Integrated pharmaco-proteogenomics defines two subgroups in isocitrate dehydrogenase wild-type glioblastoma with prognostic and therapeutic opportunities.

The prognostic and therapeutic relevance of molecular subtypes for the most aggressive isocitrate dehydrogenase 1/2 (IDH) wild-type glioblastoma (GBM) is currently limited due to high molecular heterogeneity of the tumors that impedes patient stratification. Here, we describe a distinct binary classification of IDH wild-type GBM tumors derived from a quantitative proteomic analysis of 39 IDH wild-type GBMs as well as IDH mutant and low-grade glioma controls. Specifically, GBM proteomic cluster 1 (GPC1) tumors exhibit Warburg-like features, neural stem-cell markers, immune checkpoint ligands, and a poor prognostic biomarker, FKBP prolyl isomerase 9 (FKBP9). Meanwhile, GPC2 tumors show elevated oxidative phosphorylation-related proteins, differentiated oligodendrocyte and astrocyte markers, and a favorable prognostic biomarker, phosphoglycerate dehydrogenase (PHGDH). Integrating these proteomic features with the pharmacological profiles of matched patient-derived cells (PDCs) reveals that the mTORC1/2 dual inhibitor AZD2014 is cytotoxic to the poor prognostic PDCs. Our analyses will guide GBM prognosis and precision treatment strategies.

A unique homodimeric NAD⁺-linked isocitrate dehydrogenase from the smallest autotrophic eukaryote Ostreococcus tauri.

In eukaryotes, NAD(+)-dependent isocitrate dehydrogenase (IDH) is strictly mitochondrial and is a key enzyme in the Krebs cycle. To date, all known NAD(+)-specific IDHs (NAD-IDHs) in the mitochondria are believed to be heteromeric in solution. Here, a unique homodimeric NAD-IDH from Ostreococcus tauri (OtIDH), the smallest autotrophic picoeukaryote, was unveiled. Active OtIDH has a molecular weight of ∼93 kDa with each subunit of 46.7 kDa. In the presence of Mn(2+) and Mg(2+), OtIDH displayed 42-fold and 51-fold preference for NAD(+) over NADP(+), respectively. Interestingly, OtIDH exhibited a sigmoidal kinetic behavior in response to isocitrate unlike other homodimeric homologs, and a remarkably high affinity for isocitrate (S0.5 < 10 µM) unlike other hetero-oligomeric homologs. Furthermore, its coenzyme specificity can be completely converted from NAD(+) (ancient trait) to NADP(+) (adaptive trait) by rational mutagenesis based on the evolutionary trace. Mutants D344R and D344R/M345H displayed a 15-fold and 72-fold preference for NADP(+) over NAD(+), respectively, indicating that D344 and M345 are the determinants of NAD(+) specificity. These findings also suggest that OtIDH may be an ancestral form of type II IDHs (all reported members are NADP(+)-linked enzymes) and may have evolved into NADP(+)-dependent IDH for adaptation to the increased demand of NADPH under carbon starvation.

Functional relevance of dynamic properties of Dimeric NADP-dependent Isocitrate Dehydrogenases.

BACKGROUND: Isocitrate Dehydrogenases (IDHs) are important enzymes present in all living cells. Three subfamilies of functionally dimeric IDHs (subfamilies I, II, III) are known. Subfamily I are well-studied bacterial IDHs, like that of Escherischia coli. Subfamily II has predominantly eukaryotic members, but it also has several bacterial members, many being pathogens or endosymbionts. subfamily III IDHs are NAD-dependent. The eukaryotic-like subfamily II IDH from pathogenic bacteria such as Mycobacterium tuberculosis IDH1 are expected to have regulation similar to that of bacteria which use the glyoxylate bypass to survive starvation. Yet they are structurally different from IDHs of subfamily I, such as the E. coli IDH. RESULTS: We have used phylogeny, structural comparisons and molecular dynamics simulations to highlight the similarity and differences between NADP-dependent dimeric IDHs with an emphasis on regulation. Our phylogenetic study indicates that an additional subfamily (IV) may also be present. Variation in sequence and structure in an aligned region may indicate functional importance concerning regulation in bacterial subfamily I IDHs. Correlation in movement of prominent loops seen from molecular dynamics may explain the adaptability and diversity of the predominantly eukaryotic subfamily II IDHs. CONCLUSION: This study discusses possible regulatory mechanisms operating in various IDHs and implications for regulation of eukaryotic-like bacterial IDHs such as that of M. tuberculosis, which may provide avenues for intervention in disease.

Structural basis of the substrate specificity of bifunctional isocitrate dehydrogenase kinase/phosphatase.

Isocitrate dehydrogenase kinase/phosphatase (AceK) regulates entry into the glyoxylate bypass by reversibly phosphorylating isocitrate dehydrogenase (ICDH). On the basis of the recently determined structure of the AceK-ICDH complex from Escherichia coli, we have classified the structures of homodimeric NADP(+)-ICDHs to rationalize and predict which organisms likely contain substrates for AceK. One example is Burkholderia pseudomallei (Bp). Here we report a crystal structure of Bp-ICDH that exhibits the necessary structural elements required for AceK recognition. Kinetic analyses provided further confirmation that Bp-ICDH is a substrate for AceK. We conclude that the highly stringent AceK binding sites on ICDH are maintained only in Gram-negative bacteria.

Mild reductions in mitochondrial NAD-dependent isocitrate dehydrogenase activity result in altered nitrate assimilation and pigmentation but do not impact growth.

Transgenic tomato (Solanum lycopersicum) plants were generated expressing a fragment of the mitochondrial NAD-dependent isocitrate dehydrogenase gene (SlIDH1) in the antisense orientation. The transgenic plants displayed a mild reduction in the activity of the target enzyme in the leaves but essentially no visible alteration in growth from the wild-type. Fruit size and yield were, however, reduced. These plants were characterized by relatively few changes in photosynthetic parameters, but they displayed a minor decrease in maximum photosynthetic efficiency (Fv/Fm). Furthermore, a clear reduction in flux through the tricarboxylic acid (TCA) cycle was observed in the transformants. Additionally, biochemical analyses revealed that the transgenic lines exhibited considerably altered metabolism, being characterized by slight decreases in the levels of amino acids, intermediates of the TCA cycle, photosynthetic pigments, starch, and NAD(P)H levels, but increased levels of nitrate and protein. Results from these studies show that even small changes in mitochondrial NAD-dependent isocitrate dehydrogenase activity lead to noticeable alterations in nitrate assimilation and suggest the presence of different strategies by which metabolism is reprogrammed to compensate for this deficiency.

Isolation of active and inactive forms of isocitrate dehydrogenase from Escherichia coli ML 308.

In Escherichia coli ML308 isocitrate dehydrogenase is partially inactivated during growth on acetate [Bennett, P.M. and Holms, W.H. (1975) J. Gen. Microbiol. 87, 37-51]. The active form of isocitrate dehydrogenase was purified to homogeneity from cells grown on glycerol. The key step in the procedure was chromatography on procion-red-Sepharose, from which the enzyme was specifically eluted with NADP+. Two forms of isocitrate dehydrogenase were purified to homogeneity from cells grown on acetate. One form did not bind to procion-red-Sepharose and was essentially inactive; this form could be resolved from the active form by non-denaturing gel electrophoresis. The other form was specifically eluted from procion-red-Sepharose and was partially active; analysis of this form by non-denaturing gel electrophoresis suggested that it was a mixture of the active and inactive forms. The three forms comigrated on denaturing gel electrophoresis and were identical by the criterion of one-dimensional peptide mapping. Analysis of the active and inactive forms by sedimentation equilibrium centrifugation and non-denaturing gel electrophoresis showed that they differed in charge but not in size. Amino acid analysis and two-dimensional peptide mapping showed that both forms were dimers of identical subunits. The active form of the enzyme contained no detectable alkali-labile phosphate, the inactive form contained 0.8 molecule/subunit and the partially active form contained an intermediate amount. The data suggest that the active and inactive forms of isocitrate dehydrogenase differ only in the presence of one phosphate group per subunit in the latter form; this is consistent with our results from phosphorylation of isocitrate dehydrogenase in vitro (Following paper in this journal). The nature of the partially active form of isocitrate dehydrogenase and the significance of the results are discussed.