Caenorhabditis elegans worms are frequently used to investigate how dietary restriction prolongs invertebrate lifespan. Researchers have shown that when food intake is restricted, C. elegans worms live longer; one of the pathways affected involves disruption of the insulin/insulin-like growth factor-1-like tyrosine kinase receptor, daf-2.1 Even without dietary restriction, worms with a mutation of this receptor have a longer lifespan.
Depuydt et al. (2013) used accurate mass and time liquid chromatography–tandem mass spectrometry (LC-MS/MS) to compare the proteomes of C. elegans worms undergoing dietary restriction (DR) and those bred with a daf-2 receptor mutation that abolishes signaling.2 Their aim was to uncover pathways common to each life-prolonging strategy and also to investigate where they diverged.
The researchers cultured age-synchronized populations of the mutant and control strains of C. elegans worms for two days before flash freezing and preparation of tissues for trypsin digestion. They analyzed samples using a constant-pressure capillary high-performance liquid chromatography (HPLC) system coupled online to an LTQ Orbitrap mass spectrometer (Thermo Scientific). Depuydt and colleagues then quantified differential expression of the proteins using a label-free method, identifying resulting peptide sequences by comparison with the WormBase (release WS210) data set for unambiguous matches.
The researchers found that, compared with fully fed worms, numbers of large and small ribosomal protein subunits were reduced in the proteomes of both mutant and DR individuals. Furthermore, they found that levels of tRNA synthetases were reduced in the daf-2 mutants. On closer examination, this was not due to gene transcription rates, as mRNA levels did not differ from those in the fully fed controls.
Depuydt and co-workers wondered if post-transcriptional regulation occurred at the level of protein synthesis. Worms were fed radio-labeled Escherichia coli to measure protein synthesis, and the researchers found that rates were indeed reduced in the DR and daf-2 mutant groups (P < 0.001 and P < 0.0005, respectively).
However, transmission electron microscopy showed that, despite a smaller body size, the DR and daf-2 mutant worms contained more muscle mass. Using quantitative reverse transcription polymerase chain reaction, the researchers found increased transcription rates for genes encoding muscle proteins in the daf-2 mutants but not in the DR worms.
Preservation of muscle mass was also reflected by proteomic analysis; proteins — including actin isoform ACT-1, myosin heavy chains, and the actin binding tropomyosin LEV-11 — were increased in the DR and daf-2 worm proteomes. The authors speculate that these increased protein levels help preserve muscle mass and function throughout DR stress.
Depuydt and co-authors emphasize that further investigation of aging processes at the proteomic level is necessary.
1. Kimura, K.D., et al. (1997) “daf-2, an Insulin Receptor–Like Gene That Regulates Longevity and Diapause in Caenorhabditis elegans,” Science, 277 (pp. 942–946).
2. Depuydt, G., et al. (2013) “Reduced insulin/insulin-like growth factor‐1 signaling and dietary restriction inhibit translation but preserve muscle mass in Caenorhabditis elegans,” Molecular and Cellular Proteomics (published on September 3 as Manuscript M113.027383), doi: 10.1074/mcp.M113.027383.
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