• Open Access

Attenuation of liver insoluble protein carbonyls: indicator of a longevity determinant?

Authors

  • Arunabh Bhattacharya,

    1. Barshop Institute of Longevity and Aging Studies, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA
    2. Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA
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  • Shanique Leonard,

    1. Barshop Institute of Longevity and Aging Studies, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA
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  • Suzette Tardif,

    1. Barshop Institute of Longevity and Aging Studies, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA
    2. Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA
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  • Rochelle Buffenstein,

    1. Barshop Institute of Longevity and Aging Studies, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA
    2. Department of Physiology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA
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  • Kathleen E. Fischer,

    1. Barshop Institute of Longevity and Aging Studies, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA
    2. Department of Physiology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA
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  • Arlan Richardson,

    1. Barshop Institute of Longevity and Aging Studies, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA
    2. Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA
    3. Department of Physiology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA
    4. South Texas Veterans Health Care System, San Antonio, TX 78229, USA
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  • Steven N. Austad,

    1. Barshop Institute of Longevity and Aging Studies, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA
    2. Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA
    3. Department of Molecular Medicine, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA
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  • Asish R. Chaudhuri

    1. Barshop Institute of Longevity and Aging Studies, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA
    2. South Texas Veterans Health Care System, San Antonio, TX 78229, USA
    3. Department of Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA
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Asish R. Chaudhuri, Barshop Institute for Longevity and Aging Studies, University of Texas Health Science Center at San Antonio, Texas Research Park Campus, 15355 Lambda Drive, San Antonio, TX 78245-32075, USA. Tel.: +1 210 562 6135; fax: +1 210 562 6110; e-mail: chaudhuria@uthscsa.edu
Steven N. Austad, Barshop Institute for Longevity and Aging Studies, University of Texas Health Science Center at San Antonio, Texas Research Park Campus, 15355 Lambda Drive, San Antonio, TX 78245-3207, USA. Tel.: +1 210 562 6135; fax: +1 210 562 6110; e-mail: austad@uthscsa.edu

Summary

Oxidative damage affects protein structure and function. Progressive accumulation of oxidized proteins is considered a putative mechanism of aging; however, empirical evidence supporting their role in aging is inconsistent. This inconsistency may reflect a failure to distinguish damage to particular cellular compartments. We found a significant reduction of protein carbonyls in the insoluble, but not in the soluble, fraction of liver tissues of long-lived compared with their short-lived counterpart. Of cellular components analyzed, only nuclear protein carbonyl level was uniformly reduced in long-lived compared with short-lived animals. This observation suggests that attenuated accumulation of protein carbonyls in the nucleus, where they can affect multiple aspects of gene expression and DNA repair, might contribute to the longevity in mammalian species.

The oxidative stress theory of aging and age-related disease (Harman, 1956; Bokov et al., 2004) asserts that compromised tissue function with age is because of progressively increasing oxidative damage, particularly to proteins. If the hypothesis is valid, oxidative damage should be lower in long-lived compared with short-lived animals at similar ages. However, empirical data have been conflicting and inconsistent (Van Remmen et al., 2003; Andziak et al., 2006; Perez et al., 2009; Buffenstein et al., 2008). Oxidative damage may be critical only for certain molecules or classes of molecules and/or only in some cellular compartments; assays that do not distinguish types of damage or damage to specific cellular compartments may mask informative, mechanistically important patterns.

Oxidative damage to proteins represents a reasonable candidate for a mechanism of aging because of proteins’ ubiquitous role in cellular processes, the requirement that proteins remain precisely folded, and that protein homeostasis is significantly compromised with age (Morimoto & Cuervo, 2009; Koga et al., 2010). The most common method of assessing protein oxidative damage is to measure the level of protein carbonyls (Sohal et al., 1993; Olivares-Corichi et al., 2005). Carbonylated proteins that escape degradation by the proteasome and the mitochondrial proteolytic machinery can accumulate over time to form high molecular weight insoluble aggregates (Shringarpure & Davies, 2002; Nystrom, 2005). However, few attempts have been made to measure protein carbonyls specifically in the insoluble fraction of cellular lysates, which contains cellular organelles. Thus, assessing the insoluble cellular fraction may be critical to understanding the impact of oxidative damage to proteins within organelles. Separating insoluble from soluble carbonyls may reveal functional patterns not evident when total carbonyls are assayed.

Even studies that focus on soluble carbonyls are frequently inconsistent in the range of cellular compartments they assay. Some studies combine all cellular compartments, others employ centrifugation strategies that omit the nucleus (i.e., 600–1000 g), and still others (11 000–30 000 g) include the cytosol mitochondria, lysosomes, and other small cellular organelles (Levine et al., 1990; Youngman et al., 1992; Cao & Cutler, 1995; Dubey et al., 1996; Alam et al., 1997; Goto et al., 1999; Levine, 2002; Dalle-Donne et al., 2003; Breusing et al., 2009; Ahmed et al., 2010). To reduce potential confounds, we have quantified soluble protein carbonyls strictly in the cytosol with minimal or no interference from free probe or organelles using a sensitive fluorescence-based gel assay (Chaudhuri et al., 2006). Using the same methodology with minor modification in buffer composition, we can assess total protein carbonyls in the insoluble fraction. These techniques, in combination with specific centrifugation strategies, allow us to assay insoluble protein carbonyls in specific cellular compartments as well.

Previous reports indicate that protein carbonylation increases with age and dietary restriction (DR) reduces the rate of accumulation of carbonylation compared with ad lib-fed animals (Sohal et al., 1994; Dubey et al., 1996; Nagai et al., 2000; Radak et al., 2002). Examining carbonylation in liver, older (24–30 months) mice, indeed, show increased carbonylation relative to young (8–10 months) mice in both soluble and insoluble fractions (Fig. 1A,B), but carbonyl attenuation by DR is entirely because of reduced carbonylation in the insoluble fraction (Fig. 1B). These results suggest that (i) attenuation of insoluble protein carbonyls is correlated with longevity; and (ii) soluble protein carbonyls may not be reduced in long-lived animals.

Figure 1.

 Assessment of protein carbonyl in the soluble and insoluble fractions of various short- and long-lived species. Carbonylation in (A) soluble and (B) insoluble fractions from the livers of young (4–6 months; n = 3–6) and old (24–30 months; n = 3–12) C57BL/6 mice fed ad libitum (AL, white bar) or with their caloric intake restricted by 40% (DR, gray bar). DR of this mouse genotype increases longevity by roughly 25% (Turturro et al., 1999). Note the significant reduction in carbonylation of the DR mice in the insoluble fraction only of young (< 0.01) and old (< 0.001) animals. Similarly, significantly (< 0.01–0.0001) lower carbonylation was seen in the insoluble (gray bar) fraction only of the longer-lived species in comparisons with (C) young adult wild-derived ID mice (6 months) vs. young adult naked mole-rats (2 years), (D) young adult rats (4–6 months) vs. young adult marmosets (4 years), and (E) young adult wild-derived mice (6 months) vs. young adult Brazilian free-tailed bats (young wild caught). Samples were homogenized in 50 mm sodium phosphate buffer pH 6.0 followed by centrifugation at 100, 000 g to obtain soluble and insoluble fractions. Supernatant and pellet proteins were labeled with the fluorescent probe FTC and were run on 12% SDS-PAGE. Protein carbonyls were quantified using typhoon with excitation and emission wavelengths of 488 and 520 nm, respectively. Coomassie staining was performed in the same samples for correction of unequal protein loading. Results are mean ± SEM of n = 3–12 animals per group, analyzed by one-way anova with Newman–Keuls multiple comparison test. Bars with different superscripts are significantly different at < 0.01.

To investigate the relationship between longevity and protein carbonylation in specific cellular fractions, we performed three paired comparisons using mammal species of approximately the same body mass but different longevities. We controlled for body size because the general correlation between size and longevity can confound interspecific patterns (Hulbert et al., 2007; Austad & Fischer, 1991; de Magalhaes JP et al., 2007). We compared mice (maximum longevity 4 years) with naked mole-rats (NMRs, maximum longevity 30 years) and separately with Brazilian free-tailed bats (longevity record in the wild 12 years). We also compared Norway rats (maximum longevity 4 years) with the common marmoset (maximum longevity 16 years). In all comparisons, the shorter-lived species exhibited greater carbonylation in the insoluble fraction of these liver extracts (Fig. 1C–E).

To assess whether protein oxidation in some cellular compartments was more strongly correlated with longevity than others, we employed the same four comparisons—one dietary and three interspecific—and fractionated the insoluble pellet into nuclear, mitochondrial, and microsomal [ER/Ribosome] subfractions using differential centrifugation and assessed protein carbonyls in each subfraction. Carbonylation of proteins in the nuclear fraction was reduced in all longer-lived groups (Fig. 2). Interestingly, in the mitochondrial fraction, we observed significantly increased carbonyl content in NMRs compared with all of the other species, which may explain why total carbonylation has been reported to be increased in NMRs relative to mice (Andziak et al., 2006).

Figure 2.

 Distribution of protein carbonyl in cellular subfractions of short- vs. long-lived species. The insoluble cellular organelles were fractionated from liver samples into nuclear, microsomal (ribosomal/ER), and mitochondrial fractions by differential centrifugation from young adult animals of various groups as in Fig. 1. Note that the only uniform result was reduced protein carbonylation in the insoluble nuclear fraction of all longer-lived groups. (A) AL vs. DR C57BL/6 mice, (B) wild-derived mice vs. naked mole-rats, (C) wild-derived mice vs. Brazilian free-tailed bats, (D) rats vs. marmosets. Cellular organelles were separated from the insoluble fraction by differential centrifugation: nuclear (1000 g), mitochondrial (16, 000 g), and microsomal (ribosomal/ER, 100, 000 g) followed by labeling the protein carbonyls with FTC. The labeled proteins were then subjected to 12% SDS-gel electrophoresis. Protein carbonyls were quantified using typhoon at excitation and emission wavelengths of 488 and 520 nm, respectively. Coomassie staining was performed with the same samples for correction of unequal protein loading. Results were analyzed by unpaired t-test (#< 0.05,*< 0.01). Results are mean ± SEM of n = 3 animals per group.

In summary, reduced protein carbonylation in the nucleus is correlated with increased longevity. This distinct pattern is observed at a young age, consistent with the hypothesis that the biological qualities that regulate lifespan should be evident in young adults and age-specific mortality should correlate with maximum lifespan. Protein oxidation is likely to interfere with transcriptional regulation on several levels; from damage to transcription factors and polymerases to inhibition of proteasomal degradation of regulatory proteins to modification of the interactions between DNA and histone (Breusing et al., 2009; Nakamura et al., 2010; Kwak et al., 2011; Pashikanti et al., 2011). Future studies to identify the nuclear target proteins that show elevated level of carbonyls in the short-lived species and decreased level in the long-lived species would help to understand their role in modulating chromatin structure and transcriptional regulation associated with longevity. Moreover, determining the underlined mechanisms such as activation of nuclear proteasomal function in long-lived species and consequent decreased accumulation of protein carbonylation would also highlight the importance of nuclear protein homeostasis in mammalian longevity.

Acknowledgments

This work was supported by K07 AG02506304 (A.R.C., S.N.A.) and NIH grants AG022873 (S.N.A.) and AG-022891 (R.B.). Marmoset tissues were provided by the Southwest National Primate Research Center.

Author contributions

Arunabh Bhattacharya, Asish R Chaudhuri, Steven N Austad, Rochelle Buffenstein, Suzette Tardif and Arlan Richardson designed and guided the study; Shanique Leonard performed research; Rochelle Buffenstein, Suzette Tardif, Arlan Richardson and Steven N Austad provided the animals used in the study; Arunabh Bhattacharya and Asish R Chaudhuri analyzed the data; Arunabh Bhattacharya, Kathleen E Fischer, Steven N Austad, and Asish R Chaudhuri wrote the manuscript.

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