NAD(P)HX repair deficiency causes central metabolic perturbations in yeast and human cells

• Published in: bioRxiv
• Main Authors: Becker-Kettern, Julia, Paczia, Nicole, Jean-Fran ois Conrotte, Zhu, Chenchen, Fiehn, Oliver, Jung, Paul P, Steinmetz, Lars M, Linster, Carole L
• Format: Paper
• Language: English
• Published: Cold Spring Harbor Cold Spring Harbor Laboratory Press 05.07.2018
• Subjects: Children; Cofactors; Epimerase; Fitness; Gene expression; Glyceraldehyde-3-phosphate dehydrogenase; Growth conditions; High temperature; High-performance liquid chromatography; Intracellular; Lactic acid; Metabolism; Metabolites; Mitochondria; NAD; NADH; NADP; Neurodegeneration; Plant cells; Redox properties; Serine; Yeast
• DOI: 10.1101/302257

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. Footnotes * - New Table 1: NAD(P)HX dehydratase deficiency decreases intracellular NAD+ levels and thereby changes the cellular redox state. - Old table 1 now new Table 2: Top 10 significantly changed genes with most different expression levels between wild-type and ykl151c(delta) strains. - Figure 1: NADHX accumulates during the postdiauxic phase in yeast strains lacking the NAD(P)HX dehydratase Ykl151c. --> Figure legend updated to clarify sampling points and conditions - Figure 2: NADHX levels increase at elevated temperature but not upon glyceraldehyde-3-phosphate dehydrogenase (GAPDH) overexpression. --> Panel C and D removed, Figure legend and Figure description within text updated - New Supplemental Figure S1: Identification of NADHX derivatives in yeast extracts by HPLC-UV. - Old Supplemental Figure 1 now new Supplemental Figure S2 (with structural information on NADHX derivatives added): NADHX accumulation in yeast cells deficient in NAD(P)HX dehydratase. - New Supplemental Figure S3: Identification of NADHX and NADPHX derivatives in yeast extracts by LC-MS. - New Supplemental Figure S4: NAD(P)HX repair deficiency does not affect NAD+ levels in human HAP1 cells. - Discussion section: NAD(P)HX repair deficiency leads to discrete changes in gene expression and central carbon metabolism in yeast --> updated to emphasize importance of serine metabolism in yeast - Supplemental Table S1: Tested growth conditions for NAD(P)HX repair deficient yeast strains. --> updated to clarify details on growth and stress conditions as well as fitness read out - minor (stylistic) modifications on text more detailed chemical names