DIA-Based Proteomics Identifies IDH2 as a Targetable Regulator of Acquired Drug Resistance in Chronic Myeloid Leukemia

Drug resistance is a critical obstacle to effective treatment in patients with chronic myeloid leukemia. To understand the underlying resistance mechanisms in response to imatinib mesylate (IMA) and adriamycin (ADR), the parental K562 cells were treated with low doses of IMA or ADR for 2 months to generate derivative cells with mild, intermediate, and severe resistance to the drugs as defined by their increasing resistance index. PulseDIA-based (DIA [data-independent acquisition]) quantitative proteomics was then employed to reveal the proteome changes in these resistant cells. In total, 7082 proteins from 98,232 peptides were identified and quantified from the dataset using four DIA software tools including OpenSWATH, Spectronaut, DIA-NN, and EncyclopeDIA. Sirtuin signaling pathway was found to be significantly enriched in both ADR-resistant and IMA-resistant K562 cells. In particular, isocitrate dehydrogenase (NADP(+)) 2 was identified as a potential drug target correlated with the drug resistance phenotype, and its inhibition by the antagonist AGI-6780 reversed the acquired resistance in K562 cells to either ADR or IMA. Together, our study has implicated isocitrate dehydrogenase (NADP(+)) 2 as a potential target that can be therapeutically leveraged to alleviate the drug resistance in K562 cells when treated with IMA and ADR.     

Oxidation of ethanol by methanogenic bacteria

Methanogenium organophilum, a non-autotrophic methanogen able to use primary and secondary alcohols as hydrogen donors, was grown on ethanol. Per mol of methane formed, 2 mol of ethanol were oxidized to acetate. In crude extract, an NADP⁺-dependent alcohol dehydrogenase (ADH) with a pH optimum of about 10.0 catalyzed a rapid (5 mol/min·mg protein; 22°C) oxidation of ethanol to acetaldehyde; after prolonged incubation also acetate was detectable. With NAD⁺ only 2% of the activity was observed. F₄₂₀ was not reduced. The crude extract also contained F₄₂₀: NADP⁺ oxidoreductase (0.45 mol/min·mg protein) that was not active at the pH optimum of ADH. With added acetaldehyde no net reduction of various electron acceptors was measured. However, the acetaldehyde was dismutated to ethanol and acetate by the crude extract. The dismutation was stimulated by NADP⁺. These findings suggested that not only the dehydrogenation of alcohol but also of aldehyde to acid was coupled to NADP⁺ reduction. If the reaction was started with acetaldehyde, formed NADPH probably reduced excess aldehyde immediately to ethanol and in this way gave rise to the observed dismutation. Acetate thiokinase activity (0.11 mol/min·mg) but no acetate kinase or phosphotransacetylase activity was observed. It is concluded that during growth on ethanol further oxidation of acetaldehyde does not occur via acetylCoA and acetyl phosphate and hence is not associated with substrate level phosphorylation. The possibility exists that oxidation of both ethanol and acetaldehyde is catalyzed by ADH. Isolation of a Methanobacterium-like strain with ethanol showed that the ability to use primary alcohols also occurs in genera other than Methanogenium.Non-standard abbreviations ADH alcohol dehydrogenase - Ap₅ALi₃ P¹,P⁵-Di(adenosine-5-)pentaphosphate - DTE dithioerythritol (2,3-dihydroxy-1,4-dithiolbutane) - F₄₂₀ N-(N-l-lactyl--l-glutamyl)-l-glutamic acid phosphodiester of 7,8-dimethyl-8-hydroxy-5-deazariboflavin-5-phosphate - Mg. Methanogenium - OD₅₇₈ optical density at 578 nm - PIPES 1,4-piperazine-diethanesulfonic acid - TRICINE N-(2-hydroxy-1,1-bis[hydroxymethyl]methyl)-glycine - Tris 2-amino-2-hydroxy-methylpropane-1,3-diol - U unit (mol substrate/min)     

Circadian Variations in the Affinities of Nad Kinase and Nadp Phosphatase for their Substrates, Nad+ and Nadp+ in Dividing and Nondividing Cells of the Achlorophyllous Zc Mutant of Euglena Gracilis Klebs (Strain Z)

-We have previously shown that NAD kinase and NADP phosphatase activities display circadian rhythms, in the soluble (SN) and membrane-bound (P) fractions of crude extracts of the achlorophyllous ZC mutant of the phytoflagellate Euglena gracilis (which displays circadian rhythmicity of cell division). We determined if changes in the affinity of NADP phosphatase and NAD kinase for their substrates, NADP⁺ and NAD⁺, were occurring by calculating the ratios 100(velocity found in Km conditions/velocity found in saturating conditions). The rationale was that if the affinity remained unchanged according to circadian time (CI), these values should always equal 50, independently of any changes in enzyme quantity; values greater than 50 should indicate increases in enzyme affinity, and values less than 50 decreases in affinity. Our results indicated that these values calculated for NADP phosphatase exhibited a complex pattern of rhythmicity, while those for NAD kinase displayed circadian variations strongly correlated with the rhythms in enzyme activity. The curves showed troughs at CT 00-04 both in dividing and nondividing cells and peaks at CT 18-20 or at CT 08-14 in cells sampled, respectively, from a dividing or a stationary culture. Such variations are indicative of changes in the kinetic properties of the enzyme, which may reflect modifications in its affinity either for effectors (such as Ca2⁺-calmodulin) or for its substrate, NAD⁺. This may be due to (i) the expression of different isoenzymes at different CTs; (ii) different posttranslational modifications of the enzyme; or (iii) concentrations of effectors varying in a circadian manner.     

The role of NADPH in the regulation of glucose-6-phosphate and 6-phosphogluconate dehydrogenases in rat adipose tissue

Summary Previous studies examining the regulation of the synthesis of G6PDH and 6PGDH in rat liver and adipose tissue have focused on the induction of these enzymes by different diets and some hormones. In rat liver these enzymatic activities seem to be regulated by a mechanism involving changes in the NADPH requirements. In this paper we have studied the effect of changes in the flux through different NADPH-consuming pathways on G6PDH and 6PGDH levels in adipose tissue and on the NADPH/NADP ratio. The results show that: I) an increase in the consumption of NADPH, caused by the activation of either fatty acid synthesis or detoxification systems which consume NADPH, is paralleled by an increase in the levels of these enzymes; II) when the increase in consumption of NADPH is prevented, the G6PDH and 6PGDH levels do not change.Abbreviations G6PDH Glucose-6-Phosphate Dehydrogenase - 6PGDH 6-Phosphogluconate Dehydrogenase - GR Glutathione Reductase - ME Malic Enzyme - tBHP t-Butyl Hydroperoxide - NF Nitrofurantoin - CumOOH Cumene Hydroperoxide     

The effect of chronic alcohol feeding on lipid peroxidation in microsomes: lack of relationship to hydroxyl radical generation

Chronic alcohol feeding causes microsomal induction including increased generation of hydroxyl radicals. Ethanol induced liver injury may be mediated by lipid peroxidation for which hydroxyl radicals have been proposed as major mediators. Ethanol promotes lipid peroxidation when given acutely but also may serve as a hydroxyl radical scavenger. Therefore, we studied the acute and chronic effects of alcohol on microsomal lipid peroxidation and hydroxyl radical generation. Chronic alcohol feeding in rats increased microsomal generation of hydroxyl radicals but lipid peroxidation of endogenous lipid was inversely related to hydroxyl radical generation. Ethanol (50mM) had a slight inhibitory effect on hydroxyl radical production in peroxidizing microsomes, no effect on endogenous lipid peroxidation and enhanced the lysis of RBCs added as targets of peroxidation. Enhanced microsomal generation of hydroxyl radicals following chronic alcohol feeding is not an important mediator of lipid peroxidation.     

Eubacterial isocitrate dehydrogenase with dual specificity for NAD and NADP from Rhodomicrobium vannielii

Abstract Cell-free extracts of the photosynthetic eubacterium Rhodomicrobium vannielii contained both NADP and NAD-linked isocitrate dehydrogenase activities. Apparent K _m values of 12 μM for NADP, 0.75 mM for NAD, 9.3 μM for isocitrate (NADP utilising) and 8.2 μM for isocitrate (NAD utilising) were determined in such extracts. Four lines of evidence indicated that one enzyme was responsible for the two activities; (i) non-additivity of reaction rates in the presence of both NADP and NAD (ii) the presence of one band which stained for activity with both cofactors on non-denaturing polyacrylamide gels (iii) identical heat inactivation kinetics for both activities (iv) co-elution of both activities after ion-exchange and hydrophobic interaction chromatography. This is the first report of a eubacterial isocitrate dehydrogenase with dual cofactor specificity.     

Application of High Enzyme Activities Present in Clostridium Thermoaceticum for the Efficient Regeneration of NADPH, NADP+, NADH AND NAD+

Crude extracts of Clostridium thermoaceticum DSM 521 contain various AMAPORs (artificial mediator accepting pyridine nucleotide oxidoreductases). The specific activities of this mixture of AMAPORs is about 8-9 U mg^-1 protein (µmoles mg^-1 min^-1) for NADPH and 3-4 U mg^-1 protein for NADH formation with reduced methylviologen (MV⁺⁺) as electron donor. These AMAPOR-activities are only slightly oxygen sensitive. The reoxidation of NADPH and NADH with carboxamido-methylviologen is catalysed by crude extracts with 2.0 and 1.6 U mg^-1 protein, respectively. The same crude extracts also catalyse the dehydrogenation of reduced pyridine nucleotides with suitable quinones such as anthraquinone-2,6-disulphonate. The reduced quinone can be reoxidised by dioxygen.

The K_m-values of these enzymes for the pyridine nucleotides and also for the artificial electron mediators are in a suitable range for preparative transformations.

Furthermore the crude extract of C. thermoaceticum contains about 2.5 U mg^-1 protein of an NADP⁺-dependent formate dehydrogenase (FDH), which is suitable for NADPH and/or MV⁺⁺ regeneration. The regeneration of MV⁺⁺ with FDH and formate as electron donor proceeds with a specific activity of about 5 U mg^-1 protein of the crude extract. The reduced viologen in turn reduces NAD(P)⁺ by AMAPOR. The formate dehydrogenase is sensitive to oxygen.

Examples of compounds which have been prepared by combination of AMAPORs or formate dehydrogenase with an oxidoreductase are: (S)-3-hydroxycarboxylates, esters of (S)-3-hydroxycarboxylates, (1R,2S)-1-hydroxypropane-tricarboxylate (D_s-(+)-isocitrate), L_s-(-)-isocitrate and 6-phosphogluconate.     

Mutations of surface residues in Anabaena vegetative and heterocyst ferredoxin that affect thermodynamic stability as determined by guanidine hydrochloride denaturation.

The stability properties of oxidized wild-type (wt) and site-directed mutants in surface residues of vegetative (Vfd) and heterocyst (Hfd) ferredoxins from Anabaena 7120 have been characterized by guanidine hydrochloride (Gdn-HCl) denaturation. For Vfd it was found that mutants E95K, E94Q, F65Y, F65W, and T48A are quite similar to wt in stability. E94K is somewhat less stable, whereas E94D, F65A, F65I, R42A, and R42H are substantially less stable than wt. R42H is a substitution found in all Hfds, and NMR comparison of the Anabaena 7120 Vfd and Hfd showed the latter to be much less stable on the basis of hydrogen exchange rates (Chae YK, Abildgaard F, Mooberry ES, Markley JL, 1994, Biochemistry 33:3287-3295); we also find this to be true with respect to Gdn-HCl denaturation. Strikingly, the Hfd mutant H42R is more stable than the wt Hfd by precisely the amount of stability lost in Vfd upon mutating R42 to H (2.0 kcal/mol). On the basis of comparison of the X-ray crystal structures of wt Anabaena Vfd and Hfd, the decreased stabilities of F65A and F65I can be ascribed to increased solvent exposure of interior hydrophobic groups. In the case of Vfd mutants E94K and E94D, the decreased stabilities may result from disruption of a hydrogen bond between the E94 and S47 side chains. The instability of the R42 mutants is also most probably due to decreased hydrogen bonding capabilities.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ferredoxin—ferredoxin NADP reductase interaction: Catalytic differences between the soluble and thylakoid-bound complex

Ferredoxin-NADP reductase (FNR) and ferredoxin form a complex when the former is membrane-bound as they do when both components are in solution, with the same dissociation constant. The rate constant of NADP photoreduction, first order with respect to the complex, is more than 20-times higher when FNR is membrane-bound than when the enzyme is in solution. The Arrhenius activation energy is identical in both conditions. These observations are interpreted in terms of ‘entropic catalysis’ of NADP reduction by the thylakoid-bound FNR.