Natural mutations lead to enhanced proteasomal degradation of human Ncb5or, a novel flavoheme reductase.

NADH cytochrome b5 oxidoreductase (Ncb5or) protects ß-cells against oxidative stress and lipotoxicity. The predominant phenotype of lean Ncb5or-null mouse is insulin-dependent diabetes due to ß-cell death. This suggests the putative role of NCB5OR polymorphism in human diabetes. Therefore, we aimed to investigate the effect of natural missense mutations on the expression of human NCB5OR. Protein and mRNA levels of five non-synonymous coding variants were analyzed in transfected HEK293 and HepG2 cells. Although the mRNA levels were only slightly affected by the mutations, the amount of Ncb5or protein was largely reduced upon two Glu to Gly replacements in the third exon (p.E87G, p.E93G). These two mutations remarkably and synergistically shortened the half-life of Ncb5or and their effect could be attenuated by proteasome inhibitors. Our results strongly indicate that p.E87G, p.E93G mutations lead to enhanced proteasomal degradation due to manifest conformational alterations in the b5 domain. These data provide first evidence for natural mutations in NCB5OR gene resulting in decreased protein levels and hence having potential implications in human pathology.

Decreased prereceptorial glucocorticoid activating capacity in starvation due to an oxidative shift of pyridine nucleotides in the endoplasmic reticulum.

Redox state of pyridine nucleotides of the endoplasmic reticulum (ER) lumen was determined in different nutritional conditions. NADPH-dependent cortisone reduction and NADP(+)-dependent cortisol oxidation were measured in rat liver microsomes, by utilizing the luminal 11ß-hydroxysteroid dehydrogenase type 1 activity. Cortisone reduction decreased, while cortisol oxidation increased during onward starvation, showing that the luminal NADPH/NADP(+) ratio was substantially decreased. Cortisone or metyrapone addition caused a smaller decrease in NADPH fluorescence in microsomes from starved rats. The results demonstrate that nutrient supply is mirrored by the redox state of ER luminal pyridine nucleotides.