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Evidence that the metabolite repair enzyme NAD(P)HX epimerase has a moonlighting function

ABSTRACT: NAD(P)H-hydrate epimerase (EC 5.1.99.6) is known to help repair NAD(P)H hydrates (NAD(P)HX), which are damage products existing as R and S epimers. The S epimer is reconverted to NAD(P)H by a dehydratase; the epimerase facilitates epimer interconversion. Epimerase deficiency in humans causes a lethal disorder attributed to NADHX accumulation. However, bioinformatic evidence suggests caution about this attribution by predicting that the epimerase has a second function connected to vitamin B6 (pyridoxal 5'-phosphate and related compounds). Specifically, (i) the epimerase is fused to a B6 salvage enzyme in plants, (ii) epimerase genes cluster on the chromosome with B6-related genes in bacteria, and (iii) epimerase and B6-related genes are coexpressed in yeast and Arabidopsis. The predicted second function was explored in Escherichia coli, whose epimerase and dehydratase are fused and encoded by the yjeF gene. The putative NAD(P)HX epimerase active site has a conserved lysine residue (K192 in E. coli YjeF). Changing this residue to alanine cut in-vitro epimerase activity by ≥95% but did not affect dehydratase activity. Mutant cells carrying the K192A mutation had essentially normal NAD(P)HX levels, showing that the mutation had very little or no effect on NAD(P)HX repair in vivo. However, these cells showed metabolome changes, particularly in amino acids, that exceeded those in cells lacking the entire yjeF gene. The K192A mutant cells also had lower levels of free pyridoxal 5'-phosphate than wild-type cells. These results provide strong circumstantial evidence that the epimerase has a metabolic function beyond NAD(P)HX repair and that this function involves vitamin B6.

ORGANISM(S): Escherichia Coli E. Coli

TISSUE(S): Cells

SUBMITTER: Oliver Fiehn

PROVIDER: ST000900 | MetabolomicsWorkbench | Tue Nov 14 00:00:00 GMT 2017

REPOSITORIES: MetabolomicsWorkbench

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Project description:Background and aims: In chronic HBV infection elevated ROS levels by dysfunctional mitochondria can cause increased protein oxidation, proteostasis engulfment and DNA damage in exhausted virus-specific CD8 T cells. Aim of this study was to understand how these defects are mechanistically interconnected, to further elucidate T cell exhaustion pathogenesis and to design novel T cell-based therapies. Methods: DNA damage and DNA repair mechanisms, including parylation, CD38 expression, histone acetylation and telomere length were studied by flow cytometry in dextramer-stained HBV-specific CD8 T cells from chronic HBV patients. FLU-specific CD8 cells from the same patients and healthy subjects served as controls. Correction of intracellular signaling alterations and improvement of anti-viral T cell functions by the NAD precursor NMN and by CD38 inhibition, was assessed by fluorescent probes and antibodies in flow cytometry Results: Elevated DNA damage was associated with defective DNA repair processes, including NAD-dependent parylation, in HBV-specific CD8 cells of chronic HBV patients. NAD depletion in these patients was indicated by the overexpression of CD38, the major NAD consumer, and by the significant improvement of DNA repair mechanisms, mitochondrial and proteostasis functions and epigenetic regulation by NAD supplementation, which could also improve the overall HBV-specific antiviral CD8 T cell function. Conclusions: Our study delineates a model of CD8 T cell exhaustion whereby multiple functionally interconnected intracellular defects, including telomere shortening, are causally related to NAD depletion suggesting similarities between T cell exhaustion and cell senescence. Correction of these deregulated intracellular functions by NAD supplementation can also restore anti-viral CD8 T cell activity and thus represents a promising potential therapeutic strategy for chronic HBV infection. Gene expression profiling analysis using data derived from low input RNA-seq of patients' virus-specific CD8 T cells either upon in vitro culture or ex-vivo isolated from total PBMCs

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Evidence that the metabolite repair enzyme NAD(P)HX epimerase has a moonlighting function

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