The Mechanistic Study Of Deubiquitinating Enzyme USP9X In The Regulation Of Circadian Rhythm

Background and Aims:Circadian rhythm regulates hundreds of functions and plays an important role in the synchronization of organism behaviors adapted to the internal or external environments.The regular circadian rhythm benefits the adaptation of organism and the stability of internal environment.Thus,it is significant to ensure the stable regulation of circadian rhythm for daily life activity.The generation of circadian rhythm is based on the oscillation level of circadian genes and their protein products in the transcription-translation feedback loops(TTFL).Several reports have found that some E3 ligases such as SCFFBXL3/21,SCF?-TrCP1/2,UBE3A,Siah2,and HUWE1,and deubiquitinating enzymes(DUBs),USP2 and USP7,which regulated the ubiquitination and stability of circadian proteins,and circadian behavior through ubiquitin-proteasome system(UPS).These results showed the importance of UPS in the regulation of circadian system.However,previous studies were mainly focused on the negative limb of the circadian TTFL and the function of ubiquitination.Only a couple of DUBs have been discovered to regulate core circadian proteins.Here,we studied the interactors of BMAL1 and looked for one DUB which might affect the stability and biology function of BMAL1.Methods:Firstly,co-immunopurification(Co-IP)and label-free quantitative shotgun proteomics were used for the purification and detection of circadian core protein BMAL1 interactors.USP9X,which might interact with BMAL1 was identified and their interaction was validated by pulldown and immunoblotting experiments.Then,we explored the effect of USP9X on the expression level and stability of BMAL1 protein,and its ubiquitination level after USP9X was expressed or knocked down in HEK293T cells in the presence of a proteasome inhibitor MG 132,which blocks the degradation of ubiquitinated proteins,or in the presence of CHX which could inhibit the protein biosynthesis.Moreover,dual-luciferase reporter assay was used to study the regulation of USP9X on BMAL1 transcriptional activity for a downstream gene PER1.USP9X was also knocked down in N2a cells for the detection of the transcriptional levels of other circadian genes.At last,the influence of USP9X on circadian behaviors such as circadian amplitude,period,and phase shift was explored using LumiCycle experiments.Results:The interaction between USP9X and BMAL1 which was detected by mass spectrometric analysis was further validated by immunoprecipitation(IP)and immunoblotting experiments.The biochemical experiments showed that USP9X expression up-regulated BMAL1 level,and enhanced its stability by reducing BMAL1 ubiquitination level.In contrast,USP9X knockdown could increase BMAL1 ubiquitination level and down-regulate BMAL1 protein level.In the exploration of USP9X on the biological function of BMAL1,we found that USP9X expression enhanced BMAL1 transcriptional activity,which increased the expression of BMAL1 downstream genes,whereas USP9X knockdown significantly attenuated the mRNA level of several BMAL1 downstream genes,such as PER1.The LumiCycle experiment also suggested the reduced amplitude of circadian rhythm after USP9X was knocked down.Conclusions:We detected and validated the interaction between USP9X and BMAL1 by mass spectrometric and immunoblotting analyses.USP9X regulated BMAL1 ubiquitination level,increased its expression level,and enhanced its stability.USP9X could also affect BMAL1 transcriptional activity for its downstream genes,as well as the amplitude of circadian rhythm.Our experiments expanded the previous research on the function of ubiquitination in the regulation of circadian rhythm,which provided a new direction for the regulation and stabilization of circadian behavior.