Altered proteostasis in aging and heat shock response in C. elegans revealed by analysis of the global and de novo synthesized proteome

Abstract

Protein misfolding and aggregation as a consequence of impaired protein homeostasis (proteostasis) not only characterizes numerous age-related diseases but also the aging process itself. Functionally related to the aging process are, among others, ribosomal proteins, suggesting an intimate link between proteostasis and aging. We determined by iTRAQ quantitative proteomic analysis in C. elegans how the proteome changes with age and in response to heat shock. Levels of ribosomal proteins and mitochondrial chaperones were decreased in aged animals, supporting the notion that proteostasis is altered during aging. Mitochondrial enzymes of the tricarboxylic acid cycle and the electron transport chain were also reduced, consistent with an age-associated energy impairment. Moreover, we observed an age-associated decline in the heat shock response. In order to determine how protein synthesis is altered in aging and in response to heat shock, we complemented our global analysis by determining the de novo proteome. For that, we established a novel method that enables both the visualization and identification of de novo synthesized proteins, by incorporating the non-canonical methionine analogue, azidohomoalanine (AHA), into the nascent polypeptides, followed by reacting the azide group of AHA by 'click chemistry' with an alkyne-labeled tag. Our analysis of AHA-tagged peptides demonstrated that the decreased abundance of, for example, ribosomal proteins in aged animals is not solely due to degradation but also reflects a relative decrease in their synthesis. Interestingly, although the net rate of protein synthesis is reduced in aged animals, our analyses indicate that the synthesis of certain proteins such as the vitellogenins increases with age.

Fig. 1

AHA labeling of de novo synthesized proteins in C. elegans. Schematic showing incorporation of AHA during aging and heat shock along with subsequent analysis of AHA-tagged proteins

Fig. 2

AHA-labeled proteins in C. elegans can be visualized by immunofluorescence analysis or immunoblot after ‘click chemistry’ using Chromeo-546-alkyne or biotin-alkyne tags, respectively. Animals were incubated with 0.5 or 2 mM (for immunofluorescence) or 2 mM AHA (for immunoblot) for 6 h before ‘click chemistry’ was performed at either young adult (YA), day 5 (D5) or day 10 (D10) of adulthood. All samples, including controls, were incubated with the Chromeo™-546-alkyne or biotin-alkyne tags. a Representative fluorescence images showing 2 mM AHA-labeled and unlabeled nematodes. The top panel “−AHA −Click” refers to sample where no AHA was added and ‘click chemistry’ was not performed. The bottom panel “+AHA +Click” refers to a where ‘click chemistry’ was performed on a sample where AHA had been added. b Fold changes of fluorescence intensity (in arbitrary units, measured using ImageJ software) between AHA-labeled and non-labeled nematodes at YA, D5, or D10 of adulthood, with or without heat shock treatment. In this experiment, labeling was conducted with 0.5 mM AHA and ‘click chemistry’ was performed only when AHA was added. c Representative fluorescence images at 546 nm of 0.5 mM AHA-labeled (+‘click chemistry’) non-heat shocked and heat shocked nematodes shown at YA, D5, or D10 of adulthood. d Immunoblot detection of biotin-labeled AHA-tagged proteins in YA animals in non-heat shocked and heat shocked samples. Actin was used as loading control. The rightmost two lanes are control samples indicating the absence of bands (other than a non-specific signal at 70 kDa) when no AHA is added before ‘click chemistry’, or when AHA is added but ‘click chemistry’ is not performed

Fig. 3

The proteomic profile of C. elegans changes with age. a Gene Ontology (cellular component) profile of proteins increased at D5 compared with YA. The extracellular proteins include vitellogenins. Only the major categories are shown; categories with fewer proteins are cumulatively displayed as ‘Others’. Proteins without a Gene Ontology (cellular component) classification are shown as ‘Unclassified’. b Gene Ontology (cellular component) profile of proteins decreased at D5 compared with YA. c Venn diagram of proteins increased at D5 and D10 compared with YA. d Venn diagram of proteins decreased in abundance at D5 and D10 compared with young adults (YA). Selected proteins that are increased (e) or decreased (f) in abundance at D5 and D10 compared with YA

Fig. 4

The proteomic profile of C. elegans changes with heat shock. Heat shocked samples are indicated by ‘HS’. a Gene Ontology (cellular component) profile of proteins increased in abundance in response to heat shock (34 °C/2 h) in young adults (YA). Only the major categories are shown; categories with fewer proteins are cumulatively displayed as ‘Others’. Proteins without a Gene Ontology (cellular component) classification are shown as ‘Unclassified’. b Gene Ontology (cellular component) profile of proteins decreased in abundance in response to heat shock in YA. c Venn diagram of proteins increased in abundance at YA, D5 and D10 after heat shock compared with their age-matched controls. d Venn diagram of proteins decreased in abundance at YA, D5, and D10 after heat shock compared with their age-matched controls. e Fold change of selected heat shock proteins increased in abundance at YA, D5 and D10 compared with their age-matched non-heat shocked controls. f Fold change of selected heat shock proteins increased in abundance at YA, D5, and D10 (non-heat shocked or HS) compared with YA (non-heat shocked)

Fig. 5

The Gene Ontology profile of newly synthesized proteins in C. elegans reflects that of the global proteome. Comparison of Gene Ontology (cellular component) profiles of AHA-labeled peptides and the global proteome for proteins/peptides shown to be a increased or b decreased in abundance at D5 or D10 of adulthood compared with YA. Percentages shown here are the proportions of proteins/peptides in the global proteome or AHA-labeled data set

Fig. 6

The proteomic profile of newly synthesized proteins in C. elegans changes in response to heat shock. Gene Ontology (cellular component) profile of AHA-modified peptides shown to be a increased or b decreased in abundance in response to heat shock (34 °C/2 h) in YA. Proteins without a Gene Ontology (cellular component) classification are shown as ‘Unclassified’