The Structure,Function And Stability Of IDH2 Mutants In AML And Other Tumors

Isocitrate dehydrogenase(IDH)is an important role enzyme family in the Krebs cycle.There are five IDH genes in human genome,coding three kinds of isocitrate dehydrogenase(IDH1,IDH2 and IDH3).IDH2 locates in the mitochondria,catalyzing the oxidative decarboxylation of isocitrate to generate ?-ketoglutarate(?-KG)and NADPH.IDH mutations occur frequently in a variety of tumors,including acute myeloid leukemia(AML),brain tumors,and glioma.The point mutations are occurred in the active-site arginine residues of IDH,such as IDH1/R132,IDH2/R140,and IDH2/R172.IDH mutants convert ?-KG to oncometabolite 2-hydroxyglutarate(2-HG),which dysregulates a set of ?-KG-dependent dioxygenases including: methylation of histone lysine residues,inhibition of TETs methylation,activation of CpG island of DNA methylation,stable of hypoxia-inducible factor(HIF-1?)expression,and up-regulation of vascular endothelial growth factor(VEGF)level.At present,the structure,function and stability of IDH2 mutants,which are closely related to the occurrence and proliferation of cancer,have been rarely reported.This topic will provide some clues and references for understanding the molecular mechanism of tumorigenesis and proliferation of IDH2 mutation and its diagnosis and treatment by analyzing the structure,function and stability of IDH2 in different mutants.Eleven of IDH2 mammalian recombinant vectors(pCMV6)were constructed by gene cloning,overlapping PCR,double digestion and identification techniques,namely: WT,R140 G,R140Q,R140 W,R172S,R172 K,R172M,R172 W,R172G,R172 C,and R172 P.In the present studies,the differences of function and stability between IDH2 wild type and mutants were investigated by western blotting,Co-IP,cell metabolism(enzymatic activity,ROS,glucose and lactate level),and protein degradation analysis after treatment with bortezomib or 17-AAG.And the effects of IDH2 mutations on the structure were analyzed by bioinformatics homology model.The results were compared with each other to explore the differences in structure,function and stability between IDH2 wild type and mutants.The results showed that the overexpression level of mutant R140/R172 was significantly different from wild type IDH2 in 293 T and BV2 cell lines.As compared to wild type IDH2,the mutants decreased the ability to transform NADP+ to NADPH,ROS,and lactate level,but partially increased glucose level.The PI3K/AKT/mTOR signal pathway and cyclin D1 expression were inhibited after overexpression of IDH2 mutants in 293 T cell line.These results indicate that overexpression of mutant IDH2 slows down cell metabolism and growth.Overexpression of IDH2 mutants induced the expression of TNF-? and IL-6 in BV2 cell line.In the present experiment,the stability of the overexpressed wild type and mutation IDH2 was evaluated by immunoblotting after treatment with different concentrations of the ubiquitinated protein ligase inhibitor bortezomib.The results showed that there was no effects on expression wild-type IDH2 and mutant R172 M,R172W,R172 G,R172C and R172 P.However,treatment with bortezomib resulted in a dramatic degradation of the mutants including R140 G,R140Q,R140 W,R172S and R172 K.Analysis of homology modeling structure indicates that the corresponding point mutation affects the structure of the protein.In addition,targeting HSP90 by 17-AAG did little affect expression of WT and mutation IDH2 due to lack of interaction of HSP90 and IDH2.These novle findings provide important information and clue to deeply understand the molecular mechanisms of mutation IDH2 in tumorigenesis and development in cancer cells.