Molecular mechanisms of riboflavin responsiveness in patients with ETF-QO variations and multiple acyl-CoA dehydrogenation deficiency

Abstract

Riboflavin-responsive forms of multiple acyl-CoA dehydrogenation deficiency (RR-MADD) have been known for years, but with presumed defects in the formation of the flavin adenine dinucleotide (FAD) co-factor rather than genetic defects of electron transfer flavoprotein (ETF) or electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO). It was only recently established that a number of RR-MADD patients carry genetic defects in ETF-QO and that the well-documented clinical efficacy of riboflavin treatment may be based on a chaperone effect that can compensate for inherited folding defects of ETF-QO. In the present study, we investigate the molecular mechanisms and the genotype-phenotype relationships for the riboflavin responsiveness in MADD, using a human HEK-293 cell expression system. We studied the influence of riboflavin and temperature on the steady-state level and the activity of variant ETF-QO proteins identified in patients with RR-MADD, or non- and partially responsive MADD. Our results showed that variant ETF-QO proteins associated with non- and partially responsive MADD caused severe misfolding of ETF-QO variant proteins when cultured in media with supplemented concentrations of riboflavin. In contrast, variant ETF-QO proteins associated with RR-MADD caused milder folding defects when cultured at the same conditions. Decreased thermal stability of the variants showed that FAD does not completely correct the structural defects induced by the variation. This may cause leakage of electrons and increased reactive oxygen species, as reflected by increased amounts of cellular peroxide production in HEK-293 cells expressing the variant ETF-QO proteins. Finally, we found indications of prolonged association of variant ETF-QO protein with the Hsp60 chaperonin in the mitochondrial matrix, supporting indications of folding defects in the variant ETF-QO proteins.

FAD binding overcomes defects in activity and stability displayed by cancer-associated variants of human NQO1

“…Therefore, p.R139W may have and increased NQO1 activity [19]. Taken together, this suggests that di- involve FAD-dependent enzymes [67][68][69][70][71]. Furthermore, our results 552 predict that the levels of vitamin B2 required to rescue p.R139W will 553 be lower than those required for p.P187S (see Fig.…”

Clinical and genetical heterogeneity of late-onset multiple acyl-coenzyme A dehydrogenase deficiency

“…The positive clinical effects of riboflavin treatment are well documented as are the molecular mechanisms for riboflavin responsiveness [[4],[7],[8],[29],[48],[49]]. In particular, the chaperone effects that can compensate for inherited folding defects of ETFDH, have recently been described [[49]].…”

“…In particular, the chaperone effects that can compensate for inherited folding defects of ETFDH, have recently been described [[49]]. The vast majority of late-onset MADD patients respond very well to riboflavin.…”

“…However, in the meanwhile, late-onset MADD patients without Q10 deficiency have been reported contradicting this hypothesis [[16]]. Q10 deficiency has been found to be associated with increased mitochondrial production of reactive oxygen species (ROS) due to an electron leak of misfolded variant ETFDH proteins with impaired Q10 binding affinity [[49],[51]]. Based on studies with fibroblasts of six patients with riboflavin-responsive MADD Cornelius et al have reported that Q10 treatment can decrease ROS production and may relieve oxidative stress.…”

Clinical presentation and outcome of riboflavin transporter deficiency: mini review after five years of experience

“…Riboflavin is a well-known chaperone effective in the treatment of inborn errors of metabolism such as MADD and mitochondrial disorders (Cornelius et al 2012; Carrozzo et al 2014). Only since 2010 there is insight into inborn errors of riboflavin transport (Green et al 2010; Bosch et al 2011; Bosch et al 2012; Foley et al 2014).…”