Coordinated and reversible reduction of enzymes involved in terminal oxidative metabolism in skeletal muscle mitochondria from a riboflavin‐responsive, multiple acyl‐CoA dehydrogenase deficiency patient

Gianazza, Elisabetta; Vergani, Lodovica; Wait, Robin; Brizio, Carmen; Brambilla, Daniela; Begum, Shajna; Giancaspero, Teresa Anna; Conserva, Francesca; Eberini, Ivano; Bufano, Daniela; Angelini, C.; Pegoraro, Elena; Tramontano, Anna; Barile, Maria

doi:10.1002/elps.200500687

Cited by 55 publications

(55 citation statements)

References 51 publications

(78 reference statements)

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“…Here we postulate another possibility, which may occur concomitantly to the previous one. The hypothesis is based on the fact that externally added riboflavin leads to an increase in the FAD content, as shown in yeast mitochondria (44) and human mitochondria (42,43). In this scenario, increased availability of the FAD ligand would promote its binding, which induces conformational changes which propagate to the overall structure.…”

Section: Discussionmentioning

confidence: 99%

Role of Flavinylation in a Mild Variant of Multiple Acyl-CoA Dehydrogenation Deficiency

Henriques

Rodrigues

Olsen

et al. 2009

Journal of Biological Chemistry

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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.

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Section: Discussionmentioning

confidence: 99%

Role of Flavinylation in a Mild Variant of Multiple Acyl-CoA Dehydrogenation Deficiency

Henriques

Rodrigues

Olsen

et al. 2009

Journal of Biological Chemistry

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“…In MADD several FAD-dependent dehydrogenases are impaired including those responsible for fatty acid oxidation, and the clinical features therefore resemble those seen in fatty acid oxidation disorders (Olsen et al 2007). Proteomic investigation of patient muscle mitochondria has revealed decrease or absence of approximately 10 enzymes related to flavin cofactor-dependent mitochondrial pathways, for example, medium chain acyl-CoA dehydrogenase (ACADM) and trifunctional enzyme b subunit of fatty acid β-oxidation (Gianazza et al 2006). Since the patient was riboflavin responsive, the protein levels were normalized after riboflavin treatment.…”

Section: Methylmalonic Acidemia and Etf-qo Deficiencymentioning

confidence: 99%

Mitochondrial proteomics—a tool for the study of metabolic disorders

Gregersen

Hansen

Palmfeldt

2012

J of Inher Metab Disea

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Mitochondria are important for a number of life and death processes, such as energy production, creation of reactive oxygen species, and elicitation of stress responses. These responses range from induction of protein quality control and antioxidant systems to mitochondria elimination and cell death. Mitochondrial dysfunctions are involved in pathologies associated with many diseases, for example metabolic disorders, diabetes, cancers, cardiovascular and neurodegenerative diseases as well as obesity and aging. Mitochondrial proteomics can be a powerful tool in the study of these diseases, especially since it can cover mitochondrial proteins from several metabolic pathways, such as the citric acid cycle, fatty acid oxidation, and respiratory chain, as well as protein networks involved in stress responses. The mitochondrial proteome can consist of more than 1,000 different proteins. However, it is difficult to define the precise number, since mitochondria are dynamic and difficult to purify, and because an unknown number of proteins possess dual or multiple localization, depending on cell type and physiological conditions. This review describes several quantitative studies of proteins from mitochondria isolated by centrifugation, separated by various methods (e.g., electrophoresis and nanoLC), and analyzed by advanced mass spectrometry. We illustrate the methods by showing that multiple pathways and networks are affected in cells from patients carrying gene variations affecting a mitochondrial protein. The study of cultured skin fibroblasts from patients with ethylmalonic aciduria associated with variations in the genes coding for short-chain acyl-CoA dehydrogenase (SCAD) or ETHE1 are two of the examples. The possibility of obtaining mitochondrial proteomics data from whole cell proteomics studies is also exemplified by the involvement of liver mitochondria in metabolic syndrome.

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“…It belongs to the group of lipid storage myopathies [32] caused by a reduction in activity of flavin-dependent acyl-CoA dehydrogenases, which may also be associated with a deficiency of complexes I and II of the respiratory chain [33,34]. Proteomic analysis using 2-DE and ESI-MS/MS of muscle mitochondria revealed the differential expression of several flavoenzymes, enzymes related to flavin cofactor-dependent mitochondrial pathways and mitochondrial or mitochondriaassociated calcium-binding proteins, such as NADHubiquinone oxidoreductase 75 kDa subunit, ETF-QO, ACADM, E2 component of BCKAD and the E2 component of PDF, suggesting that a number of enzymatic pathways are altered in RR-MAD [35].…”

Section: -D Gel Electrophoresis and 2-d Differential Gel Electrophormentioning

confidence: 99%

Proteomics as Applied to Inherited Metabolic Diseases

Richard¹,

Gámez²,

Ruíz-Sala³

et al. 2009

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Inherited metabolic diseases (IMD) include a broad spectrum of biochemical alterations caused by genetic defects which affect the structure and function of proteins involved in cellular metabolic pathways. Until now, more than 500 different IMD that alter the synthesis, metabolism, transport and/or storage of biochemical compounds have been identified. Molecular and biochemical techniques can be applied to determine both the genotype and phenotype of IMD patients; they allow for accurate diagnosis and application of individual based treatment strategies. The emerging genomic, proteomic and metabolomic tools using molecular, cellular and physiological approaches provide deeper insight into the pathophysiology of metabolic diseases. Mass spectrometry is a powerful and established technology, employed in IMD research and newborn screening that leads to early diagnosis of different metabolic disorders. Over the last years, the number of different proteomic techniques applied to IMD investigation has dramatically increased. The most remarkable ones include: 2-DE and LC for protein separation; and ESI-MS, ESI-MS/MS, MALDI-MS, MALDI-MS/MS, SELDI-MS, DIOS-MS for protein/metabolite identification and analysis. Proteomics offers the ability to identify novel proteins and to study their expression patterns and function, along with the potential to discover biomarkers as diagnostic tools or for the development of new therapeutic strategies. This review describes the main proteomic techniques applied to IMD diagnosis and research.

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Coordinated and reversible reduction of enzymes involved in terminal oxidative metabolism in skeletal muscle mitochondria from a riboflavin‐responsive, multiple acyl‐CoA dehydrogenase deficiency patient

Cited by 55 publications

References 51 publications

Role of Flavinylation in a Mild Variant of Multiple Acyl-CoA Dehydrogenation Deficiency

Role of Flavinylation in a Mild Variant of Multiple Acyl-CoA Dehydrogenation Deficiency

Mitochondrial proteomics—a tool for the study of metabolic disorders

Proteomics as Applied to Inherited Metabolic Diseases

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