Study On The Mechanism Of E3 Ubiquitin Ligase UHRF1 Regulating Autophagy

Autophagy is a catabolic process in eukaryotes,whose degradation station is the lysosomes.Cells initiate autophagy by sensing the lack of nutrients or stress in the external environment.Autophagy provides energy sources,like carbon,nitrogen,to resist adverse living environments by degrading excess organelles and long-lived proteins.The induction of autophagy requires the expression of a series of autophagy-related genes.The autophagic flux includes autophagosome formation,membrane vesicle fusion,and finally autophagosome-lysosome fusion.The function of lysosomes requires V-ATPase,a proton pump located on the cell vesicle membrane.The V-ATPase on the lysosome membrane transports H~+against the concentration gradient to maintain the acidic internal environment of the lysosome,which is the basis for the lysosome function.Ubiquitin-like containing PHD and Ring Finger1(UHRF1)is an epigenetic regulatory factor with multiple domains.UHRF1 interacts with several proteins and non-protein molecules through the cooperation between its multiple domains,thereby changing the DNA methylation status or histone modification codes,to regulate the expression of related genes.The RING domain gives its E3 ubiquitin ligase activity,the SRA domain makes it to bind hemi-methylated DNA,the TTD domain and PHD domain makes it to recognize histone methylation modifications.Its distribution is tissue-specific and cell-specific.In low-differentiated and rapidly proliferating cells,the expression level of UHRF1 is higher.In cells with high-differentiation and no proliferation ability,it is almost undetectable.In a variety of cancer cells,abnormal expression of UHRF1 has been found.Knockout of UHRF1 caused death during pregnancy in mouse.Current studies have shown that UHRF1 is involved in the regulation of many process,including cell proliferation and differentiation,embryonic development,DNA damage repair,cancer occurrence and development.However,whether UHRF1 is involved in the regulation of autophagy and its phosphorylation modification role remains to be explored.c-Abl is a non-receptor tyrosine kinase widely expressed in mammals,distributed in the subcellular structures including cytoplasm,nucleus,endoplasmic reticulum,mitochondria,actin cytoskeleton,lipid rafts,etc.c-Abl kinase is a multifunctional protein involved in many cellular processes,such as cell proliferation and differentiation,DNA damage response,actin dynamics,autophagy and apoptosis.The function of c-Abl is strictly regulated when participating in these physiological conditions.Under physiological conditions,the kinase activity of c-Abl is inhibited to be in a state of self-inhibition.DNA damage and other stress signals can activate c-Abl kinase.Disorders of kinase activity can lead to diseases.Previous studies in our laboratory found that c-Abl kinase can regulate the assembly of V-ATPase.In the presence of c-Abl,the expression level of V-ATPase V1B subunits is higher.Two important E3 ubiquitin ligases,UHRF1 and RNF40,were discovered,when investigating the factors which influence the stability of the V1B subunit.These results indicated that UHRF1 or RNF40 may mediated the degradation of the V1B subunit through the ubiquitin-proteasome pathway.Further immunoprecipitation experiments found that UHRF1 and V1B2 subunits have a significant interaction,but we did not detect the interaction of RNF40 and V1B2 subunit.Subsequently,we tested the ubiquitination level of the V1B subunits when we knocked down UHRF1 and overexpressed UHRF1,and determined that UHRF1 is the E3 ubiquitin ligase of the V1B subunit.In this study,the result of immunoprecipitation revealed that both UHRF1 and c-Abl can interact with V1B subunits,and c-Abl can mediate the phosphorylation of UHRF1.Redundant V-ATPase subunits,such as V1B,may be degraded through the UHRF1-mediated ubiquitination pathway when the intracellular environment is stable and the supply of intracellular lysosomes can meet the needs of maintaining cell homeostasis.When autophagy signals such as starvation are generated,c-Abl tyrosine kinase is activated to mediate the phosphorylation UHRF1.On the one hand,the interaction of V1B subunit and UHRF1 was inhibited to reduce the ubiquitination of V1B subunits,and thus facilitate the assembly and maturation of V-ATPase.On the other hand,the starve condition can reduce the stability of UHRF1 itself and promote its degradation.The degradation of UHRF1 may further promote the transcription of autophagy-related genes such as ulk1?atg5?atg12?lc3,to increase the intensity of autophagic flux and resist stress such as starvation.This study reveals a new mechanism of autophagy through lysosomal V-ATPase activity regulation and UHRF1 mediated epigenetic regulation,which is expected to provide molecular mechanisms and intervention strategies for the occurrence of tumor,neurodegenerative,and metabolic diseases.