Henriques Bárbara J, Rodrigues João V, Olsen Rikke K, Bross Peter, Gomes Cláudio M
Instituto Tecnologia Química e Biológica, Universidade Nova de Lisboa, 2780-756 Oeiras, Portugal.
J Biol Chem. 2009 Feb 13;284(7):4222-9. doi: 10.1074/jbc.M805719200. Epub 2008 Dec 16.
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 ETFbeta-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 ETFbeta-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 ETFbeta-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.
编码线粒体电子传递黄素蛋白（ETF）的α亚基和β亚基以及电子传递黄素蛋白：泛醌氧化还原酶（ETF：QO）的基因突变会导致多种酰基辅酶A脱氢酶缺乏症（MADD），这是一种脂肪酸和氨基酸代谢紊乱疾病。ETF中的点突变可能会影响折叠和/或活性，与轻度和重度MADD形式均相关。在此，我们报告了对致病变体ETFβ-D128N的构象和稳定性特性的研究，以及我们关于黄素化在调节蛋白质构象稳定性和活性方面作用的发现。包括圆二色性、可见吸收、黄素和色氨酸荧光发射在内的生化和生物物理方法的组合，使得能够分析结构变化和FAD部分。ETFβ-D128N变体保留了野生型的整体折叠，但在应激条件下其黄素结合变得不那么紧密。黄素化被证明可以提高不稳定的D128N变体蛋白的构象稳定性和生物学活性。此外，黄素的存在通过避免蛋白质不稳定来防止蛋白水解消化。一名ETFβ-D128N突变纯合的患者在病毒感染和发烧时出现了严重的疾病症状。与此一致的是，我们的结果表明，突变体在高于37摄氏度的温度下热失活可能更显著。为了在体外模拟发烧情况，在39摄氏度下测试了黄素化状态。FAD发挥了药理伴侣的作用，改善了ETF构象，并产生了更稳定和活性更高的酶。我们的结果提供了一个结构和功能框架，有助于阐明从核黄素补充中获得的细胞FAD含量增加不仅在MADD的分子发病机制中，而且在一般黄素蛋白遗传疾病中可能发挥的作用。
