A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signalling.

Mechanisms that integrate the metabolic state of a cell with regulatory pathways are necessary to maintain cellular homeostasis. Endogenous, intrinsically reactive metabolites can form functional, covalent modifications on proteins without the aid of enzymes1,2, and regulate cellular functions such as metabolism3-5 and transcription6. An important 'sensor' protein that captures specific metabolic information and transforms it into an appropriate response is KEAP1, which contains reactive cysteine residues that collectively act as an electrophile sensor tuned to respond to reactive species resulting from endogenous and xenobiotic molecules. Covalent modification of KEAP1 results in reduced ubiquitination and the accumulation of NRF27,8, which then initiates the transcription of cytoprotective genes at antioxidant-response element loci. Here we identify a small-molecule inhibitor of the glycolytic enzyme PGK1, and reveal a direct link between glycolysis and NRF2 signalling. Inhibition of PGK1 results in accumulation of the reactive metabolite methylglyoxal, which selectively modifies KEAP1 to form a methylimidazole crosslink between proximal cysteine and arginine residues (MICA). This posttranslational modification results in the dimerization of KEAP1, the accumulation of NRF2 and activation of the NRF2 transcriptional program. These results demonstrate the existence of direct inter-pathway communication between glycolysis and the KEAP1-NRF2 transcriptional axis, provide insight into the metabolic regulation of the cellular stress response, and suggest a therapeutic strategy for controlling the cytoprotective antioxidant response in several human diseases

Accuracy of prenatal diagnosis of X-linked hypohidrotic ectodermal dysplasia by tooth germ sonography

Objective: X-linked hypohidrotic ectodermal dysplasia (XLHED), a developmental disorder characterized by malformation of hair, teeth, and sweat glands, results from defective ectodysplasin A1 (EDA1) caused by EDA mutations. Inability to sweat, the major problem of XLHED which can lead to life-threatening hyperthermia, has been shown to be amenable to intrauterine therapy with recombinant EDA1. The aim of this retrospective study was to evaluate the diagnostic accuracy of tooth germ sonography to identify affected fetuses in pregnant women with EDA mutations. Methods: Tooth germ sonography was performed in 38 cases at 10 study sites between gestational weeks 18 and 28. XLHED was diagnosed if fewer than six tooth germs were detected in mandible and/or maxilla. In all subjects, diagnoses were verified postnatally by EDA sequencing and/or clinical findings (standardized clinical assessments of hair, sweating, and dentition; orthopantomograms). Estimated weights of 12 affected male fetuses and postnatal weight gain of 12 boys with XLHED were assessed using appropriate growth charts. Results: In 19 of 38 sonografic examinations of 23 male and 13 female fetuses, a prenatal diagnosis of XLHED was made. The diagnosis proved to be correct in 37 cases; one affected male fetus was missed. Specificity and positive predictive value were both 100%. Tooth counting by clinical assessment corresponded well with radiografic findings. We observed no weight deficits of subjects with XLHED in utero but occasionally during infancy. Conclusions: Tooth germ sonography is highly specific and reliable in establishing a prenatal diagnosis of XLHED

Cytosolic NADPH balancing in Penicillium chrysogenum cultivated on mixtures of glucose and ethanol

The in vivo flux through the oxidative branch of the pentose phosphate pathway (oxPPP) in Penicillium chrysogenum was determined during growth in glucose/ethanol carbon-limited chemostat cultures, at the same growth rate. Non-stationary 13C flux analysis was used to measure the oxPPP flux. A nearly constant oxPPP flux was found for all glucose/ethanol ratios studied. This indicates that the cytosolic NADPH supply is independent of the amount of assimilated ethanol. The cofactor assignment in the model of van Gulik et al. (Biotechnol Bioeng 68(6):602–618, 2000) was supported using the published genome annotation of P. chrysogenum. Metabolic flux analysis showed that NADPH requirements in the cytosol remain nearly the same in these experiments due to constant biomass growth. Based on the cytosolic NADPH balance, it is known that the cytosolic aldehyde dehydrogenase in P. chrysogenum is NAD +  dependent. Metabolic modeling shows that changing the NAD + -aldehyde dehydrogenase to NADP + -aldehyde dehydrogenase can increase the penicillin yield on substrate

Cycloheximide resistant incorporation of amino acids into a polypeptide of the cytochrome oxidase of Neurospora crassa

Radioaetive leueine was ineorporated by N eurospora crassa mitoehondria in vivo in the presence of cyeloheximide. When the membrane protein of these mitochondria was ehromatographieally separated on oleyl polymethaerylie aeid resin, & nurober of fraetions were obtained whieh differ with respeet to their eontents of radioaetivity and eytoehromes. The highest speeifie radioaetivity was found in the fraction eontaining eytoehrome aa3• This fraetion proved to be a pure and enzymatically aetive cytoehrome oxidase. Its ratio of absorbanee at 280 nm (ox)/ 443 nm (red.) was 2.1. By means of sodium dodeeylsulfate gel-electrophoresis, this enzymewas separated into five polypeptides with molecular weights of 30000, 20000, 13000, 10000, and 8000. Only the polypeptide with the molecular weight 20000 displayed a high specific radioaetivity

Incorporation of amino acids into mitochondrial protein of the flight muscle of Locusta migratoria in vitro and in vivo in the presence of cycloheximide

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