The O-GlcNAc-modication Of SKN-1 Regulates C.elegans Lifespan And Oxidative Stress Resistance

Aging is a natural and complex physiological process in cells,tissues,organs,and even individuals.Many physiological,biochemical and molecular events are proven to happen in this process,including genome damage induced by oxidative stress and imbalance of protein stability.Caenorhabditis elegans(C.elegans)is regarded as an ideal model animal for studying aging,because of its short lifespan of only 21 days.In C.elegans,SKN-1(Skinhead-1)is a major transcription factor,which plays an important role in regulating oxidative stress resistance and aging.As a homologue of Nrf1/Nrf2 protein in vertebrates,SKN-1 belongs to the family of Cap 'n' Collar(Cnc)bZIP transcription factor.It is well-known that the activity of SKN-1 is mainly regulated by phosphorylation.The phosphorylation of SKN-1 modulates its nuclear accumulation in the intestines and target gene expression.The evidence has shown that AKT-1/-2 kinase phosphorylates SKN-1 at serine 12,which prevents SKN-1 to localize to the nuclei of intestinal cells and decreases the worms' ability of oxidative stress resistance and anti-aging.In addition,phosphorylation of SKN-1 on serine 483 catalyzed by GSK-3 also inhibits the trans-localization of SKN-1 into the intestinal nuclei.In contrast,the phosphorylation of SKN-1 at serine 74 and 340 induced by P38-MAPK signaling pathway is required for the accumulation of SKN-1 in intestinal nuclei.O-GlcNAcylation is a conserved posttranslational modification from worms to mammals.As both phosphorylation and O-GlcNAcylation occur at serine and threonine residues,protein O-GlcNAcylation has been proven to have an extensive cross talk with the protein phosphorylation recently.In C.elegans,two enzymes contribute to O-GlcNAc modification of target proteins.One is O-GlcNActransferase(OGT-1),which is responsible for adding O-GlcNAc modification on the target proteins.The other is O-GlcNAcase OGA-1,which removes this modification from the proteins.It has been reported that loss of ogt-1 or oga-1 changes the lifespan of worms.However,the targets of O-GlcNAc modification involved in regulating aging remain unclear to date.In this study,we first discovered a novel post-translational modification of SKN-1,O-GlcNAc modification.We found the changes of O-GlcNAc level in C.elegans influenced the intestinal nuclear accumulation of SKN-1 and SKN-1's target genes expressions.GST-pulldown and co-IP results showed that OGT-1 interacted with SKN-1 directly.Moreover,in vitro O-GlcNAcylation assay and mass spectrometric(MS)analysis showed that SKN-1was O-GlcNAcylated at Ser470 and Thr493 by OGT-1.We then performed the mutations of Ser470 and Thr493 on SKN-1 in the wild-type and skn-1(zu67)worms respectively,and found that Ser470/Thr493 double mutations repressed the accumulation of SKN-1 in the intestinal nuclei,and the functions of SKN-1 on anti-aging and oxidative stress resistance.Moreover,in response to the oxidative stress,SKN-1 was highly O-GlcNAcylated by OGT-1,which resulted in the decrease of GSK-3-mediated phosphorylation at Ser483.Taken together,we found O-GlcNAc modification of SKN-1 positively regulates the activity of SKN-1 in maintaining the normal lifespan and the ability of oxidative stress resistance.These findings have important implications for studying the functions of O-GlcNAcylation on the homolog Nrf-2 in human aging-related diseases.