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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00018-014-1558-7) contains supplementary material, which is available to authorized users.

Bio-orthogonal labeling as a tool to visualize and identify newly synthesized proteins in Caenorhabditis elegans

“…(b) Three possible approaches to identify via AHA incorporation de novo-synthesized proteins are used in this protocol: click chemistry followed by either western blotting or fluorescence microscopy, and iTRAQ quantitative mass spectrometry. Scheme adapted from Liang et al 22 .…”

“…We therefore developed two click-chemistry protocols based on two previously established in vitro methods, which were termed bio-orthogonal noncanonical amino acid tagging (BONCAT) 41,51 and fluorescent noncanonical amino acid tagging (FUNCAT) 52 , incorporating our own work 22 . Specifically, we present a protocol detailing three complementary, mutually alternative types of in vivo analysis of de novo protein synthesis: 'click chemistry' followed either by western blotting or fluorescence microscopy, as well as iTRAQ quantitative mass spectrometry for the identification of de novo-synthesized proteins ( Figs.…”

Proteostasis in cardiac health and disease

“…Unfortunately, functional impairment of the PQC is also part of ageing, because ageing attenuates the stress response and protein degradation pathways [63][64][65][66] . Consequently, restoring PQC by overexpression of small HSPs extends lifespan in D. melanogaster 67,68 .…”

Combining Protein Ratio p-Values as a Pragmatic Approach to the Analysis of Multirun iTRAQ Experiments

“…The Stouffer z-score has been used in the context of iTRAQ in only a handful of publications. 21,10,22 In the current work we couple it with a ratio trend consistency measure and demonstrate it is conservative and favors proteins identified with high confidence. We compare the approach with the one sample t test, another simple statistical tool applicable under similar conditions, and show that they overlap significantly on a data set with large protein expression differences between sample groups, with Stouffer's approach being more conservative.…”