The Role Of The Integrity Of Zinc Finger Motifs In PML Protein Degradation By Arsenic Trioxide

Objectives:The first objective is to investigate the mechanism of zinc finger motifs in the Ring domain and B-box(1&2)motifs of PML protein on this protein degradation after exposure to arsenic trioxide(As2O3,it is hydrolyzed as arsenite,iAs?,in water,hereinafter referred to as arsenic).And we tried to confirm the necessity of the zinc finger on PML protein degradation by chelating its zinc ions or destroying the zinc finger structure in each domain.Then,we observed the nuclear bodies(NBs)formation and SUMOylation of PML mutations,the aim was to explain why mutant PML proteins are resistant to arsenic trioxide in clinical treatment.Methodology:The zinc ions present in the zinc finger motifs of PML protein were chelated by 1,10-Phenanthroline monohydrate(Zn2+ chelater),and exposed to AS2O3(i.e.,iAs?).Western blot(WB)was used to detect normal and mutant PML proteins solubility changes along with degradation following exposure to arsenic.Additionally,proteins from cells were extracted by RIPA buffer to obtain the supernatant(Soluble)and Pellet(Insoluble)fractions.Confocal laser scanning microscopy was used to observe the formation of nuclear bodies and SUMOylation.And then,cysteine or histidine residues in the Ring,B-box 1 and B-box 2 domains of PML protein were point mutated to other amino acids by site-directed mutagenesis and then mutant PML proteins(PML-? and ?)solubility changes as well as degradations were determined by Western Blot.Furthermore,we predicted zinc finger structures in each region of PML protein after point mutation by site directed mutagenesis.At last,different mutant PML genes were transfected into HEK293T cells,then the cells were treated with As2O3 to determine the PML-NBs formation and PML-SUMOylation.Confocal laser scanning microscopy was used to determine the formation of PML-NBs and the recruitment of its partner protein(e.g.,SUMO-1).Besides,we also tried to explain that why the structural integrity of zinc finger motifs in PML protein played an important role in arsenic-induced PML degradation in acute promyelocytic leukemia(APL).Results:In the first part of the study,we found that pre-treatment of the Zinc ions chelator,1,10-phenanthroline abolished the formation of PML-NBs and recruitment of SUMO-1 into its PML nuclear bodies after treatment with arsenic trioxide.In the second part,it was found that PML IV and PML V overexpressed 293T and Hela cells pretreated with Zinc ions chelator(i.e.,1,10-phenanthroline)resulted in inhibiting the PML proteins shift from the Supernatant(soluble)to Pellet(Insoluble)fraction as compared with cells treated with iAs? alone,indicating that the Zinc ions or Zinc Finger motifs are necessary for PML protein degradation by iAs?,and this data was also consistent with the result of NB4 cells.In the third part,we clearly observed that although the mutant PML proteins could form PML NBs as like normal PML NBs,but SUMOylation of PML protein was found to be delayed by its mutations.Conclusion:According to the above results,it can be concluded that the integrity of the zinc finger structures in PML played a crucial role in degradation of these PML proteins on treatment with arsenic trioxide,furthermore it can be suggested that the integrity of Zinc fingers directly influences the transfer of PML proteins from nucleoplasm to nuclear matrix and also have an effect on the formation of PML nuclear bodies.