Proteomic analysis of age-dependent changes in protein solubility identifies genes that modulate lifespan

Abstract

While it is generally recognized that misfolding of specific proteins 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 analysis was performed to identify proteins that adopt sodium dodecyl sulfate (SDS)-insoluble conformations during aging in Caenorhabditis 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 measured. 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.

Fig. 1

Accumulation of insoluble proteins in aged Caenorhabditis elegans. SDS-PAGE of the aqueous, SDS-soluble and SDS-insoluble fractions of 1 and 11-day-old adult animals. Levels of SDS-insoluble proteins are greatly enhanced in extracts from old animals as compared with extracts from young animals.

Fig. 2

Gene ontology (GO) analysis of proteins observed in the insoluble fraction of old Caenorhabditis elegans. A) Functional categories of proteins identified in the insoluble fraction of old worms. Classification is as assigned by Klusters of Orthologous Groups (KOG). B) GO annotations compared between total lysate and the insoluble fraction. Fold enrichment for GO cellular component terms for each of the two fractions is shown.

Fig. 3

A large fraction of the insoluble proteins reduce Caenorhabditis elegans lifespan. Synchronous populations of adult worms were subjected to RNAi knock-down of transcript levels of various genes encoding proteins identified in the insoluble fraction of old worms. Kaplan-Meyer survival curves were generated based upon survival at the indicated time points. A) Survival curves while knocking down transcript levels of genes encoding translation-related proteins. B) Survival curves while knocking down transcript levels of genes encoding mitochondrial proteins.

Fig. 4

The SDS-insoluble fraction is highly enriched for proteins that negatively modulate lifespan. Comparison of survival data after knock-down of genes selected from a total lysate and the insoluble fraction. Percentage of genes with no effect, extending, and decreasing lifespan is shown.