Regulation of Caenorhabditis elegans lifespan by a proteasomal E3 ligase complex

Abstract

The proteasome maintains cellular homeostasis by degrading oxidized and damaged proteins, a function known to be impaired during aging. The proteasome also acts in a regulatory capacity through E3 ligases to mediate the spatially and temporally controlled breakdown of specific proteins that impact biological processes. We have identified components of a Skp1-Cul1-F-Box E3 ligase complex that are required for the extended lifespan of Caenorhabditis elegans insulin/insulin-like growth factor-1-signaling (IIS) mutants. The CUL-1 complex functions in postmitotic, adult somatic tissues of IIS mutants to enhance longevity. Reducing IIS function leads to the nuclear accumulation of the DAF-16/FOXO transcription factor, which extends lifespan by regulating downstream longevity genes. These CUL-1 complex genes act, at least in part, by promoting the transcriptional activity of DAF-16/FOXO. Together, our findings describe a role for an important cellular pathway, the proteasomal pathway, in the genetic determination of lifespan.

Fig. 1.

cul-1 RNAi shortens the extended lifespan of daf-2 mutants. (A) Schematic representation of the CUL-1 E3 ligase complex. (B–G) Lifespan curves of long-lived mutants and wild-type worms grown as adults on control empty vector (green lines), daf-16 RNAi (blue lines), and cul-1 RNAi (red lines). (B) daf-2(mu150). Control vector: mean = 33.4 ± 0.7, n = 89/90 (number of animals that died/total; see Materials and Methods); daf-16 RNAi: mean = 17.3 ± 0.5, n = 80/83, P < 0.0001 vs. control; cul-1 RNAi: mean = 25.2 ± 0.4, n = 80/85, P < 0.0001 vs. control. (C) daf-2(e1368). Control vector: mean = 32.7 ± 0.7, n = 58/105; daf-16 RNAi: mean = 22.1 ± 0.3, n = 76/105, P < 0.0001 vs. control; cul-1 RNAi: mean = 25.1 ± 0.4, n = 69/104, P < 0.0001 vs. control. (D) daf-2(e1370). Control vector: mean = 40.8 ± 1.0, n = 72/89; daf-16 RNAi: mean = 22.7 ± 0.5, n = 67/90, P < 0.0001 vs. control; cul-1 RNAi: mean = 31.1 ± 0.9, n = 77/83, P < 0.0001 vs. control. (E) N2. Control vector: mean = 20.7 ± 0.7, n = 67/88; daf-16 RNAi: mean = 16.6 ± 0.5, n = 67/87, P < 0.0001 vs. control; cul-1 RNAi: mean = 18.8 ± 0.6, n = 84/90, P = 0.3 vs. control. (F) glp-1(e2141ts). Control vector: mean = 23.2 ± 0.4, n = 86/87; daf-16 RNAi: mean = 17.8 ± 0.4, n = 79/84, P < 0.0001 vs. control; cul-1 RNAi: mean = 23.9 ± 0.3, n = 74/76, P = 0.4 vs. control. (G) eat-2(ad1116). Control vector: mean = 27.3 ± 0.6, n = 70/96; daf-16 RNAi: mean = 24.8 ± 0.6, n = 68/96, P = 0.005 vs. control; cul-1 RNAi: mean = 25.2 ± 0.5, n = 77/102, P = 0.2 vs. control.

Fig. 2.

cul-1 acts in postmitotic adult somatic tissues to regulate the lifespan of daf-2 mutants. (A) cul-1 RNAi shortens the extended lifespan of gonad-ablated daf-2 mutants. Lifespans of gonad precursor (Z1 and Z4) ablated daf-2(e1368) mutants (solid lines) and unablated control worms (dotted lines) grown as adults on bacteria expressing dsRNA for cul-1 (red), daf-16 (blue), and control vector (green) are shown. Z1,Z4(−) control vector (green solid line): mean = 32.6 ± 1.4, n = 43/43; Z1,Z4(−) cul-1 RNAi (red solid line): mean = 23.0 ± 0.6, n = 54/54, P < 0.0001 vs. control; Z1,Z4(−) daf-16 RNAi (blue solid line): mean = 18.2 ± 0.36, n = 30/30, P < 0.0001 vs. control; unablated control vector (green dotted line): mean = 31.4 ± 1.0, n = 35/43; unablated cul-1 RNAi (red dotted line): mean = 23.1 ± 0.9, n = 39/45, P < 0.0001 vs. control; unablated daf-16 RNAi (blue dotted line): mean = 20.8 ± 0.5, n = 40/45, P < 0.0001 vs. control. Similar results were obtained with additional repetitions of this experiment (SI Table 3). (B–D) cul-1 expression in adult somatic tissues. A Pcul-1::rfp construct reveals expression in adult neurons (head neurons are shown in B), muscles (C; colocalization with Pmyo-3::gfp coinjection marker), and intestinal cells (D; composite fluorescence-differential interference contrast microscopy image). The red fluorescent protein signal appears nuclear because of the presence of a nuclear localization signal in the construct for ease of cell identification.

Fig. 3.

skr-1/2 are required for the extended lifespan of daf-2 mutants but not other long-lived mutants or wild-type worms. Lifespan experiments of long-lived mutants and wild-type worms grown as adults on control empty vector (green lines), daf-16 RNAi (blue lines), skr-1 RNAi (deep red lines), and skr-2 RNAi (bright red lines). (A) daf-2(mu150). Control vector: mean = 33.4 ± 0.7, n = 89/90; daf-16 RNAi: mean = 17.3 ± 0.5, n = 80/83, P < 0.0001 vs. control; skr-1 RNAi: mean = 22.0 ± 0.5, n = 88/91, P < 0.0001 vs. control; skr-2 RNAi: mean = 22.8 ± 0.4, n = 92/92, P < 0.0001 vs. control. (B) N2. Control vector: mean = 20.7 ± 0.7, n = 67/88; daf-16 RNAi: mean = 16.6 ± 0.5, n = 67/87, P < 0.0001 vs. control; skr-1 RNAi: mean = 20.7 ± 0.7, n = 82/86, P = 0.71 vs. control; skr-2 RNAi: mean = 21.4 ± 0.9, n = 67/81, P = 0.19 vs. control. (C) glp-1(e2141ts). Control vector: mean = 22.4 ± 1.0, n = 82/85; daf-16 RNAi: mean = 16.1 ± 0.3, n = 88/88, P < 0.0001 vs. control; skr-1 RNAi: mean = 21.8 ± 0.8, n = 88/91, P = 0.19 vs. control; skr-2 RNAi: mean = 22.1 ± 0.7, n = 92/92, P = 0.1 vs. control. (D) eat-2(ad1116). Control vector: mean = 27.3 ± 0.6, n = 70/96; skr-1 RNAi: mean = 23.8 ± 1.1, n = 66/94, P = 0.2 vs. control; skr-2 RNAi: mean = 27.3 ± 0.6, n = 67/93, P = 0.8 vs. control.

Fig. 4.

Effects of RNAi inactivation of F-Box adaptors on lifespan. Lifespan experiments with long-lived mutants and wild-type worms subjected as adults to feeding RNAi for control vector (green lines), daf-16 (blue lines), and genes encoding the following F-Box adaptor proteins: lin-23 (red lines), F59B2.8 (orange lines), fbxa-121 (pink lines), and phi-3 (gray lines). (A) daf-2(mu150). Control vector: mean = 37.8 ± 0.5, n = 80/91; daf-16 RNAi: mean = 17.8 ± 0.3, n = 92/92, P < 0.0001 vs. control; lin-23 RNAi: mean = 26.7 ± 0.3, n = 78/84, P < 0.0001 vs. control; F59B2.8 RNAi: mean = 24.4 ± 0.7, n = 86/87, P < 0.0001 vs. control; fbxa-121 RNAi: mean = 25.8 ± 0.9, n = 81/86, P < 0.0001 vs. control; phi-3 RNAi: mean = 27.0 ± 0.4, n = 84/84, P < 0.0001 vs. control. (B) N2. Control vector: mean = 18.6 ± 0.8, n = 70/88; daf-16 RNAi: mean = 15.6 ± 0.4, n = 86/90, P < 0.0001 vs. control; lin-23 RNAi: mean = 18.6 ± 0.6, n = 82/94, P = 0.68 vs. control, P < 0.0001 vs. daf-16 RNAi; F59B2.8 RNAi: mean = 17.5 ± 0.5, n = 74/89, P = 0.07 vs. control, P = .06 vs. daf-16 RNAi; fbxa-121 RNAi: mean = 18.3 ± 0.7, n = 59/86, P = 0.64 vs. control, P = 0.02 vs. daf-16 RNAi; phi-3 RNAi: mean = 15.7 ± 0.5, n = 80/91, P = .007 vs. control, P = 0.31 vs. daf-16 RNAi. (C) glp-1(e2141ts). Control vector: mean = 22.4 ± 1.0, n = 82/85; daf-16 RNAi: mean = 16.1 ± 0.3, n = 88/88, P < 0.0001 vs. control; lin-23 RNAi: mean = 17.9 ± 0.4, n = 85/90, P < 0.0001 vs. control; F59B2.8 RNAi: mean = 16.8 ± 0.3, n = 85/88, P < 0.0001 vs. control; fbxa-121 RNAi: mean = 17.0 ± 0.4, n = 90/90, P < 0.0001 vs. control; phi-3 RNAi: mean = 22.2 ± 0.7, n = 92/94, P = 0.2 vs. control. (D) eat-2(ad1116). Control vector: mean = 27.3 ± 0.6, n = 70/96; lin-23 RNAi: mean = 23.0 ± 0.9, n = 85/90, P < 0.02 vs. control; F59B2.8 RNAi: mean = 16.6 ± 0.4, n = 64/96, P < 0.0001 vs. control; fbxa-121 RNAi: mean = 20.2 ± 0.9, n = 90/90, P < 0.0001 vs. control; phi-3 RNAi: mean = 29.5 ± 0.7, n = 74/94, P = 0.008 vs. control.

Fig. 5.

RNAi of cul-1 complex genes affects DAF-16 transcriptional activity. (A) daf-16(mu86); daf-2(e1370); Pdaf-16::daf-16^AM::gfp worms were subjected to RNAi treatment from day 1 of adulthood. Empty vector control (green curve): mean = 34.6 ± 0.6, n = 94/102; daf-16 RNAi (blue curve): mean = 22.7 ± 0.7; n = 84/90, P < 0.0001 vs. control; cul-1 RNAi (red curve): mean = 24.3 ± 0.3, n = 95/104, P < 0.0001 vs. control. (B–G) daf-2(e1370); Psod-3::gfp worms subjected to RNAi of cul-1 complex genes from the L4 stage through day 2 of adulthood. sod-3 expression is increased in daf-2 mutants (B) in a daf-16-dependent manner (C). This increased expression is significantly reduced in RNAi of cul-1 (D), skr-1/2 (E), and lin-23 (F). A milder, variable effect was observed on phi-3 RNAi (G). Data from multiple trials are shown in SI Table 5. Bar graph representation is found in SI Fig. 7.

Fig. 6.

A mutation in the PAPP domain of lin-23 shortens the extended lifespan of daf-2 mutants. (A and B) The lin-23(ot1) mutation shortens the extended lifespan of daf-2 mutants. (A) daf-2(e1368) (green curve): mean = 36.3 ± 1.1, n = 43/72; lin-23(ot1); daf-2(e1368) line #2 (bright red curve): mean = 21.6 ± 0.7, n = 46/68, P < 0.0001 vs. control; lin-23(ot1); daf-2(e1368) line #1 (deep red curve): mean = 23.7 ± 0.6, n = 56/86, P < 0.0001 vs. control. (B) daf-2(e1370) (green curve): mean = 55.2 ± 2.0, n = 58/86; lin-23(ot1); daf-2(e1370) line #1 (deep red curve): mean = 37.4 ± 2.2, n = 41/79, P < 0.0001 vs. control; lin-23(ot1); daf-2(e1370) line #2 (bright red curve): mean = 38.8 ± 1.6, n = 57/90, P < 0.0001 vs. control. In the above experiments, worms were grown at 20°C on normal food (OP50) for their entire life. (C) lin-23(ot1) mutants display significantly reduced lifespan extension in response to daf-2 RNAi as compared with wild-type worms. lin-23(ot1) mutants grown at 20°C on normal OP50 bacteria from hatching until L4 and then shifted to feeding RNAi bacteria expressing dsRNA for the control empty vector (bright red curve, mean = 19.1 ± 0.5, n = 62/106) and daf-2 (deep red curve, mean = 24.3 ± 1.5, n = 68/105, P < 0.0001 vs. control). N2 worms grown under similar conditions on control empty vector (bright green curve, mean = 20.7 ± 0.6, n = 58/111) and daf-2 RNAi (deep green curve, mean = 39.7 ± 1.4, n = 74/106, P < 0.0001 vs. control). Similar results were obtained when the worms were grown at 20°C during development and transferred to 25°C as adults, except with daf-2(e1370). lin-23(ot1) extended the lifespan of daf-2(e1370) mutants at 25°C (SI Table 6).