RESUMO
Defects in iron-sulphur [Fe-S] cluster biogenesis are increasingly recognized as causing neurological disease. Mutations in a number of genes that encode proteins involved in mitochondrial [Fe-S] protein assembly lead to complex neurological phenotypes. One class of proteins essential in the early cluster assembly are ferredoxins. FDX2 is ubiquitously expressed and is essential in the de novo formation of [2Fe-2S] clusters in humans. We describe and genetically define a novel complex neurological syndrome identified in two Brazilian families, with a novel homozygous mutation in FDX2. Patients were clinically evaluated, underwent MRI, nerve conduction studies, EMG and muscle biopsy. To define the genetic aetiology, a combination of homozygosity mapping and whole exome sequencing was performed. We identified six patients from two apparently unrelated families with autosomal recessive inheritance of a complex neurological phenotype involving optic atrophy and nystagmus developing by age 3, followed by myopathy and recurrent episodes of cramps, myalgia and muscle weakness in the first or second decade of life. Sensory-motor axonal neuropathy led to progressive distal weakness. MRI disclosed a reversible or partially reversible leukoencephalopathy. Muscle biopsy demonstrated an unusual pattern of regional succinate dehydrogenase and cytochrome c oxidase deficiency with iron accumulation. The phenotype was mapped in both families to the same homozygous missense mutation in FDX2 (c.431C > T, p.P144L). The deleterious effect of the mutation was validated by real-time reverse transcription polymerase chain reaction and western blot analysis, which demonstrated normal expression of FDX2 mRNA but severely reduced expression of FDX2 protein in muscle tissue. This study describes a novel complex neurological phenotype with unusual MRI and muscle biopsy features, conclusively mapped to a mutation in FDX2, which encodes a ubiquitously expressed mitochondrial ferredoxin essential for early [Fe-S] cluster biogenesis.
Assuntos
Ferredoxinas/genética , Ferredoxinas/fisiologia , Adolescente , Adulto , Brasil , Criança , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Homozigoto , Humanos , Ferro/metabolismo , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/fisiologia , Leucoencefalopatias/metabolismo , Masculino , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Doenças Musculares/genética , Mialgia/genética , Atrofia Óptica/genética , Linhagem , Fenótipo , Succinato Desidrogenase/metabolismo , Síndrome , Sequenciamento do ExomaRESUMO
Iron-sulfur [Fe-S] clusters are inorganic prosthetic groups that play essential roles in all living organisms. In vivo [Fe-S] cluster biogenesis requires enzymes involved in iron and sulfur mobilization, assembly of clusters, and delivery to their final acceptor. In these systems, a cysteine desulfurase is responsible for the release of sulfide ions, which are incorporated into a scaffold protein for subsequent [Fe-S] cluster assembly. Although three machineries have been shown to be present in Proteobacteria for [Fe-S] cluster biogenesis (NIF, ISC, and SUF), only the SUF machinery has been found in Firmicutes. We have recently described the structural similarities and differences between Enterococcus faecalis and Escherichia coli SufU proteins, which prompted the proposal that SufU is the scaffold protein of the E. faecalis sufCDSUB system. The present work aims at elucidating the biological roles of E. faecalis SufS and SufU proteins in [Fe-S] cluster assembly. We show that SufS has cysteine desulfurase activity and cysteine-365 plays an essential role in catalysis. SufS requires SufU as activator to [4Fe-4S] cluster assembly, as its ortholog, IscU, in which the conserved cysteine-153 acts as a proximal sulfur acceptor for transpersulfurization reaction.
Assuntos
Liases de Carbono-Enxofre/metabolismo , Cisteína/metabolismo , Enterococcus faecalis/enzimologia , Proteínas Ferro-Enxofre/fisiologia , Enxofre/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Liases de Carbono-Enxofre/química , Liases de Carbono-Enxofre/genética , Liases de Carbono-Enxofre/isolamento & purificação , Clonagem Molecular , Cisteína/química , Enterococcus faecalis/química , Enterococcus faecalis/genética , Enterococcus faecalis/metabolismo , Ativação Enzimática , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Modelos Moleculares , Ligação Proteica , Especificidade por Substrato , Enxofre/químicaRESUMO
Saccharomyces cerevisiae lacking copper-zinc superoxide dismutase (sod1) shows a series of defects, including reduced rates of aerobic growth in synthetic glucose medium and reduced ability to grow by respiration in glycerol-rich medium. In this work, we observed that addition of iron improved the respiratory growth of the sod1 mutant and in glucose medium total intracellular iron content was higher in the sod1 mutant than in wild type cells. Transcription of the high affinity iron transporter gene, FET3, was enhanced in the sod1 mutant, suggesting that iron transport systems were up-regulated. An sod1/fet3 double mutant showed increased sensitivity to oxygen and increased transcription of FET4, an alternative, low affinity, iron transporter. We propose that this increased iron demand in the sod1 mutant may be a reflection of the cells' efforts to reconstitute iron-sulfur cluster-containing enzymes that are continuously inactivated in conditions of excess superoxide.