The Skp1 Homologs SKR-1/2 Are Required for the Caenorhabditis elegans SKN-1 Antioxidant/Detoxification Response Independently of p38 MAPK

Abstract

SKN-1/Nrf are the primary antioxidant/detoxification response transcription factors in animals and they promote health and longevity in many contexts. SKN-1/Nrf are activated by a remarkably broad-range of natural and synthetic compounds and physiological conditions. Defining the signaling mechanisms that regulate SKN-1/Nrf activation provides insights into how cells coordinate responses to stress. Nrf2 in mammals is regulated in part by the redox sensor repressor protein named Keap1. In C. elegans, the p38 MAPK cascade in the intestine activates SKN-1 during oxidative stress by promoting its nuclear accumulation. Interestingly, we find variation in the kinetics of p38 MAPK activation and tissues with SKN-1 nuclear accumulation among different pro-oxidants that all trigger strong induction of SKN-1 target genes. Using genome-wide RNAi screening, we identify new genes that are required for activation of the core SKN-1 target gene gst-4 during exposure to the natural pro-oxidant juglone. Among 10 putative activators identified in this screen was skr-1/2, highly conserved homologs of yeast and mammalian Skp1, which function to assemble protein complexes. Silencing of skr-1/2 inhibits induction of SKN-1 dependent detoxification genes and reduces resistance to pro-oxidants without decreasing p38 MAPK activation. Global transcriptomics revealed strong correlation between genes that are regulated by SKR-1/2 and SKN-1 indicating a high degree of specificity. We also show that SKR-1/2 functions upstream of the WD40 repeat protein WDR-23, which binds to and inhibits SKN-1. Together, these results identify a novel p38 MAPK independent signaling mechanism that activates SKN-1 via SKR-1/2 and involves WDR-23.

NHR‐49/HNF4 integrates regulation of fatty acid metabolism with a protective transcriptional response to oxidative stress and fasting

“…Although the transcription factors DAF‐16, HSF‐1, HLH‐30, and HIF‐1 activate a variety of stress/pathogen‐protective responses, SKN‐1 is especially important to maintain redox balance and defend against oxidative stress caused by xenobiotics that target GSH and cause protein unfolding (Blackwell et al., 2015; Miranda‐Vizuete & Veal, 2016). Nevertheless, distinct responses are initiated in response to different oxidants (Blackwell et al., 2015; Wu, Deonarine, Przybysz, Strange & Choe, 2016). For example, the organic peroxide tert‐butyl‐hydroperoxide (tBOOH) elicits a transcriptional response that is largely skn‐1 ‐independent (Oliveira et al., 2009).…”

A Genetic Analysis of the Caenorhabditis elegans Detoxification Response

“…Finally, loss of xrep-4 activity had no effect on constitutive gst-4::gfp in the skn-1(gof) mutants ( Figure 5C), whereas expression was strongly eliminated by targeting skn-1 itself by RNAi (Supplemental Figure 2B), confirming a previous report (Paek et al, 2012). Different exogenous toxins have been shown to elicit the Phase II detoxification response through distinct pathways (Wu et al, 2016), although all converge on the regulation of SKN-1; our results demonstrate that XREP-4 also functions through SKN-1. Taken together, our findings place XREP-4 at an upstream nodal point that senses and/or triggers the Phase II detoxification pathway in response to both endogenous and exogenous toxins, with the primary response limited to either bodywall muscle or pharyngeal, hypodermal, and intestinal tissues, respectively.…”

“…SKR-1 and -2, nearly identical proteins, are related to the SCF ubiquitin ligase complex member Skp-1, a known Fbox interacting protein (Nayak et al, 2002;Yamanaka et al, 2002). SKR-1/2 have been linked to the regulation of gst-4 expression via WDR-23 and SKN-1 (Wu et al, 2016), and WDR-23 has been shown to interact with a CUL-4 SCF ubiquitin ligase complex to regulate nuclear SKN-1 levels and activity (Choe et al, 2009). …”

“…Treatment with oxidative stressors like sodium arsenite activates p38 MAP kinase and genetic interference with the p38 pathway prevents both SKN-1 nuclear accumulation and impairs resistance to oxidative stress (Blackwell et al, 2015). However, recent evidence suggests that the WDR-23 dependent localization of SKN-1 may be independent of p38 MAPK signaling (Wu et al, 2016). …”

“…We XREP-4 has been shown to interact with SKR-1, a protein encoded by the skrubiquitination/protein degradation (Boxem et al, 2008). A genome-wide RNAi screen to identify novel regulators that are required for activation of gst-4 during exposure to the electrophile juglone identified skr-1/2 as the only members of this multigene family that were required in this assay (Wu et al, 2016). Based on these observations, we carried out RNAi inactivation of skr-1/2 and found that, like xrep-4 inactivation, gst-4::gfp expression was blocked by skr-1/2 depletion in the alh-6 endogenous stress mutant background.…”