122 Aging, protein solubility and lifespan in C. elegans, P. Reis-Rodrigues et al. (A) Translation, ribosomal structure and biogenesis Energy production and conversion Posttranslational modification, protein turnover, chaperones na Lipid metabolism Amino acid transport and metabolism cytoskeleton Extracellular structures General function prediction only Cell division and chromosome partitioning Intracellular trafficking, secretion, and vesicular transport RNA processing and modification DNA replication, recombination and repair Function unknown Carbohydrate transport and metabolism Chromatin structure and dynamics Nucleotide transport and metabolism Secondary metabolites biosynthesis, transport and catabolism Signal transduction mechanisms Cell envelope biogenesis, outer membrane Defense mechanisms Inorganic ion transport and metabolism Nuclear structure Transcription (B) Small ribosomal subunit Ribosome Ribosomal subunit Ribonucleoprotein complex Proton-transporting two-sector ATPase complex Proton-transporting ATP synthase complex t Protein complex n e Proteasome core complex n o p Proteasome complex m o Organelle part c r a Organelle membrane l u l l Organelle e C Non-membrane-bound organelle - m Mitochondrion r e T Macromolecular complex y g Intracellular part o l o Intracellular organelle part t n O Intracellular organelle e n Intracellular non-membrane-bound organelle e G Intracellular Endoplasmic reticulum Cytosol Cytoskeleton Cytoplasmic part Cytoplasm 0 10 20 Fold enrichment Total lysate Insoluble fraction 30 40 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. we randomly selected 28 proteins previously identiﬁed in a total lysate and selectively knocked down transcript levels of the respective genes. Survival analysis revealed that 18% of the RNAi treatments extended life- span and 14% reduced it (Fig. 4, Table S3). Taken together, our survival data show that by fractionating a total lysate according to solubility, there is a signiﬁcant enrichment for proteins that contribute to a normally short lifespan in an SDS-insoluble fraction. Discussion Aging is a major risk factor for a wide range of diseases including cancer, diabetes, and neurodegeneration. The accumulation of insoluble protein- aceous aggregates is a shared feature in the most common neurodegen- erative diseases (Alzheimer’s, Parkinson’s, Huntington’s and amyotrophic lateral sclerosis), and these have been sometimes referred to as protein- opathies. We hypothesized that processes related to protein aggregation occurring during normal aging (in the absence of disease) may share fea- tures with pathologic processes underlying late-onset neurodegenera- tion. We thus considered a biochemical characterization of protein homeostasis and protein aggregation during normal aging important in understanding the cellular pathology of late-onset neurodegeneration. Using an unbiased approach, we have identiﬁed which proteins share biochemical characteristics of known aggregation-prone proteins impli- cated in neurodegeneration and critically whether these proteins have any connection to normal aging. We set out to identify proteins that exhibited age-dependent changes solubility in adult C. elegans. By analy- sis and comparison of protein extracts from young and old worms, we were able to quantify the relative abundance of SDS-insoluble proteins in each sample thus generating a sample of the proteins that become SDS insoluble during normal aging. We found that no one protein or class of proteins were represented in the insoluble protein of older worms. Indeed a wide variety of biochemical and physiological functions are implied by homology across 203 proteins identiﬁed. However, there is a notable enrichment for proteins encoding ribosomal subunits. 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