Monitoring Newly Synthesized Proteins over the Adult Life Span of Caenorhabditis elegans.

Feb. 16, 2015

Little is known regarding how the synthesis and degradation of individual proteins changes during the life of an organism. Such knowledge is vital to understanding the aging process. To fill this knowledge gap, we monitored newly synthesized proteins on a proteome scale in Caenorhabditis elegans over time during adulthood using a SILAC-based label-chase approach. For most proteins, the rate of appearance of newly synthesized protein was high during the first 5 days of adulthood, slowed down between the fifth and the 11th days, and then increased again after the 11th day. However, the magnitude of appearance rate differed significantly from protein to protein. For example, the appearance of newly synthesized protein was fast for proteins involved in embryonic development, transcription regulation, and lipid binding/transport, with >70% of these proteins newly synthesized by day 5 of adulthood, whereas it was slow for proteins involved in cellular assembly and motility, such as actin and myosin, with <70% of these proteins newly synthesized even on day 16. The late-life increase of newly synthesized protein was especially high for ribosomal proteins and ATP synthases. We also investigated the effect of RNAi-mediated knockdown of the rpl-9 (ribosomal protein), atp-3 (ATP synthase), and ril-1 (RNAi-induced longevity-1 ) genes and found that inhibiting the expression of atp-3 and ril-1 beginning in late adulthood is still effective to extend the life span of C. elegans.

Figure: Strategy for monitoring the appearance of newly synthesized proteins in a proteome scale. a) A single nematode was propagated on heavy Lys (13C6-Lys)-labeled E. coli for two generations, and then age-synchronized adult worms were transferred to light Lys (12C6-Lys)- labeled E. coli plates on day 1 and harvested at various time points. Proteins extracted from these samples were digested with Lys-C and analyzed by LC-MS/MS. The resulting data were then analyzed to identify and quantify the proteins present. b) Hypothetical mass spectra of a peptide from worms at different ages. The 12C6-Lys labeled 'light' peak (L) increases relative to the 13C6Lys labeled 'heavy' (H) peak as the worm ages, thus indicating increase of the newly synthesized protein from which the peptide originated.

Results from: Vukoti K, Yu X, Sheng Q, Saha S, Feng Z, Hsu AL, Miyagi M. J Proteome Res. 2015 Feb 16. [Epub ahead of print]