novo synthesized proteome.
      ,
      • Copes N.
      • Edwards C.
      • Chaput D.
      • Saifee M.
      • Barjuca I.
      • Nelson D.
      • Paraggio A.
      • Saad P.
      • Lipps D.
      • Stevens Jr, S.M.
      • Bradshaw P.C.
      Metabolome and proteome changes with aging in Caenorhabditis elegans.
      ,
      • Dong M.Q.
      • Venable J.D.
      • Au N.
      • Xu T.
      • Park S.K.
      • Cociorva D.
      • Johnson J.R.
      • Dillin A.
      • Yates 3rd., J.R.
      Quantitative mass spectrometry identifies insulin signaling targets in C. elegans.
      ,
      • Walther D.M.
      • Kasturi P.
      • Zheng M.
      • Pinkert S.
      • Vecchi G.
      • Ciryam P.
      • Morimoto R.I.
      • Dobson C.M.
      • Vendruscolo M.
      • Mann M.
      • Hartl F.U.
      Widespread proteome remodeling and aggregation in aging C. elegans.
      ,
      • Narayan V.
      • Ly T.
      • Pourkarimi E.
      • Murillo A.B.
      • Gartner A.
      • Lamond A.I.
      • Kenyon C.
      Deep proteome analysis identifies age-related processes in C. elegans.
      ) have been used to profile the changes in gene expression and protein abundance levels of aging C. elegans, respectively. Overall, these studies report an age-dependent decline in ribosomal proteins and an increase of proteasome complexes and small heat shock proteins (
      • Liang V.
      • Ullrich M.
      • Lam H.
      • Chew Y.L.
      • Banister S.
      • Song X.
      • Zaw T.
      • Kassiou M.
      • Gotz J.
      • Nicholas H.R.
      Altered proteostasis in aging and heat shock response in C. elegans revealed by analysis of the global and de novo synthesized proteome.
      ,
      • Walther D.M.
      • Kasturi P.
      • Zheng M.
      • Pinkert S.
      • Vecchi G.
      • Ciryam P.
      • Morimoto R.I.
      • Dobson C.M.
      • Vendruscolo M.
      • Mann M.
      • Hartl F.U.
      Widespread proteome remodeling and aggregation in aging C. elegans.
      ), corroborating the link between the aging process and proteostasis.
      Recently, we found a drastic decrease of protein turnover rates in aging C. elegans using a classical 35S pulse-chase labeling (
      • Depuydt G.
      • Shanmugam N.
      • Rasulova M.
      • Dhondt I.
      • Braeckman B.P.
      Increased protein stability and decreased protein turnover in the Caenorhabditis elegans Ins/IGF-1 daf-2 mutant.
      ). Liang et al. (2014) profiled changes in the pool of de novo synthesized proteins in old versus young worms, whereas another recent study has used a single pulse-chase SILAC technique to estimate protein appearance at proteomic scale during adult lifespan (
      • Vukoti K.
      • Yu X.
      • Sheng Q.
      • Saha S.
      • Feng Z.
      • Hsu A.L.
      • Miyagi M.
      Monitoring newly synthesized proteins over the adult life span of Caenorhabditis elegans.
      ). A similar approach was used by another group, which reports protein half-lives for developing L4 and day 5 old worms (
      • Visscher M.
      • De Henau S.
      • Wildschut M.H.
      • van Es R.M.
      • Dhondt I.
      • Michels H.
      • Kemmeren P.
      • Nollen E.A.
      • Braeckman B.P.
      • Burgering B.M.
      • Vos H.R.
      • Dansen T.B.
      Proteome-wide changes in protein turnover rates in C. elegans models of longevity and age-related disease.
      ). Although these studies point out shifts in protein synthesis rates in old worms, the gradual change in turnover of individual proteins with increasing chronological age has not been investigated previously. Do aging worms display a heterogeneous decline in proteome turnover or do specific subsets of proteins exhibit distinct trends in protein turnover with age?
      To address this question, we analyzed the change of individual protein half-lives in aging worms using a multiple-pulse metabolic 15N-labeling method. Hereto, subsamples of an aging C. elegans cohort were taken daily and pulse-chased individually. These series of timed samples were subsequently analyzed with high-resolution mass spectrometry to estimate protein half-lives. Our data indicates that turnover of aging proteome slows down partially, although this pattern cannot be generalized for all proteins. Instead, it seems that most protein turnover rates are affected in a heterogeneous way as clear patterns are absent within functionally or spatially related protein groups, which indicates a nonorchestrated collapse of proteome management in aging worms. On the other hand, some distinct protein pools do show specific tendencies in protein turnover with age, such as the proteasomal proteins which maintain their fast turnover rates and the consistent slowdown observed for all tubulins and vitellogenins. In summary, increased heterogeneity in protein turnover occur during normal aging in C. elegans.

      DISCUSSION

      The overall downturn in protein turnover is a common observation in senescent organisms, including nematodes (
      • Ryazanov A.G.
      • Nefsky B.S.
      Protein turnover plays a key role in aging.
      ,
      • Rattan S.I.
      Synthesis, modifications, and turnover of proteins during aging.
      ,
      • Rothstein M.
      • Sharma H.K.
      Altered enzymes in the free-living nematode, Turbatrix aceti, aged in the absence of fluorodeoxyuridine.
      ). Because of increased protein dwell times with age, it is likely that proteins have ample time to undergo oxidation, aggregation and misallocation. It is still unclear whether the age-related decrease in protein turnover is consistent over the entire proteome or whether groups of functionally related proteins show specific patterns. Using a pulse labeling method, we have monitored the change in individual protein turnover in aging C. elegans. Our findings are consistent with a previous study applying a single SILAC-based label-chase approach in aging worms over time (
      • Vukoti K.
      • Yu X.
      • Sheng Q.
      • Saha S.
      • Feng Z.
      • Hsu A.L.
      • Miyagi M.
      Monitoring newly synthesized proteins over the adult life span of Caenorhabditis elegans.
      ) and a second study testing protein half-lives of young developing animals and day 5 old worms (
      • Visscher M.
      • De Henau S.
      • Wildschut M.H.
      • van Es R.M.
      • Dhondt I.
      • Michels H.
      • Kemmeren P.
      • Nollen E.A.
      • Braeckman B.P.
      • Burgering B.M.
      • Vos H.R.
      • Dansen T.B.
      Proteome-wide changes in protein turnover rates in C. elegans models of longevity and age-related disease.
      ). However, our experimental setup with daily pulses and chases provided much more detail on individual protein turnover dynamics over adult age. We report the gradual slowdown of the turnover of a considerable part of the aging proteome, whereas only a minor fraction shows increased turnover with age. One may assume that worms tend to eat less E. coli with increasing age, resulting in a reduced uptake of 15N. Because differences in feeding rate and/or label uptake can confound the turnover estimates, we checked the atomic proportion of 15N in newly synthesized peptides over time. Overall, we observed a slight, but very slow decline of 15N proportion (70% at day 1, 65% at day 7), which indicates no substantial confounding effect of label incorporation in aging worms (supplemental Fig. S2). Disparate protein turnover changes within the majority of functionally and spatially related protein groups indicate the heterogeneous impact of aging on protein turnover. Notably, these trends are consistently observed across the studied aging cohorts, indicating that functionally or spatially related proteins become less stable with advancing age. Therefore, it is likely that the variable change of protein turnover might be one of the important components underlying the age-dependent deterioration. Conceivably, the protein synthesis machinery itself might be a key player driving this dysregulation, as it shows an escalation in variation of turnover with age. This idea is further corroborated in overall decline in ribosomal protein abundances with age, with a prominent imbalance in the relative ribosomal subunit stoichiometry (
      • Walther D.M.
      • Kasturi P.
      • Zheng M.
      • Pinkert S.
      • Vecchi G.
      • Ciryam P.
      • Morimoto R.I.
      • Dobson C.M.
      • Vendruscolo M.
      • Mann M.
      • Hartl F.U.
      Widespread proteome remodeling and aggregation in aging C. elegans.
      ). Thus, dysregulation of protein turnover of the translation machinery in addition with slower turnover of the aggregation-prone folding chaperones (
      • Walther D.M.
      • Kasturi P.
      • Zheng M.
      • Pinkert S.
      • Vecchi G.
      • Ciryam P.
      • Morimoto R.I.
      • Dobson C.M.
      • Vendruscolo M.
      • Mann M.
      • Hartl F.U.
      Widespread proteome remodeling and aggregation in aging C. elegans.
      ), likely promote the ultimate collapse of the aging proteome.
      Nevertheless, consistent trends over time within functional subsets of proteins do exist. Our data shows high maintenance of the degradation apparatus in aged worms, which highly contrasts the hampered turnover of the translation machinery. Moreover, an up-regulation in proteasomal subunits has been observed earlier in aging C. elegans (
      • Walther D.M.
      • Kasturi P.
      • Zheng M.
      • Pinkert S.
      • Vecchi G.
      • Ciryam P.
      • Morimoto R.I.
      • Dobson C.M.
      • Vendruscolo M.
      • Mann M.
      • Hartl F.U.
      Widespread proteome remodeling and aggregation in aging C. elegans.
      ). Notwithstanding their fast turnover rates, these components likely get trapped into aggregates with age. Thus, the sustained protein turnover of proteasomal subunits at advanced age is likely the ongoing endeavor to keep up the degradation machinery to further deal with the proteome imbalance. However, chronic proteotoxicity will eventually exceed the proteasomal capacity (
      • Hipp M.S.
      • Park S.H.
      • Hartl F.U.
      Proteostasis impairment in protein-misfolding and -aggregation diseases.
      ).
      No uniform slowdown in turnover of enzymes involved in energy metabolism could be observed, because only one-fourth shows a significant decline in protein turnover with age. As mitochondria are main producers of ROS, proteins with slow turnover are liable to oxidative modification, resulting in the accumulation of altered enzymes. Although the turnover of only minor fraction of mitochondrial proteins slows down with age, these damage-prone enzymes may be the driving force of the functional decline in metabolic performance with age (
      • Braeckman B.P.
      • Houthoofd K.
      • De Vreese A.
      • Vanfleteren J.R.
      Apparent uncoupling of energy production and consumption in long-lived Clk mutants of Caenorhabditis elegans.
      ,
      • Shoyama T.
      • Ozaki T.
      • Ishii N.
      • Yokota S.
      • Suda H.
      Basic principle of the lifespan in the nematode C. elegans.
      ). Interestingly, fatty-acid β-oxidation proteins, that primarily reside in the intestine (
      • Ashrafi K.
      Obesity and the regulation of fat metabolism.
      ), exhibit increased turnover with age, which is also reflected in the tissue enrichment analysis. These findings may point to a shift from carbohydrate metabolism to fatty-acid β-oxidation in the intestine of old worms. Alternatively, the fatty-acid β-oxidation machinery may need improved turnover compared with enzymes involved in carbohydrate metabolism because of its specific location in the mitochondria and peroxisomes, which are well-known ROS generation sites that may impose excessive oxidative damage to residing proteins (
      • Adachi H.
      • Fujiwara Y.
      • Ishii N.
      Effects of oxygen on protein carbonyl and aging in Caenorhabditis elegans mutants with long (age-1) and short (mev-1) life spans.
      ,
      • Nguyen A.T.
      • Donaldson R.P.
      Metal-catalyzed oxidation induces carbonylation of peroxisomal proteins and loss of enzymatic activities.
      ).
      Tubulins and vitellogenins show consistent slowdown of turnover rates with age. Because the latter groups have opposite aggregation propensities, protein aggregation cannot be the sole underlying mechanism responsible for the deceleration of protein turnover with age and vice versa. Microtubules are responsible for a variety of functions, including cellular transport (
      • Cooper G.M.
      ). Aged cells frequently display the accumulation of cell organelles probably because of insufficient organelle transport along disrupted microtubules (
      • Schatten H.
      • Chakrabarti A.
      • Hedrick J.
      Centrosome and microtubule instability in aging Drosophila cells.
      ). Hence, we presume that attenuation of tubulin turnover may underlie the changing microtubule organization and dynamics during aging. On the other hand, protein aggregation might be a driving force in slowing down the turnover of vitellogenins, as these macromolecules, irrelevant to post-reproductive worms, accumulate throughout the body cavity in old worms (
      • Herndon L.A.
      • Schmeissner P.J.
      • Dudaronek J.M.
      • Brown P.A.
      • Listner K.M.
      • Sakano Y.
      • Paupard M.C.
      • Hall D.H.
      • Driscoll M.
      Stochastic and genetic factors influence tissue-specific decline in ageing C. elegans.
      ). Vitellogenins, trapped into accumulating protein aggregates over time, likely show increased protein half-lives with age as they become less susceptible for protein degradation in aging worms.
      Aging nematodes are characterized by a progressive locomotory decline (
      • Bolanowski M.A.
      • Russell R.L.
      • Jacobson L.A.
      Quantitative measures of aging in the nematode Caenorhabditis elegans. I. Population and longitudinal studies of two behavioral parameters.
      ). Muscle-specific proteins are very stable proteins, showing barely any turnover during a worm's lifespan (this study and previous findings (
      • Dhondt I.
      • Petyuk V.A.
      • Cai H.
      • Vandemeulebroucke L.
      • Vierstraete A.
      • Smith R.D.
      • Depuydt G.
      • Braeckman B.P.
      FOXO/DAF-16 activation slows down turnover of the majority of proteins in C. elegans.
      )). Therefore, the lack of protein turnover may be associated with the age-related structural loss of sarcomere integrity (
      • Herndon L.A.
      • Schmeissner P.J.
      • Dudaronek J.M.
      • Brown P.A.
      • Listner K.M.
      • Sakano Y.
      • Paupard M.C.
      • Hall D.H.
      • Driscoll M.
      Stochastic and genetic factors influence tissue-specific decline in ageing C. elegans.
      ). Interestingly, in pharyngeal muscle cells, proteins with increased and fast turnover rates are relatively over-represented, compared with other muscle tissues. One possibility is that the pharyngeal muscle cells are, besides general deterioration, more susceptible to microbial attacks than body wall muscles (
      • Chow D.K.
      • Glenn C.F.
      • Johnston J.L.
      • Goldberg I.G.
      • Wolkow C.A.
      Sarcopenia in the Caenorhabditis elegans pharynx correlates with muscle contraction rate over lifespan.
      ). Hence, proteins with higher turnover rates might be more flexible to cope with this additional stress.
      In conclusion, our data suggests that senescent C. elegans is characterized by a variety of changes in protein turnover rates. Diverging dwell times of proteins involved in translation, consistently observed over three independent biological replicates, are likely responsible for a collapse of the translation machinery over time. Intriguingly, we found that aging worms seem to preserve their (protein) quality control mechanisms, especially the UPS and antioxidant machinery, possibly to cope with the increasing proteotoxic and oxidative stress with age. However, this maintenance fights a losing battle, eventually resulting in the ultimate collapse of the proteome.

      Data Availability

      The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (
      • Vizcaino J.A.
      • Deutsch E.W.
      • Wang R.
      • Csordas A.
      • Reisinger F.
      • Rios D.
      • Dianes J.A.
      • Sun Z.
      • Farrah T.
      • Bandeira N.
      • Binz P.A.
      • Xenarios I.
      • Eisenacher M.
      • Mayer G.
      • Gatto L.
      • Campos A.
      • Chalkley R.J.
      • Kraus H.J.
      • Albar J.P.
      • Martinez-Bartolome S.
      • Apweiler R.
      • Omenn G.S.
      • Martens L.
      • Jones A.R.
      • Hermjakob H.
      ProteomeXchange provides globally coordinated proteomics data submission and dissemination.
      ) via the PRIDE partner repository with the data set identifier PXD002901 and 10.6019/PXD002901.

      Acknowledgments

      We thank the Gems laboratory for providing the strain GA153. We are grateful to Caroline Vlaeminck and Renata Coopman for technical support with the SILeNCe experiments. We thank Andy Vierstraete for his technical assistance in data-processing. Preliminary experiments were performed by Dr. Filip Mathijssens, who was a postdoctoral member of the Braeckman lab.

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