Aging Cell (2012) 11, pp120–127 Doi: 10.1111/j.1474-9726.2011.00765.x Proteomic analysis of age-dependent changes in protein solubility identiﬁes genes that modulate lifespan Pedro Reis-Rodrigues, Gregg Czerwieniec, Theodore W. Peters, Uday S. Evani, Silvestre Alavez, Emily A. Gaman, Maithili Vantipalli, Sean D. Mooney, Bradford W. Gibson*, Gordon J. Lithgow* and Robert E. Hughes* The Interdisciplinary Research Consortium on Geroscience, The Buck Institute for Research on Aging, 8001 Redwood Blvd., Novato, CA 94949, USA Summary While it is generally recognized that misfolding of speciﬁc pro- teins can cause late-onset disease, the contribution of protein aggregation to the normal aging process is less well understood. To address this issue, a mass spectrometry-based proteomic analy- sis was performed to identify proteins that adopt sodium dodecyl sulfate (SDS)-insoluble conformations during aging in Caenor- habditis elegans. SDS-insoluble proteins extracted from young and aged C. elegans were chemically labeled by isobaric tagging for relative and absolute quantiﬁcation (iTRAQ) and identiﬁed by liquid chromatography and mass spectrometry. Two hundred and three proteins were identiﬁed as being signiﬁcantly enriched in an SDS-insoluble fraction in aged nematodes and were largely absent from a similar protein fraction in young nematodes. The SDS-insoluble fraction in aged animals contains a diverse range of proteins including a large number of ribosomal proteins. Gene ontology analysis revealed highly signiﬁcant enrichments for energy production and translation functions. Expression of genes encoding insoluble proteins observed in aged nematodes was knocked down using RNAi, and effects on lifespan were mea- sured. 41% of genes tested were shown to extend lifespan after RNAi treatment, compared with 18% in a control group of genes. These data indicate that genes encoding proteins that become insoluble with age are enriched for modiﬁers of lifespan. This demonstrates that proteomic approaches can be used to identify genes that modify lifespan. Finally, these observations indicate that the accumulation of insoluble proteins with diverse functions may be a general feature of aging. Key words: C. elegans; lifespan; aging; protein solubility; pro- tein aggregation. Introduction Longevity and aging phenotypes are under the inﬂuence of a large num- ber of functionally diverse genes. Genes modulating lifespan are likely to l lCorrespondence Bradford W. Gibson; Gordon J. Lithgow; Robert E. Hughes, The Buck Institute e for Research on Aging, 8001 Redwood Blvd., Novato, CA 94945, USA. Tel.: 415 C209 2032; 415 209 2094; 415 209 2069; fax: 415 209 2231; e-mail: bgibson@ buckinstitute.org; glithgow@buckinstitute.org; rhughes@buckinstitute.org gAccepted for publication 19 October 2011 nRe-use of this article is permitted in accordance with the Terms and Condi- i tions set out at http://wileyonlinelibrary.com/onlineopen#OnlineOpen_Terms g A120 affect multiple pathways and in many cases exert general effects on cellu- lar homeostasis. Longevity of the nematode Caenorhabditis elegans is inﬂuenced by hundreds of genes encoding a range of signaling and meta- bolic processes including an insulin-like signaling pathway that regulates the FOXO transcription factor DAF-16 (Kenyon et al., 1993; Kimura et al., 1997; Ogg et al., 1997). Together with the stress response transcription factor, heat shock factor 1 (HSF-1), DAF-16 regulates the formation pro- tein aggregates in C. elegans and also extend lifespan (Kenyon et al., 1993; Lin et al., 1997; Henderson & Johnson, 2001; Hsu et al., 2003; Cohen et al., 2006). Many of the targets of these transcription factors, such as the heat shock proteins, help maintain other proteins in func- tional states or target them for degradation. Over-expression of even one HSP-encoding gene was sufﬁcient to confer stress resistance and lifespan extension in C. elegans and Drosophila melanogaster (Yokoyama et al., 2002; Walker & Lithgow, 2003; Morrow et al., 2004; Wang et al., 2004). This suggested that the maintenance of protein conformation is required for lifespan extension and that a loss of protein homeostasis could contribute to aging. Protein homeostasis plays an important role in a range of age-related diseases, and a number of toxic conformations of mutant human proteins are associated with disease pathology (Balch et al., 2008). When genes encoding such proteins are expressed in invertebrate models, the proteins often form insoluble aggregates that are associated with cellular toxicity and tissue dysfunction. For example, the expansion of polyglutamine (pol- yQ) residues in various proteins is associated with neurodegenerative dis- ease in humans such as Huntington’s disease and the spinocerebellar ataxias. Toxic effects of expressing mutant polyQ-containing proteins have been observed in model organisms as well. For example, when expanded polyQ proteins are expressed in C. elegans, a generalized impairment of protein homeostasis is observed (Satyal et al., 2000; Mor- ley et al., 2002). Likewise, when the A3-42 b-amyloid peptide (Ab) is expressed in C. elegans muscle, Ab aggregates form and the worms become paralyzed (Link, 1995; Drake et al., 2003; McColl et al., 2009). Although protein aggregation is well studied in the context of neurode- generative diseases, its contribution to normal aging is poorly deﬁned. However, models of protein aggregation have been studied during nor- mal aging (Ben-Zvi et al., 2009). It is notable that expression of proteins prone to aggregation accelerated the aggregation of other proteins, highlighting the limiting capacities of the protein homeostasis machinery (Morimoto, 2008). It has been hypothesized that a loss in protein homeo- stasis during aging may lead to impaired protein solubility and cellular dysfunction (Ben-Zvi et al., 2009). As modifying expression of genes encoding proteins linked to protein homeostasis can modulate lifespan in the context of age-related disease (Morimoto, 2008), we tested whether global changes in protein confor- mation could inﬂuence lifespan in the context of normal aging (Morimoto & Cuervo, 2009). Using an unbiased biochemical and proteomic approach, we determined which proteins exhibit altered solubility during aging. We have made a comparative quantitative analysis of SDS-insolu- ble protein extracts from young and old adult C. elegans hermaphrodites and have identiﬁed a wide range of insoluble proteins that are signiﬁ- cantly enriched in older nematodes. When we reduce the expression of a cohort of these individual proteins by RNA interference (RNAi), we frequently observe an extended lifespan. These results are consistent with Published 2011. This article is a U.S. Government work and is in the public domain in the USA. Aging Cell ª 2011 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland