O-GlcNAcylation,Phosphorylation And Acetylation Regulate USP1 Binding To MET1 And ECR

Research background,existing scientific questions and research significanceRetinoid X Receptor(RXR)is a type of receptor in the nucleus,which can directly or indirectly regulate gene expression by forming homologous/heterodimer complexes with other receptors,and participate in various metabolic and physiological processes such as glucose metabolism and cholesterol transport.Therefore,RXR is considered as a potential drug target for treating diseases such as diabetes,neurological diseases and tumors.However,the specific mechanism by which RXR binds to different receptors is still unclear.Post-Translational Modifications(PTMs)occur during or after protein translation and can be modifications to the charge and conformation of the protein through the covalent binding of the donor group to the protein,so as to quickly adjust the function of the protein and complete the cell’s response to internal and external signals.Adapt to changes in the physiological state of the body.At present,more than 600 posttranslational modifications have been discovered.Among them,post-translational modifications such as O-linked N-acetylglucosamine(O-GlcNAc)modification(OGlcNAcylation),phosphorylation,acetylation,succinylation,ubiquitination and methylation have been extensively studied.Therefore,it is worth considering whether the binding of RXR to different receptors is regulated by post-translational modifications,what are the relationships between these modifications,and what are their upstream regulatory mechanisms.Metamorphosis is an important mode of insect adaptation to the environment.Metamorphosis development is regulated cooperatively by Juvenile hormone(JH)and 20-hydroxyecdysone(20E).During feeding,JHⅢ binds to methoprene tolerant protein 1(MET1)and phosphorylated heat shock protein 90(Hsp90)and ultraspiracle(USP)form a complex that binds to the JH response element(kJHRE)and promotes the expression of Krhl and other genes to maintain larval status.In the metamorphosis stage,20E titer increases,binds with ecdysone nuclear receptor(ECR).forms transcription complex with USP,and binds with ecdysone response element(ECRE).The downstream gene expression of 20E signaling pathway was activated to promote the development of larvae metamorphosis.USP,which can bind to both MET1 and ECR,is the direct homolog of RXR in insects and receives JH and 20E signaling pathways respectively.Previous laboratory work demonstrated that a species of USP was identified in the genome of Helicoverpa armigera,which was highly similar to other species USP1,so it was named USP1.Helicoverpa armigera USP1 Ser21 is phosphorylated by PKC and Lys303 is acetylated.both of which are related to the interaction of USP1 and ECR.It can be seen that USP1 can not only bind to a variety of receptors to form dimers mediating different hormone signaling pathways,but also has a variety of post-translational modifications,which is an ideal model to explore the regulation of post-translational modifications on the binding of RXR to different receptors.Based on this,in this paper,the metamorphosis development process of complete metamorphosis insect Helicoverpa armigera(cotton bollworm)was taken as the research object to explore the selection of different post-translational modifications regulating USP1 interacting proteins,the relationship between various modifications and the upstream regulation mechanism.The results of this study can improve the object selection mechanism of RXR forming heterodimers,and provide reference for drug development and pest control,which is of great significance in theory and practice.ResultsWestern blot results showed that the USP1 O-GlcNAcylation was higher in feeding stage,while the phosphorylation and acetylation modification were higher in metamorphosis stage.20E induced the decrease of O-GlcNAcylation and the increase of phosphorylation at Ser21 in USP1.Interference with β-N-acetylglucosamine glycoside hydrolase(OGA),which is responsible for removing O-GlcNAc,reduces the O-GlcN Acylation level in USP 1.Co-immunoprecipitation showed that OGICNAcylated USP1 was bound to MET1.phosphorylated USP1 was less bound to MET1 and more bound to ECR.After the phosphorylation modification was removed by mutation at Serine 21,the acetylation level of USP 1 remained unchanged,while the phosphorylation level of USP1 remained unchanged after mutation at lysine 303.Interference of several acetylation enzymes was performed to detect changes in the acetylation state of USP1.The results showed that compared with E1A binding protein p300(Ep300)gene.K(lysine)acetyltransferase 2A(KAT2A)gene.K(lysine)acetyltransferase 5(KAT5)gene.knockdown of Mitochondrial Acetyl-CoA Acetyltransferase 1(ACAT1)gene significantly inhibited 20E-induced acetylation of USP1.ACAT1 expression levels did not change during feeding and metamorphosis,but the level of succinvlation was reduced.When lysine at 238 was mutated to arginine,it could bind to USP1 and acetylate it.When it was mutated to glutamic acid,it could not bind to USP1 and could not acetylate USP1.Interfering SIR2-Related Enzyme 5(SIRT5),ACAT1 succinylation cannot be removed.ConclusionUSP1 is O-GlycNacylated during larval feeding stage and interacts with MET1.Phosphorylation and acetylation occur during metamorphosis and interact with ECR.Both O-GlcNAcylation and phosphorylation occurred at Ser21 of USP1.and the relationship between the two is negative.Phosphorylation and acetylation are independent of each other.During the metamorphosis phase,OGA causes USP1 to remove O-GlcNAc and thus phosphorylation,while SIRT5 causes ACAT1 to be desuccinylated and USP1 to undergo acetylation.