Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, POB 260, 1444 Budapest, Hungary.
Biochimie. 2013 Jul;95(7):1403-10. doi: 10.1016/j.biochi.2013.03.004. Epub 2013 Mar 19.
NADH cytochrome b5 oxidoreductase (Ncb5or) protects β-cells against oxidative stress and lipotoxicity. The predominant phenotype of lean Ncb5or-null mouse is insulin-dependent diabetes due to β-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.
烟酰胺腺嘌呤二核苷酸（NADH）细胞色素 b5 氧化还原酶（Ncb5or）可保护β细胞免受氧化应激和脂毒性的侵害。瘦素型 Ncb5or 基因敲除小鼠的主要表型为胰岛素依赖性糖尿病，原因是β细胞死亡。这表明 NCB5OR 多态性可能与人类糖尿病有关。因此，我们旨在研究天然错义突变对人 NCB5OR 表达的影响。在转染的 HEK293 和 HepG2 细胞中分析了五个非同义编码变异体的蛋白和 mRNA 水平。尽管突变仅略微影响 mRNA 水平，但在第三个外显子中两个 Glu 突变为 Gly（p.E87G，p.E93G）会大大降低 Ncb5or 蛋白的含量。这两个突变显著协同地缩短了 Ncb5or 的半衰期，并且其作用可以通过蛋白酶体抑制剂减弱。我们的研究结果强烈表明，p.E87G、p.E93G 突变导致 b5 结构域明显的构象改变，从而导致蛋白酶体降解增强。这些数据首次为 NCB5OR 基因的天然突变导致蛋白水平降低提供了证据，这可能对人类病理学具有潜在影响。
