In vitro visualization and characterization of wild type and mutant IDH homo- and heterodimers using Bimolecular...

Mutations in the metabolic enzyme isocitrate dehydrogenase (IDH) were recently found in ~80% of WHO grade II-III gliomas and secondary glioblastomas. These mutations reduce the enzyme's ability to convert isocitrate to α-ketoglutarate and, instead, confer a novel gain-of-function resulting in the conversion of α-ketoglutarate to 2-hydroxglutarate (2-HG). However, IDH mutations exist in a heterozygous state such that a functional wild type allele is retained. Recent data suggest that the ability of mutant IDH1, but not mutant IDH2, to produce 2-HG is dependent on the activity of the retained wild type allele. In this study, we aimed to further our understanding of the interaction and function of wild type and mutant IDH heterodimers utilizing Bimolecular Fluorescence Complementation (BiFC). Dimerization of wild type and mutant IDH monomers conjugated to the N- and C-terminus of Venus protein, respectively, is directly proportional to the amount of fluorescence emitted and can be used as an approach to visualize and assess IDH dimerization. Thus, we utilized this method to visualize IDH homo- and heterodimers and to examine their cellular physiology based on subcellular localization, NADPH production, and 2-HG levels. Our results demonstrate that wild type and mutant IDH1 or IDH2 heterodimers display similar physiological characteristics to that of mutant IDH1 or IDH2 homodimers with the exception of their ability to generate NADPH. IDH1 heterodimers consistently generate NADPH whereas IDH2 heterodimers do not. However, the presence of mutant IDH1 or IDH2 in homo- or heterodimer configurations consistently generates equivalent levels of 2-HG. Our data suggest that the wild type protein is not required for the generation of 2-HG.