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.

Cells continually degrade and replace damaged proteins. However, the high energetic demand of protein turnover generates reactive oxygen species that compromise the long-term health of the proteome. Thus, the relationship between aging, protein turnover, and energetic demand remains unclear. Here, we used a proteomic approach to measure rates of protein turnover within primary fibroblasts isolated from a number of species with diverse life spans including the longest-lived mammal, the bowhead whale. We show that organismal life span is negatively correlated with turnover rates of highly abundant proteins. In comparison with mice, cells from long-lived naked mole rats have slower rates of protein turnover, lower levels of ATP production, and reduced reactive oxygen species levels. Despite having slower rates of protein turnover, naked mole rat cells tolerate protein misfolding stress more effectively than mouse cells. We suggest that in lieu of a rapid constitutive turnover, long-lived species may have evolved more energetically efficient mechanisms for selective detection and clearance of damaged proteins.

Cells continually degrade and replace damaged and old proteins. However, the high energetic demand of protein turnover generates reactive oxygen species (ROS) that compromise the long-term health of the proteome. Thus, the relationship between aging, protein turnover and energetic demand remains unclear. Here, we used a proteomic approach to measure rates of protein turnover within primary fibroblasts isolated from a number of species with diverse lifespans including the longest-lives rodent, the naked mole rat and the longest-lived mammal, the bowhead whale. We show that organismal lifespan is negatively correlated with turnover rates of highly abundant proteins. In comparison to mice, cells from long-lived naked mole rats have slower rates of protein turnover, lower levels of ATP production and reduced ROS levels. Despite having slower rates of protein turnover, naked mole rat cells tolerate protein misfolding stress more effectively than mouse cells. We suggest that in lieu of rapid constitutive turnover, longlived species may have evolved more energetically efficient mechanisms for selective detection and clearance of damaged proteins.

The folding of most proteins occurs during the course of their translation while their tRNA-bound C-termini are embedded in the ribosome. How the close proximity of nascent proteins to the ribosome influences their folding thermodynamics remains poorly understood. Here, we have developed a mass spectrometry-based approach for determining the stabilities of nascent polypeptide chains using methionine oxidation as a folding probe. This approach enables quantitative measurements sub-global folding stabilities of ribosome nascent chains (RNCs) within complex protein mixtures and extracts. To validate the methodology, we analyzed the folding thermodynamics of three model proteins (DHFR, CheY and DinB) in soluble and ribosome-bound states. The data indicated that the ribosome can significantly alter the stability of nascent polypeptides. Ribosome-induced stability modulations were highly variable among different folding domains and were dependent on localized charge distributions within nascent polypeptides. The results implicated electrostatic interactions between the ribosome surface and nascent polypeptides as the cause of ribosome-induced stability modulations. The study establishes a robust proteomic methodology for analyzing localized stabilities within ribosome-bound nascent polypeptides and sheds light on how the ribosome influences the thermodynamics of protein folding.

The folding of most proteins occurs during the course of their translation while their tRNA-bound C termini are embedded in the ribosome. How the close proximity of nascent proteins to the ribosome influences their folding thermodynamics remains poorly understood. Here, we have developed a mass spectrometry–based approach for determining the stabilities of nascent polypeptide chains using methionine oxidation as a folding probe. This approach enables quantitative measurement subglobal folding stabilities of ribosome nascent chains within complex protein mixtures and extracts. To validate the methodology, we analyzed the folding thermodynamics of three model proteins (dihydrofolate reductase, chemotaxis protein Y, and DNA polymerase IV) in soluble and ribosome-bound states. The data indicate that the ribosome can significantly alter the stability of nascent polypeptides. Ribosome-induced stability modulations were highly variable among different folding domains and were dependent on localized charge distributions within nascent polypeptides. The results implicated electrostatic interactions between the ribosome surface and nascent polypeptides as the cause of ribosome-induced stability modulations. The study establishes a robust proteomic methodology for analyzing localized stabilities within ribosome-bound nascent polypeptides and sheds light on how the ribosome influences the thermodynamics of protein folding.