Cystic fibrosis is mostly caused by the F508del mutation, which impairs CFTR protein from exiting the endoplasmic reticulum due to misfolding. VX-809 is a small molecule that rescues F508del-CFTR localization, which recently went into clinical trial but with unknown mechanism of action (MoA). Herein, we assessed if VX-809 is additive or synergistic with genetic revertants of F508del-CFTR, other correctors, and low temperature to determine its MoA. We explored and integrated those various agents in combined treatments, showing how they add to each other to identify their complementary MoA upon correction of F508del-CFTR. Our experimental and modeling data, while compatible with putative binding of VX-809 to NBD1:ICL4 interface, also indicate scope for further synergistic F508del-CFTR correction by other compounds at distinct conformational sites/cellular checkpoints, thus suggesting requirement of combined therapies to fully rescue F508del-CFTR.

Mutations in the genes encoding the ␣-subunit and ␤-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␤-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␤-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␤-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°C. To mimic a situation of fever in vitro, the flavinylation status was tested at 39°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.

Riboflavin, commonly known as vitamin B2, is the precursor of flavin cofactors. It is present in our typical diet, and inside the cells it is metabolized to FMN and FAD. As a result of their rather unique and flexible chemical properties these flavins are among the most important redox cofactors present in a large series of different enzymes. A problem in riboflavin metabolism or a low intake of this vitamin will have consequences on the level of FAD and FMN in the cell, resulting in disorders associated with riboflavin deficiency. In a few number of cases, riboflavin deficiency is associated with impaired oxidative folding, cell damage and impaired heme biosynthesis. More relevant are several studies referring reduced activity of enzymes such as dehydrogenases involved in oxidative reactions, respiratory complexes and enzymes from the fatty acid β-oxidation pathway. The role of this vitamin in mitochondrial metabolism, and in particular in fatty acid oxidation, will be discussed in this review. The basic aspects concerning riboflavin and flavin metabolism and deficiency will be addressed, as well as an overview of the role of the different flavoenzymes and flavin chemistry in fatty acid β-oxidation, merging clinical, cellular and biochemical perspectives. A number of recent studies shedding new light on the cellular processes and biological effects of riboflavin supplementation in metabolic disease will also be overviewed. Overall, a deeper understanding of these emerging roles of riboflavin intake is essential to design better therapies.

No abstract

Protein misfolding is a hallmark of a number of metabolic diseases, in which fatty acid oxidation defects are included. The latter result from genetic deficiencies in transport proteins and enzymes of the mitochondrial β-oxidation, and milder disease conditions frequently result from conformational destabilization and decreased enzymatic function of the affected proteins. Small molecules which have the ability to raise the functional levels of the affected protein above a certain disease threshold are thus valuable tools for effective drug design. In this work we have investigated the effect of mitochondrial cofactors and metabolites as potential stabilizers in two β-oxidation acyl-CoA dehydrogenases: short chain acyl-CoA dehydrogenase and the medium chain acyl-CoA dehydrogenase as well as glutaryl-CoA dehydrogenase, which is involved in lysine and tryptophan metabolism. We found that near physiological concentrations (low micromolar) of FAD resulted in a spectacular enhancement of the thermal stabilities of these enzymes and prevented enzymatic activity loss during a 1h incubation at 40°C. A clear effect of the respective substrate, which was additive to that of the FAD effect, was also observed for short- and medium-chain acyl-CoA dehydrogenase but not for glutaryl-CoA dehydrogenase. In conclusion, riboflavin may be beneficial during feverish crises in patients with short- and medium-chain acyl-CoA dehydrogenase as well as in glutaryl-CoA dehydrogenase deficiencies, and treatment with substrate analogs to butyryl- and octanoyl-CoAs could theoretically enhance enzyme activity for some enzyme proteins with inherited folding difficulties.