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See detailNAD(P)HX dehydratase (NAXD) deficiency: a novel neurodegenerative disorder exacerbated by febrile illnesses
Van Bergen, Nicole; Guo, Yiran; Rankin, Julia et al

in Brain: a Journal of Neurology (2019), 142(1), 50-58

Physical stress, including high temperatures, may damage the central metabolic nicotinamide nucleotide cofactors [NAD(P)H], generating toxic derivatives [NAD(P)HX]. The highly conserved enzyme NAD(P)HX ... [more ▼]

Physical stress, including high temperatures, may damage the central metabolic nicotinamide nucleotide cofactors [NAD(P)H], generating toxic derivatives [NAD(P)HX]. The highly conserved enzyme NAD(P)HX dehydratase (NAXD) is essential for intracellular repair of NAD(P)HX. Here we present a series of infants and children who suffered episodes of febrile illness-induced neurodegeneration or cardiac failure and early death. Whole-exome or whole-genome sequencing identified recessive NAXD variants in each case. Variants were predicted to be potentially deleterious through in silico analysis. Reverse-transcription PCR confirmed altered splicing in one case. Subject fibroblasts showed highly elevated concentrations of the damaged cofactors S-NADHX, R-NADHX and cyclic NADHX. NADHX accumulation was abrogated by lentiviral transduction of subject cells with wild-type NAXD. Subject fibroblasts and muscle biopsies showed impaired mitochondrial function, higher sensitivity to metabolic stress in media containing galactose and azide, but not glucose, and decreased mitochondrial reactive oxygen species production. Recombinant NAXD protein harbouring two missense variants leading to the amino acid changes p.(Gly63Ser) and p.(Arg608Cys) were thermolabile and showed a decrease in Vmax and increase in KM for the ATP-dependent NADHX dehydratase activity. This is the first study to identify pathogenic variants in NAXD and to link deficient NADHX repair with mitochondrial dysfunction. The results show that NAXD deficiency can be classified as a metabolite repair disorder in which accumulation of damaged metabolites likely triggers devastating effects in tissues such as the brain and the heart, eventually leading to early childhood death. [less ▲]

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See detailNAD(P)HX repair deficiency causes central metabolic perturbations in yeast and human cells
Becker-Kettern, Julia UL; Paczia, Nicole UL; Conrotte, Jean-François UL et al

in FEBS Journal (2018)

NADHX and NADPHX are hydrated and redox inactive forms of the NADH and NADPH cofactors, known to inhibit several dehydrogenases in vitro. A metabolite repair system that is conserved in all domains of ... [more ▼]

NADHX and NADPHX are hydrated and redox inactive forms of the NADH and NADPH cofactors, known to inhibit several dehydrogenases in vitro. A metabolite repair system that is conserved in all domains of life and that comprises the two enzymes NAD(P)HX dehydratase and NAD(P)HX epimerase, allows reconversion of both the S- and R-epimers of NADHX and NADPHX to the normal cofactors. An inherited deficiency in this system has recently been shown to cause severe neurometabolic disease in children. Although evidence for the presence of NAD(P)HX has been obtained in plant and human cells, little is known about the mechanism of formation of these derivatives in vivo and their potential effects on cell metabolism. Here, we show that NAD(P)HX dehydratase deficiency in yeast leads to an important, temperature-dependent NADHX accumulation in quiescent cells with a concomitant depletion of intracellular NAD+ and serine pools. We demonstrate that NADHX potently inhibits the first step of the serine synthesis pathway in yeast. Human cells deficient in the NAD(P)HX dehydratase also accumulated NADHX and showed decreased viability. In addition, those cells consumed more glucose and produced more lactate, potentially indicating impaired mitochondrial function. Our results provide first insights into how NADHX accumulation affects cellular functions and pave the way for a better understanding of the mechanism(s) underlying the rapid and severe neurodegeneration leading to early death in NADHX repair deficient children. [less ▲]

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See detailStudy of Metabolite Repair in Eukaryotic Cells: Metabolic origin and fate of D-2-hydroxyglutarate in yeast and effect of NAD(P)HX repair deficiency on yeast and human cells
Becker-Kettern, Julia UL

Doctoral thesis (2017)

Abnormal metabolites, which are useless and can even be toxic, are constantly generated inside the cell by unwanted chemical reactions or by enzymatic side reactions. Metabolite repair enzymes clean the ... [more ▼]

Abnormal metabolites, which are useless and can even be toxic, are constantly generated inside the cell by unwanted chemical reactions or by enzymatic side reactions. Metabolite repair enzymes clean the metabolite pool from these molecules. The proportion of proteins annotated as metabolite repair enzymes is currently very small but accumulating evidence suggests that a bigger part might be hidden among proteins of unknown function. The aim of this thesis was to study two of these metabolite repair systems and their physiological relevance in more detail as their importance is well illustrated through implication in disease processes. D-2-hydroxyglutaric aciduria, a severe human neurometabolic disorder, can be caused by a deficiency in the metabolite repair enzyme D-2-hydroxyglutarate (D-2HG) dehydrogenase. Higher levels of D-2HG have also been observed in cancerous cells with a mutated form of isocitrate dehydrogenase. Strikingly, in the model organism Saccharomyces cerevisiae, 2-hydroxyglutarate metabolism had remained completely unexplored. We elucidated the metabolic pathways involved in D-2HG formation and degradation in yeast using bioinformatics, metabolomics, yeast genetics, and classical biochemical tools. We discovered that Dld3, currently annotated as a D-lactate dehydrogenase, actually degrades D-2HG to α-ketoglutarate while reducing pyruvate to D-lactate, thereby acting as a transhydrogenase. We also demonstrated that the yeast phosphoglycerate dehydrogenases Ser3 and Ser33 are major sources for D-2HG formation. These findings paved the way to integrate 2HG and its associated genes into the yeast metabolic network and might help, on the long-term, to better understand underlying mechanisms in human disease as well. Other recently identified metabolite repair enzymes, NAD(P)HX dehydratase and NAD(P)HX epimerase (encoded in yeast by the YKL151C and YNL200C genes, respectively), specifically act on NADHX and NADPHX, hydrated and inactive forms of the central NADH and NADPH cofactors. Although extensively biochemically characterized, the physiological importance of these two enzymes still remains largely unclear. Only very recently, case reports were published indicating a correlation between NAD(P)HX repair deficiency and severe neuropathological symptoms starting in early childhood upon events of febrile illnesses and rapidly leading to a fatal outcome. We systematically analyzed extracts of NAD(P)HX repair deficient yeast and human cells using HPLC and LC-MS/MS methods. This enabled us to demonstrate that NADHX and NADPHX can be formed intracellularly. In the yeast system, NADHX accumulation, which could be modulated by the cultivation temperature, was accompanied by a decrease in intracellular NAD+ levels. Furthermore, we showed that NADHX interferes with serine metabolism by inhibiting the first step of the main synthesis pathway of this amino acid. In the human cell system, NAD(P)HX dehydratase deficiency led, as in yeast, to intracellular NADHX accumulation, but also to a marked decrease in cell viability after prolonged cultivation times. This is, to our knowledge, the first report about the effect of NADHX accumulation on cellular metabolism. Expanding our experimental strategy of combined transcriptomics and metabolomics approaches to the human cell model might ultimately lead to the discovery of the disease-causing cellular process. The findings in both projects led to an unexpected connection between NAD(P)HX and 2HG metabolism via the yeast homologues of 3-phoshpoglycerate dehydrogenase, Ser3 and Ser33. Both proteins catalyze the oxidation of 3-phosphoglycerate to 3-phosphohydroxypyruvate in the initial step of de novo serine biosynthesis with a concomitant reduction of α-ketoglutarate to D-2-hydroxyglutarate. By acting as transhydrogenases, they substantially, even though not exclusively, contribute to D-2HG formation in yeast. The very same enzymes were strongly inhibited in vitro and, as suggested by our findings, also in vivo by the presence of NADHX, leading to serine depletion in NAD(P)HX repair deficient cells. [less ▲]

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See detailSaccharomyces cerevisiae Forms D-2-Hydroxyglutarate and Couples its Degradation to D-Lactate Formation via a Cytosolic Transhydrogenase.
Becker-Kettern, Julia UL; Paczia, Nicole UL; Conrotte, Jean-Francois et al

in The Journal of Biological Chemistry (2016)

The D or L form of 2-hydroxyglutarate (2HG) accumulates in certain rare neurometabolic disorders and high D-2HG levels are also found in several types of cancer. Although 2HG has been detected in ... [more ▼]

The D or L form of 2-hydroxyglutarate (2HG) accumulates in certain rare neurometabolic disorders and high D-2HG levels are also found in several types of cancer. Although 2HG has been detected in Saccharomyces cerevisiae, its metabolism in yeast has remained largely unexplored. Here we show that S. cerevisiae actively forms the D enantiomer of 2HG. Accordingly, the S. cerevisiae genome encodes two homologs of the human D-2HG dehydrogenase: Dld2, which, as its human homolog, is a mitochondrial protein, and the cytosolic protein Dld3. Intriguingly, we found that a dld3Delta knockout strain accumulates millimolar levels of D-2HG, while a dld2Delta knockout strain displayed only very moderate increases in D-2HG. Recombinant Dld2 and Dld3, both currently annotated as D-lactate dehydrogenases, efficiently oxidized D-2HG to alpha-ketoglutarate. Depletion of D-lactate levels in the dld3Delta, but not in the dld2Delta mutant, led to the discovery of a new type of enzymatic activity, carried by Dld3, to convert D-2HG to alpha-ketoglutarate, namely an FAD-dependent transhydrogenase activity using pyruvate as a hydrogen acceptor. We also provide evidence that Ser3 and Ser33, which are primarily known for oxidizing 3-phosphoglycerate in the main serine biosynthesis pathway, in addition reduce alpha-ketoglutarate to D-2HG using NADH and represent major intracellular sources of D-2HG in yeast. Based on our observations, we propose that D-2HG is mainly formed and degraded in the cytosol of S. cerevisiae cells in a process that couples D-2HG metabolism to the shuttling of reducing equivalents from cytosolic NADH to the mitochondrial respiratory chain via the D-lactate dehydrogenase Dld1. [less ▲]

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