Cross-species Comparison of Proteome Turnover Kinetics

The constitutive process of protein turnover plays a key role in maintaining cellular homeostasis. Recent technological advances in mass spectrometry have enabled the measurement of protein turnover kinetics across the proteome. However, it is not known if turnover kinetics of individual proteins are highly conserved or if they have evolved to meet the physiological demands of individual species. Here, we conducted systematic analyses of proteome turnover kinetics in primary dermal fibroblasts isolated from eight different rodent species. Our results highlighted two trends in the variability of proteome turnover kinetics across species. First, we observed a decrease in cross-species correlation of protein degradation rates as a function of evolutionary distance. Second, we observed a negative correlation between global protein turnover rates and maximum lifespan of the species. We propose that by reducing the energetic demands of continuous protein turnover, long-lived species may have evolved to lessen the generation of reactive oxygen species and the corresponding oxidative damage over their extended lifespans. The constitutive process of protein turnover plays a key role in maintaining cellular homeostasis. Recent technological advances in mass spectrometry have enabled the measurement of protein turnover kinetics across the proteome. However, it is not known if turnover kinetics of individual proteins are highly conserved or if they have evolved to meet the physiological demands of individual species. Here, we conducted systematic analyses of proteome turnover kinetics in primary dermal fibroblasts isolated from eight different rodent species. Our results highlighted two trends in the variability of proteome turnover kinetics across species. First, we observed a decrease in cross-species correlation of protein degradation rates as a function of evolutionary distance. Second, we observed a negative correlation between global protein turnover rates and maximum lifespan of the species. We propose that by reducing the energetic demands of continuous protein turnover, long-lived species may have evolved to lessen the generation of reactive oxygen species and the corresponding oxidative damage over their extended lifespans. Within a cell, proteins are in a state of flux and are continually degraded and re-synthesized (1.Goldberg A.L. St John A.C. Intracellular protein degradation in mammalian and bacterial cells: Part 2.Ann. Rev. Biochem. 1976; 45: 747-803Crossref PubMed Scopus (808) Google Scholar). The process of protein turnover plays a critical quality control function in cells. Over time, proteins tend to become damaged by a number of stochastic mechanisms including oxidation, nitrosylation, and aggregation (2.Stadtman E.R. Levine R.L. Free radical-mediated oxidation of free amino acids and amino acid residues in proteins.Amino Acids. 2003; 25: 207-218Crossref PubMed Scopus (1390) Google Scholar). The process of turnover ensures that damaged proteins are perpetually replaced by a nascent pool of undamaged, functional proteins. Additionally, protein turnover plays an important role in the regulation of protein expression levels. The constant turnover of proteins allows their steady-state levels to adjust in response to changes in synthesis rates (3.Davies K.J. Protein damage and degradation by oxygen radicals. I. general aspects.J. Biol. Chem. 1987; 262: 9895-9901Abstract Full Text PDF PubMed Google Scholar, 4.Ryazanov A.G. Nefsky B.S. Protein turnover plays a key role in aging.Mech. Ageing Development. 2002; 123: 207-213Crossref PubMed Scopus (79) Google Scholar). Recent advances in quantitative proteomics and mass spectrometry have enabled the measurement of protein turnover kinetics on proteome-wide scales (5.Pratt J.M. Petty J. Riba-Garcia I. Robertson D.H. Gaskell S.J. Oliver S.G. Beynon R.J. Dynamics of protein turnover, a missing dimension in proteomics.Mol. Cell. Proteomics. 2002; 1: 579-591Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar, 6.Price J.C. Guan S. Burlingame A. Prusiner S.B. Ghaemmaghami S. Analysis of proteome dynamics in the mouse brain.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 14508-14513Crossref PubMed Scopus (247) Google Scholar, 7.Claydon A.J. Beynon R. Proteome dynamics: revisiting turnover with a global perspective.Mol. Cell. Proteomics. 2012; 11: 1551-1565Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 8.Cambridge S.B. Gnad F. Nguyen C. Bermejo J.L. Kruger M. Mann M. Systems-wide proteomic analysis in mammalian cells reveals conserved, functional protein turnover.J. Proteome Res. 2011; 10: 5275-5284Crossref PubMed Scopus (177) Google Scholar, 9.Schwanhausser B. Busse D. Li N. Dittmar G. Schuchhardt J. Wolf J. Chen W. Selbach M. Global quantification of mammalian gene expression control.Nature. 2011; 473: 337-342Crossref PubMed Scopus (4058) Google Scholar, 10.Toyama B.H. Savas J.N. Park S.K. Harris M.S. Ingolia N.T. Yates 3rd, J.R. Hetzer M.W. Identification of long-lived proteins reveals exceptional stability of essential cellular structures.Cell. 2013; 154: 971-982Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar). These studies have shown that turnover rates are highly variable within the proteome, with protein half-lives ranging from minutes to years. Several factors can influence the turnover rates of proteins in vivo. In some proteins, identities of N-terminal residues appear to have a strong influence on half-lives, a phenomenon referred to as the “N-end rule” (11.Bachmair A. Finley D. Varshavsky A. In vivo half-life of a protein is a function of its amino-terminal residue.Science. 1986; 234: 179-186Crossref PubMed Scopus (1372) Google Scholar). The presence of longer sequence domains, termed “degrons,” have also been shown to affect protein turnover. For example, sequences rich in proline, glutamic acid, serine, and threonine have been shown to act as robust degradative markers (12.Rogers S. Wells R. Rechsteiner M. Amino acid sequences common to rapidly degraded proteins: the PEST hypothesis.Science. 1986; 234: 364Crossref PubMed Scopus (1957) Google Scholar, 13.Reverte C.G. Ahearn M.D. Hake L.E. CPEB degradation during Xenopus oocyte maturation requires a PEST domain and the 26S proteasome.Developmental Biol. 2001; 231: 447-458Crossref PubMed Scopus (76) Google Scholar). In addition to sequence determinants, physical properties of proteins such as isoelectric points, surface areas, thermodynamic stabilities and molecular weights can influence half-lives (14.Dice J.F. Hess E.J. Goldberg A.L. Studies on the relationship between the degradative rates of proteins in vivo and their isoelectric points.Biochem. J. 1979; 178: 305-312Crossref PubMed Scopus (30) Google Scholar, 15.Dice J.F. Goldberg A.L. Relationship between in vivo degradative rates and isoelectric points of proteins.Proc. Natl. Acad. Sci. U.S.A. 1975; 72: 3893-3897Crossref PubMed Scopus (129) Google Scholar, 16.Miller S. Lesk A.M. Janin J. Chothia C. The accessible surface area and stability of oligomeric proteins.Nature. 1987; 328: 834-836Crossref PubMed Scopus (307) Google Scholar). However, none of these determinants are universally applicable to the entirety of the proteome and it is currently not possible to predict the half-life of a protein based solely on its sequence and structure. The turnover rate of a protein is not only dependent on its sequence and structure, but also on the relative activity and selectivity of proteolytic pathways within the cell. Hence, proteins of identical sequence can have vastly different half-lives within different cell types, tissues and environmental conditions (6.Price J.C. Guan S. Burlingame A. Prusiner S.B. Ghaemmaghami S. Analysis of proteome dynamics in the mouse brain.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 14508-14513Crossref PubMed Scopus (247) Google Scholar, 7.Claydon A.J. Beynon R. Proteome dynamics: revisiting turnover with a global perspective.Mol. Cell. Proteomics. 2012; 11: 1551-1565Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Within a cell, proteins can be degraded by several proteolytic pathways and proteases. In eukaryotes, the two major degradation pathways with broad selectivity are autophagy and the ubiquitin proteasome system (UPS) 1The abbreviations used are: UPS, ubiquitin proteasome system; AGC, automatic gain control; CID, collision induced dissociation; SILAC, stable isotopic labeling in cell culture; CV, coefficient of variation; LC-MS/MS, liquid chromatography tandem mass spectrometry; PRIDE, proteomics identifications; rs, Spearman's rank correlation coefficient; r, Pearson's correlation coefficient; GO, gene ontology; mRNA, messenger ribonucleic acid; ROS, reactive oxygen species; DTT, dithiothreitol; FBS, fetal bovine serum; EMEM, Eagle's minimum essential medium; BCA, bicinchoninic assay; PBS, phosphate buffer saline; MEM, minimum essential medium; TCEP, Tris(2-carboxyethyl)phosphine; IAA, iodoacetamide; RT, room temperature; TFA, trifluoric acid; mTOR, mammalian target of rapamycin; IGF-1, insulin growth factor-1; ATP, adenosine triphosphate; DNA, deoxyribonucleic acid; REVIGO, reduce and visualize gene ontology; PSM, peptide spectral match. 1The abbreviations used are: UPS, ubiquitin proteasome system; AGC, automatic gain control; CID, collision induced dissociation; SILAC, stable isotopic labeling in cell culture; CV, coefficient of variation; LC-MS/MS, liquid chromatography tandem mass spectrometry; PRIDE, proteomics identifications; rs, Spearman's rank correlation coefficient; r, Pearson's correlation coefficient; GO, gene ontology; mRNA, messenger ribonucleic acid; ROS, reactive oxygen species; DTT, dithiothreitol; FBS, fetal bovine serum; EMEM, Eagle's minimum essential medium; BCA, bicinchoninic assay; PBS, phosphate buffer saline; MEM, minimum essential medium; TCEP, Tris(2-carboxyethyl)phosphine; IAA, iodoacetamide; RT, room temperature; TFA, trifluoric acid; mTOR, mammalian target of rapamycin; IGF-1, insulin growth factor-1; ATP, adenosine triphosphate; DNA, deoxyribonucleic acid; REVIGO, reduce and visualize gene ontology; PSM, peptide spectral match. (17.Klionsky D.J. Emr S.D. Autophagy as a Regulated Pathway of Cellular Degradation.Science. 2000; 290: 1717Crossref PubMed Scopus (2969) Google Scholar). These two pathways are believed to have distinct functions in maintaining protein homeostasis. Autophagy has been shown to be in the degradation of damaged protein long-lived proteins, of during of and protein In the is believed to be the degradation of and proteins and of damaged proteins D. D.J. The of Res. PubMed Scopus Google Scholar, D. I. A. The role of protein mechanisms in and PubMed Scopus Google Scholar). the primary mechanisms of the relative of autophagy and can a major role in the half-lives of proteins in vivo. are the turnover kinetics of individual proteins conserved across a proteomic studies have in the results have been For example, a of and observed in protein turnover rates between the two species R. N. F. Global proteome turnover analyses of the S. and S. Full Text Full Text PDF PubMed Scopus Google Scholar). an analysis of two cell and from and mouse tissues a correlation in protein turnover rates S.B. Gnad F. Nguyen C. Bermejo J.L. Kruger M. Mann M. Systems-wide proteomic analysis in mammalian cells reveals conserved, functional protein turnover.J. Proteome Res. 2011; 10: 5275-5284Crossref PubMed Scopus (177) Google Scholar). In a of in vivo turnover rates in two mouse and in two also correlation A.J. D. J.L. Beynon R.J. Proteome dynamics: in the kinetics of in Cell. Proteomics. Full Text Full Text PDF PubMed Scopus Google Scholar). However, to a systematic cross-species of protein turnover rates a of has not been conducted in a Here, we have used isotopic labeling and quantitative proteomics to protein turnover kinetics in primary dermal fibroblasts isolated from eight different rodent species. 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Ghaemmaghami S. of degradation dynamics in response to growth Natl. Acad. Sci. U.S.A. PubMed Scopus Google and is based on the Protein synthesis is a process with to protein degradation a constant rate that is the protein protein degradation can be as a process with to protein protein of cell not during the and the system is on these we can the rate of proteins and of proteins: is the rate constant protein is the rate constant protein degradation and is the rate constant cell We the and the protein labeling are with to steady-state protein the observed labeling is conducted in the rate of cell is Hence, and a rate the relationship between and half-life of a protein is as a global cross-species of protein turnover fibroblasts isolated from eight different rodent and A. 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Global quantification of mammalian gene expression control.Nature. 2011; 473: 337-342Crossref PubMed Scopus (4058) Google Scholar). studies of in protein half-lives species S.B. Gnad F. Nguyen C. Bermejo J.L. Kruger M. Mann M. Systems-wide proteomic analysis in mammalian cells reveals conserved, functional protein turnover.J. Proteome Res. 2011; 10: 5275-5284Crossref PubMed Scopus (177) Google Scholar, R. N. F. Global proteome turnover analyses of the S. and S. Full Text Full Text PDF PubMed Scopus Google Scholar, A.J. D. J.L. Beynon R.J. Proteome dynamics: in the kinetics of in Cell. Proteomics. Full Text Full Text PDF PubMed Scopus Google Scholar). 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