Fitting wwp-1 in the dietary restriction network

Abstract

One of the most universal environmental conditions that impacts longevity is dietary restriction (DR). DR is a reduction in caloric uptake without malnutrition; DR can increase health and life span in many organisms, from yeast to invertebrates and mammals. Studies in C. elegans have identified novel genetic regulators of DR, including transcription factors (TFs) and environmental sensors, such as pha-4, skn-1, ampk, and hsf-1. In addition, our group uncovered for the first time a role for the ubiquitin pathway in DR-mediated longevity by identifying the C. elegans wwp-1 HECT E3 ligase and its associated E2 ubiquitin conjugating enzyme, ubc-18, as positive regulators of lifespan in response to DR. 1 E2 and E3 enzymes facilitate the ubiquitylation of substrate proteins to alter their function in various ways. Therefore, the identification of targets of UBC-18/WWP-1 would yield an understanding of how this E2/E3 complex regulates the response to nutrient intake and longevity in worms.To identify substrates of WWP-1 that regulate DR-induced longevity, we performed a targeted RNAi screen by selecting worm orthologues of known ubiquitylated substrates of the mammalian WWP-1 orthologues-WWP1, WWP2, and ITCH. 2 None of the 12 genes we analyzed was able to extend lifespan in wild-type (WT) worms when knocked-down. However, we found that loss of one gene, the Kr€ uppel-like TF klf-1, was able to suppress the extended longevity of a DR genetic model (eat-2 mutant animals) while not affecting the lifespan of WT worms. Like wwp-1, we found that klf-1 was essential for the DR-longevity response, and that overexpression of klf-1 in the intestines, a site where longevity cues are expressed, led to lifespan extension dependent on wwp-1. Using biochemical approaches, we were able to demonstrate a direct physical interaction between WWP-1 and KLF-1 that results in the multi-monoubiquitylation of KLF-1 both in vitro and in vivo. 2 Together, our data support a model in which modulation of KLF-1 by WWP-1 regulates DR-mediated longevity. While the mechanism through which KLF-1 ubiquitylation drives lifespan extension is not known, it is reasonable to speculate that multi-monoubiquitylation modifies KLF-1 transcriptional output, possibly altering how nutrient stores are utilized in the worm. Given that wwp-1 is evolutionarily conserved and that mammalian WWP-1 orthologues target multiple KLFs, 3 we can speculate that a similar signaling pathway regulates DR-induced longevity in higher organisms.Many mutations or treatments that prolong lifespan in C. elegans also increase resistance to environmental stressors such as heat, UV, and oxidative conditions. Previously, we found that loss of wwp-1 decreased stress resistance in the adult worm. 1 To our surprise, reduced klf-1 expression did not affect the stress response in worms. Also in unpublished results, we found that loss of pha-4, which we had shown to be downstream of both wwp-1 and klf-1, 1,2 caused no change in stress resistance in worms, much like loss of klf-1. This would sugges...