Role of Flavinylation in a Mild Variant of Multiple Acyl-CoA Dehydrogenation Deficiency A MOLECULAR RATIONALE FOR THE EFFECTS OF RIBOFLAVIN SUPPLEMENTATION
Role of Flavinylation in a Mild Variant of Multiple Acyl-CoA Dehydrogenation Deficiency A MOLECULAR RATIONALE FOR THE EFFECTS OF RIBOFLAVIN SUPPLEMENTATION
复制标题
DOI：
10.1074/jbc.m805719200
复制
发表时间：
2009-02-13
影响因子：
4.8
通讯作者：
Gomes, Claudio M.
中科院分区：
文献类型：
--
作者：
Henriques, Barbara J.;Rodrigues, Joao V.;Gomes, Claudio M.
编码线粒体电子转移黄素蛋白（ETF）的α亚基和β亚基以及电子转移黄素蛋白：泛醌氧化还原酶（ETF：QO）的基因的突变导致多酰基辅酶A脱氢缺乏症（MADD），这是脂肪酸和氨基酸代谢的障碍。ETF中的点突变可能损害折叠和/或活性，与轻度和重度形式的MADD相关。在这里，我们报告的调查的构象和稳定性的致病变体ETF β-D128 N，和我们的研究结果的影响，黄素化调节蛋白质构象稳定性和活性。生物化学和生物物理学方法的组合，包括圆二色性，可见吸收，黄素，色氨酸荧光发射允许的结构变化和FAD部分的分析。ETF beta-D128 N变体保留了野生型的整体折叠，但在压力条件下，其黄素的结合变得不那么紧密。黄素化显示出改善不稳定的D128 N变体蛋白的构象稳定性和生物活性。此外，黄素的存在防止蛋白水解消化，避免蛋白质不稳定。一名ETF β-D128 N突变纯合子患者出现了与病毒感染和发热相关的严重疾病症状。与此一致，我们的研究结果表明，突变体的热失活可能在37摄氏度以上的温度下更相关。为了在体外模拟发热的情况，在39 ℃下测试黄素化状态。FAD发挥药理学伴侣的作用，改善ETF构象，并产生更稳定和活性的酶。我们的研究结果提供了一个结构和功能的框架，可以帮助阐明的作用，从核黄素补充获得的细胞FAD含量增加可能发挥的分子发病机制，不仅MADD，但一般的黄素蛋白遗传疾病。
Mutations in the genes encoding the alpha-subunit and beta-subunit of the mitochondrial electron transfer flavoprotein (ETF) and the electron transfer flavoprotein: ubiquinone oxidoreductase ( ETF: QO) cause multiple acyl-CoA dehydrogenation deficiency ( MADD), a disorder of fatty acid and amino acid metabolism. Point mutations in ETF, which may compromise folding, and/or activity, are associated with both mild and severe forms of MADD. Here we report the investigation on the conformational and stability properties of the disease-causing variant ETF beta-D128N, and our findings on the effect of flavinylation in modulating protein conformational stability and activity. A combination of biochemical and biophysical methods including circular dichroism, visible absorption, flavin, and tryptophan fluorescence emission allowed the analysis of structural changes and of the FAD moiety. The ETF beta-D128N variant retains the overall fold of the wild type, but under stress conditions its flavin becomes less tightly bound. Flavinylation is shown to improve the conformational stability and biological activity of a destabilized D128N variant protein. Moreover, the presence of flavin prevented proteolytic digestion by avoiding protein destabilization. A patient homozygous for the ETF beta-D128N mutation developed severe disease symptoms in association with a viral infection and fever. In agreement, our results suggest that heat inactivation of the mutant may be more relevant at temperatures above 37 degrees C. To mimic a situation of fever in vitro, the flavinylation status was tested at 39 degrees C. FAD exerts the effect of a pharmacological chaperone, improving ETF conformation, and yielding a more stable and active enzyme. Our results provide a structural and functional framework that could help to elucidate the role that an increased cellular FAD content obtained from riboflavin supplementation may play in the molecular pathogenesis of not only MADD, but genetic disorders of flavoproteins in general.