Increased Protein Stability and Decreased Protein Turnover in the Caenorhabditis elegans Ins/IGF-1 daf-2 Mutant

Abstract

In Caenorhabditis elegans, cellular proteostasis is likely essential for longevity. Autophagy has been shown to be essential for lifespan extension of daf-2 insulin/IGF mutants. Therefore, it can be hypothesized that daf-2 mutants achieve this phenotype by increasing protein turnover. However, such a mechanism would exert a substantial energy cost. By using classical ³⁵S pulse-chase labeling, we observed that protein synthesis and degradation rates are decreased in young adults of the daf-2 insulin/IGF mutants. Although reduction of protein turnover may be energetically favorable, it may lead to accumulation and aggregation of damaged proteins. As this has been shown not to be the case in daf-2 mutants, another mechanism must exist to maintain proteostasis in this strain. We observed that proteins isolated from daf-2 mutants are more soluble in acidic conditions due to increased levels of trehalose. This suggests that trehalose may decrease the potential for protein aggregation and increases proteostasis in the daf-2 mutants. We postulate that daf-2 mutants save energy by decreasing protein turnover rates and instead stabilize their proteome by trehalose.

Keywords: Caenorhabditis; Protein metabolism; Radiolabeling.; Trehalose.

Figure 1.

(A) Relative rate of ³⁵S incorporation in worm proteins with age after feeding ³⁵S-labeled bacteria to controls (glp-4 daf-16; daf-2(e1370)) and the insulin/IGF-1 receptor mutant glp-4; daf-2(e1370). (B) Relative rate of ³⁵S removal in the trichloroacetic acid–precipitated protein fraction over age. *p < .05, **p < .005, ***p < .0005 (linear regression model). Averages ± SEM are from four independent replicate experiments.

Figure 2.

(A) Percentage of total ³⁵S activity remaining soluble in the presence of trichloroacetic acid (TCA) (10% final concentration) measured after an 8-h chase period as a function of adult age. Mean values ± SEM of three to six independent experiments. (B) Percentage of total proteinaceous material remaining soluble in the presence of TCA (10% final concentration) as determined with a bicinchoninic acid assay. Mean values ± SEM of three independent replicates. (C) Total amino acid content determined by HPLC analysis of free- (solid stacked bars) and bound- (hatched stacked bars) amino acids in the sTCA fraction. Bound amino acids originate from complete acid hydrolysis of peptides and proteins that remained soluble in the sTCA fraction. Full bar height is indicative for total amino acid content (free amino acids, peptides, and proteins) in the TCA-soluble (sTCA) fraction. Worm samples of (B) and (C) were taken at the second day of adulthood. Mean values ± SEM of three independent replicates are shown. *p < .05, **p < .005, and ***p < .0005 for Student’s two-sided unpaired t-test.

Figure 3.

(A) The effect of trehalose, dimethyl sulfoxide (DMSO), and glycerol on protein solubility in the presence of 10% trichloroacetic acid (TCA). (B) The effect of adding 5mM trehalose to the culture medium on both protein solubility and worm trehalose concentration. (C) Protein solubility and trehalose content of worms fed either RNAi bacteria expressing tps-1 and tps-2 or empty vector (L4440; *p < .05, **p < .005, ***p < .0005). Asterisks on top of bars refer to comparison with the control (CTR in [A] and glp-4; daf-2 L4440 in [C]), whereas asterisks on brackets compare the connected bars.