Study On The Role Of OGT Mediated O-GlcNAcylation Of NSL3 In MtDNA Transcription Regulation

Glycosylation modification of O-GlcNAc plays an important role in energy substances and catabolism,which is an important post translational modification of proteins by adding or removing the nucleotide sugar donor UDP-GlcNAc to or from serine or threonine residues.UDP-GlcNAc is generated through the hexosamine biosynthesis pathway(HBP),which combines glucose,amino acids,fatty acids,and nucleotide metabolism.Therefore,the level of O-GlcNAcylation is closely dependent on the concentration of the nucleotide sugar donor UDP-GlcNAc.That is,fluctuations in the concentration of UDP-GlcNAc in cells are determined by changes in the nutrient levels entering the cells.Therefore,OGlcNAcylation modification is also considered to be a sensitive nutritional sensor.In cells,O-GlcNAc chemical groups are added by OGT(O-GlcNAc transfer)and removed by OGA(O-GlcNAcase).Both OGT and OGA enzymes synergistically regulate the level of O-GlcNAcylation modification in cells.It has been reported that NSL(non specific lethal)histone acetyltransferase complexes can acetylate the Lys-5,Lys-8,and Lys-16 sites on histone H4.Interestingly,OGT participates in the composition of the NSL complex as a subunit component.In our previous studies,we found that OGT not only can bind to the key subunit NSL3 in the NSL complex,but also can glycosylate the NSL3T755 site,thereby stabilizing the structure and overall enzyme activity of the NSL complex.In addition,it has been reported that NSL3 can assist MOF in entering mitochondria and binding to the D-loop region of mitochondrial DNA(mtDNA),thereby affecting mtDNA transcription.This thesis aims to explore the mechanism of O-GlcNAcylation modification of NSL3 in mtDNA transcription regulation by studying the correlation between OGlcNAcylation modification of NSL3 and mtDNA transcription.The main work and results of this paper are as follows:1.O-GlcNAcylation,as an intracellular nutrient receptor,is extremely sensitive to changes in intracellular glucose levels.In order to investigate the response of OGlcNAcylation modification of NSL3 to cellular nutrition levels,we tested the OGlcNAcylation modification level of NSL3 in 293 T cells by changing the glucose concentration in the cell culture medium.The results showed that the O-GlcNAcylation modification level of NSL3 changes due to changes in glucose concentration.The results showed that the level of O-GlcNAcylation modification in NSL3 increased with increasing glucose concentration,suggesting that O-GlcNAcylation modification in NSL3 is closely related to glucose metabolism.2.According to literature reports,NSL3,as a key subunit in the NSL complex,participates in regulating mtDNA transcription by assisting MOF to enter mitochondria.This paper explores the effect of O-GlcNAcylation modification of NSL3 on mtDNA transcription regulation.Firstly,we used immunofluorescence staining and cell subcomponent separation experiments to clarify the location of NSL complexes in mitochondria.At the same time,we used O-GlcNAc glycosylase OGA activity inhibitor TMG to increase the level of O-GlcNAcylation modification in the whole cell.Through subcomponent separation experiments,it was confirmed that increasing the level of OGlcNAcylation modification can promote the recruitment of NSL3 in mitochondria;Secondly,by overexpressing wild-type NSL3 plasmid and NSL3Thr755 glycosylation site mutant plasmid,it was demonstrated that O-GlcNAcylation modification of NSL3 can also promote the entry of NSL3 into mitochondria;Finally,in order to further explore the impact of O-GlcNAcylation modification of NSL3 on its transcription in mtDNA,we used realtime quantitative PCR experiments to prove that increasing the level of O-GlcNAcylation modification in the whole cell or increasing the level of O-GlcNAcylation modification of NSL3 can promote mtDNA transcription,suggesting that mtDNA transcription is at least partially dependent on O-GlcNAcylation modification of NSL3.3.In order to clarify the effect of O-GlcNAcylation modification of NSL3 on mitochondrial function and cell growth status,we conducted JC-1 staining and cell scratch experiments after transfecting wild-type NSL3 and NSL3 Thr755 mutant plasmids in Hela cells.The results showed that wild-type NSL3 group could stabilize mitochondrial membrane potential and inhibit cell migration ability,It is suggested that O-GlcNAcylation modification at NSL3 Thr755 site plays an important role in stabilizing mitochondrial membrane potential and maintaining mitochondrial related biological functions.4.The glycolytic metabolic pathway(Warburg effect)is often preferred in rapidly proliferating cancer cells.The above experimental results confirm that O-GlcNAcylation modification of NSL3 can inhibit the migration ability of cancer cells.We conducted protein expression analysis using the GEPIA database and found that the expression of NSL3 varies significantly among different cancers.Analysis of the patient survival profile found that low expression of NSL3 can promote cancer prognosis,suggesting that NSL3 also plays an important role in the occurrence and development of cancer.In summary,we speculate that NSL3 may sense the external nutritional environment through O-GlcNAcylation modification,and affect the recruitment of NSL3 in mitochondria and regulate the transcription of mtDNA through the stability of OGlcNAcylation modified proteins at the Thr755 site of NSL3.The experimental results of this paper provide a new theoretical basis for regulating mtDNA transcription,and also provide new effective and feasible potential targets for the treatment of mitochondrial related diseases.