The impact of genotype on the phenotype of Mycobacterium tuberculosis ΔsufR mutants.

Iron-sulphur (FeS) cluster biogenesis is a tightly regulated process in vivo. In Mycobacterium tuberculosis (Mtb), SufR functions as a transcriptional repressor of the operon encoding the primary FeS cluster biogenesis system. Previously, three independently isolated mutants (ΔRv1460stop_1.19, ΔRv1460stop _5.19 and ΔRv1460stop _5.20) harbouring the same deletion in sufR, displayed different growth kinetics in OADC supplemented 7H9 media. To investigate this discrepancy, we performed whole genome sequencing of the 3 mutants and the wild-type progenitor. Single nucleotide polymorphisms (SNPs) were identified in 3 genes in the ΔRv1460stop_1.19 mutant and one gene in the ΔRv1460stop_5.20 mutant. Phenotyping of the ΔRv1460stop_5.19 mutant, which had no additional SNPs, revealed increased susceptibility to clofazimine, DMNQ and menadione, while uptake and survival in THP-1 cells were not significantly different from the wild-type strain. Given that these results differ from those reported for other sufR deletion mutants (ΔSufRMTB and MtbΔSufR), they suggest that the position of the sufR deletion and the genotype of the progenitor strain impact the resulting phenotype.

The C-terminal domain of the ferric uptake regulator (Fur) binds a [2Fe-2S] cluster to sense the intracellular free iron content in Escherichia coli.

Escherichia coli ferric uptake regulator (Fur) binds a [2Fe-2S] cluster, not a mononuclear iron, when the intracellular free iron content is elevated in E. coli cells. Here we report that the C-terminal domain (residues 83-148) of E. coli Fur (Fur-CTD) is sufficient to bind the [2Fe-2S] cluster in response to elevation of the intracellular free iron content in E. coli cells. Deletion of gene fur in E. coli cells increases the intracellular free iron content and promotes the [2Fe-2S] cluster binding in the Fur-CTD in the cells grown in LB medium under aerobic growth conditions. When the Fur-CTD is expressed in wild type E. coli cells grown in M9 medium supplemented with increasing concentrations of iron, the Fur-CTD also progressively binds a [2Fe-2S] cluster with a maximum occupancy of about 36%. Like the E. coli Fur-CTD, the CTD of the Haemophilus influenzae Fur can also bind a [2Fe-2S] cluster in wild type E. coli cells grown in M9 medium supplemented with increasing concentrations of iron, indicating that binding of the [2Fe-2S] cluster in the C-terminal domain is highly conserved among Fur proteins. The results suggest that the Fur-CTD can be used as a physiological probe to assess the intracellular free iron content in bacteria.

Disrupting the plastidic iron-sulfur cluster biogenesis pathway in Toxoplasma gondii has pleiotropic effects irreversibly impacting parasite viability.

Like many other apicomplexan parasites, Toxoplasma gondii contains a plastid harboring key metabolic pathways, including the sulfur utilization factor (SUF) pathway that is involved in the biosynthesis of iron-sulfur clusters. These cofactors are crucial for a variety of proteins involved in important metabolic reactions, potentially including plastidic pathways for the synthesis of isoprenoid and fatty acids. It was shown previously that impairing the NFS2 cysteine desulfurase, involved in the first step of the SUF pathway, leads to an irreversible killing of intracellular parasites. However, the metabolic impact of disrupting the pathway remained unexplored. Here, we generated another mutant of this pathway, deficient in the SUFC ATPase, and investigated in details the phenotypic consequences of TgNFS2 and TgSUFC depletion on the parasites. Our analysis confirms that Toxoplasma SUF mutants are severely and irreversibly impacted in division and membrane homeostasis, and suggests a defect in apicoplast-generated fatty acids. However, we show that increased scavenging from the host or supplementation with exogenous fatty acids do not fully restore parasite growth, suggesting that this is not the primary cause for the demise of the parasites and that other important cellular functions were affected. For instance, we also show that the SUF pathway is key for generating the isoprenoid-derived precursors necessary for the proper targeting of GPI-anchored proteins and for parasite motility. Thus, we conclude plastid-generated iron-sulfur clusters support the functions of proteins involved in several vital downstream cellular pathways, which implies the SUF machinery may be explored for new potential anti-Toxoplasma targets.

Hypoketotic hypoglycemia without neuromuscular complications in patients with SLC25A32 deficiency.

Mitochondrial flavin adenine dinucleotide (FAD) transporter deficiencies are new entities recently reported to cause a neuro-myopathic phenotype. We report three patients from two unrelated families who presented primarily with hypoketotic hypoglycemia. They all had acylcarnitine profiles suggestive of multiple acyl-CoA dehydrogenase deficiency (MADD) with negative next-generation sequencing of electron-transfer flavoprotein genes (ETFA, ETFB, and ETFDH). Whole exome sequencing revealed a homozygous c.272 G > T (p.Gly91Val) variant in exon 2 of the SLC25A32 gene. The three patients shared the same variant, and they all demonstrated similar clinical and biochemical improvement with riboflavin supplementation. To date, these are the first patients to be reported with hypoketotic hypoglycemia without the neuromuscular phenotype previously reported in patients with SLC25A32 deficiency.

Clinical, pathological and genetic features and follow-up of 110 patients with late-onset MADD: a single-center retrospective study.

To observe a long-term prognosis in late-onset multiple acyl-coenzyme-A dehydrogenation deficiency (MADD) patients and to determine whether riboflavin should be administrated in the long-term and high-dosage manner, we studied the clinical, pathological and genetic features of 110 patients with late-onset MADD in a single neuromuscular center. The plasma riboflavin levels and a long-term follow-up study were performed. We showed that fluctuating proximal muscle weakness, exercise intolerance and dramatic responsiveness to riboflavin treatment were essential clinical features for all 110 MADD patients. Among them, we identified 106 cases with ETFDH variants, 1 case with FLAD1 variants and 3 cases without causal variants. On muscle pathology, fibers with cracks, atypical ragged red fibers (aRRFs) and diffuse decrease of SDH activity were the distinctive features of these MADD patients. The plasma riboflavin levels before treatment were significantly decreased in these patients as compared to healthy controls. Among 48 MADD patients with a follow-up of 6.1 years on average, 31 patients were free of muscle weakness recurrence, while 17 patients had episodes of slight muscle weakness upon riboflavin withdrawal, but recovered after retaking a small-dose of riboflavin for a short-term. Multivariate Cox regression analysis showed vegetarian diet and masseter weakness were independent risk factors for muscle weakness recurrence. In conclusion, fibers with cracks, aRRFs and diffuse decreased SDH activity could distinguish MADD from other genotypes of lipid storage myopathy. For late-onset MADD, increased fatty acid oxidation and reduced riboflavin levels can induce episodes of muscle symptoms, which can be treated by short-term and small-dose of riboflavin therapy.

ETF dehydrogenase advances in molecular genetics and impact on treatment.

Electron transfer flavoprotein dehydrogenase, also called ETF-ubiquinone oxidoreductase (ETF-QO), is a protein localized in the inner membrane of mitochondria, playing a central role in the electron-transfer system. Indeed, ETF-QO mediates electron transport from flavoprotein dehydrogenases to the ubiquinone pool. ETF-QO mutations are often associated with riboflavin-responsive multiple acyl-CoA dehydrogenase deficiency (RR-MADD, OMIM#231680), a multisystem genetic disease characterized by various clinical manifestations with different degrees of severity. In this review, we outline the clinical features correlated with ETF-QO deficiency and the benefits obtained from different treatments, such as riboflavin, L-carnitine and/or coenzyme Q10 supplementation, and a diet poor in fat and protein. Moreover, we provide a detailed summary of molecular and bioinformatic investigations, describing the mutations identified in ETFDH gene and highlighting their predicted impact on enzymatic structure and activity. In addition, we report biochemical and functional analysis, performed in HEK293 cells and patient fibroblasts and muscle cells, to show the relationship between the nature of ETFDH mutations, the variable impairment of enzyme function, and the different degrees of RR-MADD severity. Finally, we describe in detail 5 RR-MADD patients carrying different ETFDH mutations and presenting variable degrees of clinical symptom severity.

Clinical characteristics and gene mutation analysis of an adult patient with ETFDH­related multiple acyl­CoA dehydrogenase deficiency.

Multiple acyl­CoA dehydrogenase deficiency (MADD) is a rare autosomal recessive disorder of fatty acid metabolism caused by defects in electron transfer flavoprotein (ETF) or electron transfer flavoprotein dehydrogenase (ETFDH). These defects are mainly classified into the neonatal and late­onset types, based on their clinical manifestations. ETFDH gene mutations are generally considered to be associated with the late­onset type. The present study reported an adult woman with late­onset MADD accompanied with biochemical and muscle biopsy findings indicating metabolic disorders. Gene sequencing analysis showed that the c.1514T>C homozygous mutation in the region of the 12th exon of the ETFDH gene, which led to the amino acid substitution p.I505T (isoleucine > threonine), resulting in defective ETFDH protein function. The results of family verification revealed that the homozygous mutation originated from her parents. The female patient was treated with a large dose of vitamin B2, L­carnitine and coenzyme Q10, and the symptoms were significantly relieved. The c.1514T>C mutation in the ETFDH gene, was considered as a novel pathogenic mutation that had not been previously reported. Therefore, it was hypothesized that this mutation was responsible for the clinical characteristics of the adult female patient. Overall, this novel mutation could expand the spectrum of the ETFDH gene mutation and provide the basis for the etiological and prenatal diagnosis of MADD.

Neonatal-onset multiple acyl-CoA dehydrogenase deficiency (MADD) in the ETFDH gene: A case report and a literature review.

RATIONALE: Multiple acyl-CoA dehydrogenase deficiency (MADD) is a rare inborn error of metabolism affecting fatty acid, amino acid, and choline metabolism. The clinical manifestation of MADD is heterogeneous, from severe neonatal forms to mild late-onset forms. PATIENT CONCERNS: Here, we report a patient who presented with severe hypoglycemia and exercise intolerance suggestive of MADD. Serum tandem mass spectrometry analysis indicated elevated levels of various acyl carnitines at 25 days of age. Exome sequencing of the proband revealed compound heterozygous mutations, c. 413T>G (p.Leu138Arg) and c.1667C > G (p.Pro556Arg), in the ETFDH gene as the probable causative mutations. DIAGNOSES: Based on the patient's clinical presentation and test results, the patient was diagnosed with MADD. INTERVENTIONS: A high-calorie and reduced-fat diet was given together with oral supplements of L-carnitine (150 mg/day). OUTCOMES: He passed away at the age of 4 months because of severe respiratory distress accompanied by muscle weakness. LESSONS: He passed away at the age of 4 months because of severe respiratory distress accompanied by muscle weakness. Clinicians should consider MADD in the differential diagnosis when patients present with muscle weakness and biochemical abnormalities. Gene testing plays a critical role in confirming the diagnosis of MADD and may not only prevent the need for invasive testing but also allow for timely initiation of treatment.

Heterologous expression of the gene for chlorite dismutase from Ideonella dechloratans is induced by an FNR-type transcription factor.

Regulation of the expression of the gene for chlorite dismutase (cld), located on the chlorate reduction composite transposon of the chlorate reducer Ideonella dechloratans, was studied. A 200 bp upstream sequence of the cld gene, and mutated and truncated versions thereof, was used in a reporter system in Escherichia coli. It was found that a sequence within this upstream region, which is nearly identical to the canonical FNR-binding sequence of E. coli, is necessary for anaerobic induction of the reporter gene. Anaerobic induction was regained in an FNR-deficient strain of E. coli when supplemented either with the fnr gene from E. coli or with a candidate fnr gene cloned from I. dechloratans. In vivo transcription of the suggested fnr gene of I. dechloratans was demonstrated by qRT-PCR. Based on these results, the cld promoter of I. dechloratans is suggested to be a class II-activated promoter regulated by an FNR-type protein of I. dechloratans. No fnr-type genes have been found on the chlorate reduction composite transposon of I. dechloratans, making anaerobic upregulation of the cld gene after a gene transfer event dependent on the presence of an fnr-type gene in the recipient.

Unraveling the role of thiosulfate sulfurtransferase in metabolic diseases.

Thiosulfate sulfurtransferase (TST, EC 2.8.1.1), also known as Rhodanese, is a mitochondrial enzyme which catalyzes the transfer of sulfur in several molecular pathways. After its initial identification as a cyanide detoxification enzyme, it was found that its functions also include sulfur metabolism, modification of iron­sulfur clusters and the reduction of antioxidants glutathione and thioredoxin. TST deficiency was shown to be strongly related to the pathophysiology of metabolic diseases including diabetes and obesity. This review summarizes research related to the enzymatic properties and functions of TST, to then explore the association between the effects of TST on mitochondria and development of diseases such as diabetes and obesity.

A NAC-type transcription factor confers aluminium resistance by regulating cell wall-associated receptor kinase 1 and cell wall pectin.

Transcriptional regulation is important for plants to respond to toxic effects of aluminium (Al). However, our current knowledge to these events is confined to a few transcription factors. Here, we functionally characterized a rice bean (Vigna umbellata) NAC-type transcription factor, VuNAR1, in terms of Al stress response. We demonstrated that rice bean VuNAR1 is a nuclear-localized transcriptional activator, whose expression was specifically upregulated by Al in roots but not in shoot. VuNAR1 overexpressing Arabidopsis plants exhibit improved Al resistance via Al exclusion. However, VuNAR1-mediated Al exclusion is independent of the function of known Al-resistant genes. Comparative transcriptomic analysis revealed that VuNAR1 specifically regulates the expression of genes associated with protein phosphorylation and cell wall modification in Arabidopsis. Transient expression assay demonstrated the direct transcriptional activation of cell wall-associated receptor kinase 1 (WAK1) by VuNAR1. Moreover, yeast one-hybrid assays and MEME motif searches identified a new VuNAR1-specific binding motif in the promoter of WAK1. Compared with wild-type Arabidopsis plants, VuNAR1 overexpressing plants have higher WAK1 expression and less pectin content. Taken together, our results suggest that VuNAR1 regulates Al resistance by regulating cell wall pectin metabolism via directly binding to the promoter of WAK1 and induce its expression.

Adolescent Hyperuricemia with Lipid Storage Myopathy: A Clinical Study.

BACKGROUND In this study, we investigated the clinical and pathological features of patients with lipid storage myopathy (LSM) complicated with hyperuricemia, to improve clinicians' understanding of metabolic multi-muscular disorder with metabolic disorders, and to reduce the risk of missed diagnosis of LSM. MATERIAL AND METHODS From January 2005 to December 2017, 8 patients underwent muscle biopsy and diagnosed by muscle pathology and genetic testing in our hospital. All 8 patients were in compliance with LSM diagnosis. We collected data on the patient's clinical performance, adjuvant examination, treatment, and outcomes to provide a comprehensive report and description of LSM patients with hyperuricemia. RESULTS All patients were diagnosed as having ETFDH gene mutations. The main clinical manifestations of patients were chronic limb and trunk weakness, limb numbness, and muscle pain. The serum creatine kinase (CK) values in all patients were higher than normal values. Electromyography showed 3 cases of simple myogenic damage and 3 cases of neurogenic injury. Hematuria metabolic screening showed that 2 patients had elevated glutaric aciduria, and 1 patient had elevated fatty acyl carnitine in the blood. All patients were given riboflavin treatment, and the clinical symptoms were significantly improved, and 3 patients returned to normal uric acid levels after treatment. Pathological staining showed an abnormal deposition of lipid droplets in muscle fibers. CONCLUSIONS If an adolescent hyperuricemia patient has abnormal limb weakness, exercise intolerance, and elevated serum CK values, clinicians need to be highly alert to the possibility of LSM. Early diagnosis and treatment of LSM should improve the clinical symptoms and quality of life and reduce complications.

Molecular and Clinical Investigations on Portuguese Patients with Multiple acyl-CoA Dehydrogenase Deficiency.

BACKGROUND: Multiple Acyl-CoA Dehydrogenase Deficiency (MADD) is a congenital rare metabolic disease with broad clinical phenotypes and variable evolution. This inborn error of metabolism is caused by mutations in the ETFA, ETFB or ETFDH genes, which encode for the mitochondrial ETF and ETF:QO proteins. A considerable group of patients has been described to respond positively to riboflavin oral supplementation, which constitutes the prototypic treatment for the pathology. OBJECTIVES: To report mutations in ETFA, ETFB and ETFDH genes identified in Portuguese patients, correlating, whenever possible, biochemical and clinical outcomes with the effects of mutations on the structure and stability of the affected proteins, to better understand MADD pathogenesis at the molecular level. METHODS: MADD patients were identified based on the characteristic urinary profile of organic acids and/or acylcarnitine profiles in blood spots during newborn screening. Genotypic, clinical and biochemical data were collected for all patients. In silico structural analysis was employed using bioinformatic tools carried out in an ETF:QO molecular model for the identified missense mutations. RESULTS: A survey describing clinical and biochemical features of eight Portuguese MADD patients was made. Genotype analysis identified five ETFDH mutations, including one extension (p.X618QextX*14), two splice mutations (c.34+5G>C and c.405+3A>T) and two missense mutations (ETF:QO-p.Arg155Gly and ETF:QO-p.Pro534Leu), and one ETFB mutation (ETFß- p.Arg191Cys). Homozygous patients containing the ETFDH mutations p.X618QextX*14, c.34+5G>C and ETF:QO-p.Arg155Gly, all presented severe (lethal) MADD phenotypes. However, when any of these mutations are in heterozygosity with the known ETF:QO-p.Pro534Leu mild variant, the severe clinical effects are partly and temporarily attenuated. Indeed, the latter destabilizes an ETF-interacting loop, with no major functional consequences. However, the position 155 in ETF:QO is localized at the ubiquinone binding and membrane interacting domain, and is thus expected to perturb protein structure and membrane insertion, with severe functional effects. Structural analysis of molecular models is therefore demonstrated to be a valuable tool to rationalize the effects of mutations in the context of the clinical phenotype severity. CONCLUSION: Advanced molecular diagnosis, structural analysis and clinical correlations reveal that MADD patients harboring a severe prognosis mutation in one allele can actually revert to a milder phenotype by complementation with a milder mutation in the other allele. However, such patients are nevertheless in a precarious metabolic balance which can revert to severe fatal outcomes during catabolic stress or secondary pathology, thus requiring strict clinical follow-up.

Improved subtyping affords better discrimination of Trichomonas gallinae strains and suggests hybrid lineages.

Trichomonas gallinae is a protozoan pathogen that causes avian trichomonosis typically associated with columbids (canker) and birds of prey (frounce) that predate on them, and has recently emerged as an important cause of passerine disease. An archived panel of DNA from North American (USA) birds used initially to establish the ITS ribotypes was reanalysed using Iron hydrogenase (FeHyd) gene sequences to provide an alphanumeric subtyping scheme with improved resolution for strain discrimination. Thirteen novel subtypes of T. gallinae using FeHyd gene as the subtyping locus are described. Although the phylogenetic topologies derived from each single marker are complementary, they are not entirely congruent. This may reflect the complex genetic histories of the isolates analysed which appear to contain two major lineages and several that are hybrid. This new analysis consolidates much of the phylogenetic signal generated from the ITS ribotype and provides additional resolution for discrimination of T. gallinae strains. The single copy FeHyd gene provides higher resolution genotyping than ITS ribotype alone. It should be used where possible as an additional, single-marker subtyping tool for cultured isolates.

Rhodiola crenulata attenuates apoptosis and mitochondrial energy metabolism disorder in rats with hypobaric hypoxia-induced brain injury by regulating the HIF-1α/microRNA 210/ISCU1/2(COX10) signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Rhodiola crenulata, a traditional Tibetan medicine, has shown promise in the treatment of hypobaric hypoxia (HH)-induced brain injury. However, the underlying mechanisms remain unclear. This study investigated the protective effects of R. crenulata aqueous extract (RCAE) on HH-induced brain injury in rats. MATERIALS AND METHODS: An animal model of high-altitude hypoxic brain injury was established in SD rats using an animal decompression chamber for 24 h. Serum and hippocampus levels of superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), oxidized glutathione (GSSG), and lactate dehydrogenase (LDH) were then determined using commercial biochemical kits. Neuron morphology and vitality were also evaluated using H&E and Nissl staining, and TUNEL staining was used to examine apoptosis. Gene and protein expression of HIF-1α, microRNA 210, ISCU1/2, COX10, Apaf-1, cleaved Caspase-3, Caspase-3, Bax, Bcl-2, and Cyto-c were determined by western blot, immunohistochemical and qRT-PCR analysis. RESULTS: RCAE administration attenuated HH-induced brain injury as evidenced by decreased levels of MDA, LDH, and GSSG, increased GSH and SOD, improvements in hippocampus histopathological changes, increased cell vitality and ATP level, and reduced apoptotic cell numbers. RCAE treatment also enhanced HIF-1α, ISCU1/2, COX10, and Bcl-2 protein expression, while dramatically inhibiting expression of Apaf-1, Bax, Cyto-c, and cleaved Caspase-3. Treatment also increased gene levels of HIF-1α, microRNA 210, ISCU1/2, and COX10, and decreased Caspase-3 gene production. CONCLUSIONS: RCAE attenuated HH-induced brain injury by regulating apoptosis and mitochondrial energy metabolism via the HIF-1α/microRNA 210/ISCU1/2 (COX10) signaling pathway.

Acute-onset multiple acyl-CoA dehydrogenase deficiency mimicking Guillain-Barré syndrome: two cases report.

BACKGROUND: Multiple acyl-CoA dehydrogenase deficiency (MADD) showed great clinical heterogeneity and poses a challenge to diagnosis. Guillain-Barré syndrome (GBS) is an acute-onset autoimmune-mediated peripheral neuropathy. However, no patients of acute-onset MADD mimicking the GBS phenotype are reported previously. CASE PRESENTATION: Two patients displayed acute-onset limb weakness, areflexia, and length-dependent sensory disturbances, which clinically indicate the diagnosis of GBS, but electrophysiological and cerebrospinal fluid results threw doubtful points to the initial diagnosis. The muscle biopsy showed lipid storage disorder; and compound heterozygous mutations in the electron transfer flavoprotein dehydrogenase (ETFDH) gene were found in the two patients through targeted next generation sequencing, which provided the definite diagnostic evidences of late-onset MADD. Muscle weakness was quickly improved by riboflavin supplementation, but sensory disturbances required a long-term treatment. DISCUSSION: The present two cases have demonstrated that MADD can mimic GBS. Taking into consideration the significant differences of therapeutic regimen and prognosis, MADD should be included in the differential diagnosis of GBS.

Characterization of two ETFDH mutations in a novel case of riboflavin-responsive multiple acyl-CoA dehydrogenase deficiency.

BACKGROUND: Deficiency of electron transfer flavoprotein dehydrogenase (ETFDH) is associated with multiple acyl-CoA dehydrogenase deficiency (MADD). This disorder is an autosomal recessive lipid storage myopathy (LSM) that exhibits a wide range of clinical features, including myopathy, weakness and multisystem dysfunctions. Many patients with late onset of MADD improve when treated with riboflavin and are also referred to as RR-MADD (riboflavin-responsive multiple Acyl-CoA dehydrogenase disorder). METHODS: In this study, we report the clinical and genetic characterization of a novel RR-MADD patient. Biochemical data were obtained from analysis of muscle and plasma samples. DNA and RNA were extracted from peripheral blood, and sequence analysis and expression study of ETFDH gene were performed. Finally, the impact of mutations on ETFDH folding was evaluated using bioinformatic tools. RESULTS: Patient initially presented with vomiting, muscle weakness, and acidosis. Muscle biopsy revealed typical myopathological patterns of lipid storage myopathy and blood acylcarnitine profiles showed a combined elevation of long and medium chain acylcarnitines, supporting the diagnosis of RR-MADD. Molecular analysis of ETFDH gene revealed two heterozygous mutations, a novel splice variation in intron 10, c.1285 + 1G > A, and the previously reported c.560C > T missense mutation. RT-PCR analysis showed an alteration of ETFDH RNA splicing which in turn should lead to the production of a truncated protein. The in silico prediction analysis of ETFDH tridimensional structure demonstrated that the missense mutation resulted in instability and loss of protein activation, while the splice site variation induced a dramatic conformational change of the truncated protein. After MCT diet supplemented with carnitine and riboflavin, the patient showed significant biochemical and clinical improvement, in spite of severe molecular defect. CONCLUSION: This case report extends the spectrum of ETFDH mutations in MADD, providing further evidence that patients presenting at least one missense mutation in the FAD-binding domain may respond to either carnitine or riboflavin treatment, due to the recovery of some enzymatic activity.

Bfd, a New Class of [2Fe-2S] Protein That Functions in Bacterial Iron Homeostasis, Requires a Structural Anion Binding Site.

Mobilization of iron from bacterioferritin (BfrB) requires specific interactions with a [2Fe-2S] ferredoxin (Bfd). Blocking the BfrB:Bfd interaction results in irreversible iron accumulation in BfrB and iron deficiency in the cytosol [Eshelman, K., et al. (2017) Metallomics 9, 646-659]. The only known Bfd structure, which was obtained in complex with BfrB (Protein Data Bank entry 4E6K ), indicated a new fold and suggested that the stability of Bfd is aided by an anion binding site consisting of R26, R29, and K46. We investigated the Bfd fold using site-directed mutagenesis, X-ray crystallography, and biochemistry in solution. The X-ray structure, which is nearly identical to that of Bfd in the BfrB:Bfd complex, shows that the [2Fe-2S] cluster preorganizes residues at the BfrB:Bfd interface into a structure complementary to the Bfd binding site on BfrB. Studies in solution showed rapid loss of the [2Fe-2S] cluster at a low ionic strength but higher stability with an increasing ionic strength, thus supporting a structural anion binding site. Structures of the R26E and R26E/K46Y mutants are nearly identical to that of Bfd, except for a new network of hydrogen bonds stabilizing the region encompassing the former anion binding site. The stability of the R26E and R26E/K46Y mutants, which is weakly and completely independent of solution ionic strength, respectively, corroborates that Bfd requires an anion binding site. The mutations, which caused only small changes to the strength of the BfrB:Bfd interaction and mobilization of iron from BfrB, indicate that the anion binding site in Bfd serves primarily a structural role.

ETFDH Mutations and Flavin Adenine Dinucleotide Homeostasis Disturbance Are Essential for Developing Riboflavin-Responsive Multiple Acyl-Coenzyme A Dehydrogenation Deficiency.

OBJECTIVE: Riboflavin-responsive multiple acyl-coenzyme A dehydrogenation deficiency (RR-MADD) is an inherited fatty acid metabolism disorder mainly caused by genetic defects in electron transfer flavoprotein-ubiquinone oxidoreductase (ETF:QO). The variant ETF:QO protein folding deficiency, which can be corrected by therapeutic dosage of riboflavin supplement, has been identified in HEK-293 cells and is believed to be the molecular mechanism of this disease. To verify this hypothesis in vivo, we generated Etfdh (h)A84T knockin (KI) mice. METHODS: Tissues from these mice as well as muscle biopsies and fibroblasts from 7 RR-MADD patients were used to examine the flavin adenine dinucleotide (FAD) concentration and ETF:QO protein amount. RESULTS: All of the homozygous KI mice (Etfdh (h)A84T/(h)A84T , KI/KI) were initially normal. After being given a high-fat and vitamin B2 -deficient (HF-B2 D) diet for 5 weeks, they developed weight loss, movement ability defects, lipid storage in muscle and liver, and elevated serum acyl-carnitine levels, which are clinically and biochemically similar to RR-MADD patients. Both ETF:QO protein and FAD concentrations were significantly decreased in tissues of HF-B2 D-KI/KI mice and in cultured fibroblasts from RR-MADD patients. After riboflavin treatment, ETF:QO protein increased in proportion to elevated FAD concentrations, but not related to mRNA levels. These results were further confirmed in cultured fibroblasts from RR-MADD patients. INTERPRETATION: For the first time, we successfully developed a RR-MADD mice model and confirmed that FAD homeostasis disturbances played a crucial role on the pathomechanism of RR-MADD in this mouse model and culture cells from patients. Supplementation of riboflavin may stabilize variant ETF:QO protein by rebuilding FAD homeostasis. Ann Neurol 2018;84:667-681.

Coenzyme Q10 serves to couple mitochondrial oxidative phosphorylation and fatty acid ß-oxidation, and attenuates NLRP3 inflammasome activation.

Multiple acyl-CoA dehydrogenase deficiency (MADD), an autosomal recessive metabolic disorder of fatty acid metabolism, is mostly caused by mutations in the ETFA, ETFB or ETFDH genes that result in dysfunctions in electron transfer flavoprotein (ETF) or electron transfer flavoprotein-ubiquinone dehydrogenase (ETFDH). In ß-oxidation, fatty acids are processed to generate acyl-CoA, which is oxidised by flavin adenine dinucleotide and transfers an electron to ETF and, through ETFDH, to mitochondrial respiratory complex III to trigger ATP synthesis. Coenzyme Q10 (CoQ10) is believed to be a potential treatment that produces symptom relief in some MADD patients. CoQ10 acts as a key regulator linking ETFDH and mitochondrial respiratory complex III. Our aim is to investigate the effectiveness of CoQ10 in serving in the ETF/ETFDH system to improve mitochondrial function and to reduce lipotoxicity. In this study, we used lymphoblastoid cells with an ETFDH mutation from MADD patients. ETFDH dysfunction caused insufficient ß-oxidation, leading to increasing lipid droplet and lipid peroxide accumulation. In contrast, supplementation with CoQ10 significantly recovered mitochondrial function and concurrently decreased the generation of reactive oxygen species and lipid peroxides, inhibited the accumulation of lipid droplets and the formation of the NOD-like receptor family pyrin domain-containing three (NLRP3) inflammasome, and reduced interleukin-1ß release and cell death. These results clarify the causal role of CoQ10 in coupling the electron transport chain with ß-oxidation, which may promote the development of CoQ10-directed therapies for MADD patients.