Metabolic remodeling in frataxin-deficient yeast is mediated by Cth2 and Adr1.

Frataxin is a mitochondrial protein involved in iron metabolism whose deficiency in humans causes Friedreich ataxia. We performed transcriptomic and proteomic analyses of conditional Yeast Frataxin Homologue (Yfh1) mutants (tetO7-YFH1) to investigate metabolic remodeling upon Yfh1 depletion. These studies revealed that Yfh1 depletion leads to downregulation of many glucose-repressed genes. Most of them were Adr1 targets, a key transcription factor required for growth in non-fermentable carbon sources. Using a GFP-tagged Adr1, we observed that Yfh1 depletion promotes the export of Adr1 from the nucleus to the cytosol without affecting its protein levels. This effect was also observed upon H2O2 treatment, but not by iron overload/starvation, indicating the presence of a regulatory pathway involved in Adr1 export and inactivation upon stress conditions. We also observed that CTH2, a gene involved in the mRNA degradation of several iron-containing enzymes, was induced upon Yfh1 depletion. Accordingly, decreased levels of aconitase and succinate dehydrogenase were observed. Nevertheless, their levels were maintained in a Δcth2 mutant even in the absence of Yfh1. From these results we can conclude that, in addition to altering iron homeostasis, frataxin depletion involves drastic metabolic remodeling governed by Adr1 and Cth2 that finally leads to downregulation of iron-sulfur proteins and other proteins involved in respiratory metabolism.

Inhibition of mammalian mtDNA transcription acts paradoxically to reverse diet-induced hepatosteatosis and obesity.

The oxidative phosphorylation system1 in mammalian mitochondria plays a key role in transducing energy from ingested nutrients2. Mitochondrial metabolism is dynamic and can be reprogrammed to support both catabolic and anabolic reactions, depending on physiological demands or disease states. Rewiring of mitochondrial metabolism is intricately linked to metabolic diseases and promotes tumour growth3-5. Here, we demonstrate that oral treatment with an inhibitor of mitochondrial transcription (IMT)6 shifts whole-animal metabolism towards fatty acid oxidation, which, in turn, leads to rapid normalization of body weight, reversal of hepatosteatosis and restoration of normal glucose tolerance in male mice on a high-fat diet. Paradoxically, the IMT treatment causes a severe reduction of oxidative phosphorylation capacity concomitant with marked upregulation of fatty acid oxidation in the liver, as determined by proteomics and metabolomics analyses. The IMT treatment leads to a marked reduction of complex I, the main dehydrogenase feeding electrons into the ubiquinone (Q) pool, whereas the levels of electron transfer flavoprotein dehydrogenase and other dehydrogenases connected to the Q pool are increased. This rewiring of metabolism caused by reduced mtDNA expression in the liver provides a principle for drug treatment of obesity and obesity-related pathology.

Mitochondrial DNA copy number in human disease: the more the better?

Most of the genetic information has been lost or transferred to the nucleus during the evolution of mitochondria. Nevertheless, mitochondria have retained their own genome that is essential for oxidative phosphorylation (OXPHOS). In mammals, a gene-dense circular mitochondrial DNA (mtDNA) of about 16.5 kb encodes 13 proteins, which constitute only 1% of the mitochondrial proteome. Mammalian mtDNA is present in thousands of copies per cell and mutations often affect only a fraction of them. Most pathogenic human mtDNA mutations are recessive and only cause OXPHOS defects if present above a certain critical threshold. However, emerging evidence strongly suggests that the proportion of mutated mtDNA copies is not the only determinant of disease but that also the absolute copy number matters. In this review, we critically discuss current knowledge of the role of mtDNA copy number regulation in various types of human diseases, including mitochondrial disorders, neurodegenerative disorders and cancer, and during ageing. We also provide an overview of new exciting therapeutic strategies to directly manipulate mtDNA to restore OXPHOS in mitochondrial diseases.

FBXL4 deficiency increases mitochondrial removal by autophagy.

Pathogenic variants in FBXL4 cause a severe encephalopathic syndrome associated with mtDNA depletion and deficient oxidative phosphorylation. To gain further insight into the enigmatic pathophysiology caused by FBXL4 deficiency, we generated homozygous Fbxl4 knockout mice and found that they display a predominant perinatal lethality. Surprisingly, the few surviving animals are apparently normal until the age of 8-12 months when they gradually develop signs of mitochondrial dysfunction and weight loss. One-year-old Fbxl4 knockouts show a global reduction in a variety of mitochondrial proteins and mtDNA depletion, whereas lysosomal proteins are upregulated. Fibroblasts from patients with FBXL4 deficiency and human FBXL4 knockout cells also have reduced steady-state levels of mitochondrial proteins that can be attributed to increased mitochondrial turnover. Inhibition of lysosomal function in these cells reverses the mitochondrial phenotype, whereas proteasomal inhibition has no effect. Taken together, the results we present here show that FBXL4 prevents mitochondrial removal via autophagy and that loss of FBXL4 leads to decreased mitochondrial content and mitochondrial disease.

Nitric oxide prevents Aft1 activation and metabolic remodeling in frataxin-deficient yeast.

Yeast frataxin homolog (Yfh1) is the orthologue of human frataxin, a mitochondrial protein whose deficiency causes Friedreich Ataxia. Yfh1 deficiency activates Aft1, a transcription factor governing iron homeostasis in yeast cells. Although the mechanisms causing this activation are not completely understood, it is assumed that it may be caused by iron-sulfur deficiency. However, several evidences indicate that activation of Aft1 occurs in the absence of iron-sulfur deficiency. Besides, Yfh1 deficiency also leads to metabolic remodeling (mainly consisting in a shift from respiratory to fermentative metabolism) and to induction of Yhb1, a nitric oxide (NO) detoxifying enzyme. In this work, we have used conditional Yfh1 mutant yeast strains to investigate the relationship between NO, Aft1 activation and metabolic remodeling. We have observed that NO prevents Aft1 activation caused by Yfh1 deficiency. This phenomenon is not observed when Aft1 is activated by iron scarcity or impaired iron-sulfur biogenesis. In addition, analyzing key metabolic proteins by a targeted proteomics approach, we have observed that NO prevents the metabolic remodeling caused by Yfh1 deficiency. We conclude that Aft1 activation in Yfh1-deficient yeasts is not caused by iron-sulfur deficiency or iron scarcity. Our hypothesis is that Yfh1 deficiency leads to the presence of anomalous iron species that can compromise iron bioavailability and activate a signaling cascade that results in Aft1 activation and metabolic remodeling.

Iron in Friedreich Ataxia: A Central Role in the Pathophysiology or an Epiphenomenon?

Friedreich ataxia is a neurodegenerative disease with an autosomal recessive inheritance. In most patients, the disease is caused by the presence of trinucleotide GAA expansions in the first intron of the frataxin gene. These expansions cause the decreased expression of this mitochondrial protein. Many evidences indicate that frataxin deficiency causes the deregulation of cellular iron homeostasis. In this review, we will discuss several hypotheses proposed for frataxin function, their caveats, and how they could provide an explanation for the deregulation of iron homeostasis found in frataxin-deficient cells. We will also focus on the potential mechanisms causing cellular dysfunction in Friedreich Ataxia and on the potential use of the iron chelator deferiprone as a therapeutic agent for this disease.

Analysis of oxidative stress-induced protein carbonylation using fluorescent hydrazides.

Protein carbonyl detection has been commonly used to analyze the degree of damage to proteins under oxidative stress conditions. Most laboratories rely on derivatization of carbonyl groups with dinitrophenylhydrazine followed by Western blot analysis using antibodies against the dinitrophenyl moiety. This paper describes a protein carbonyl detection method based on fluorescent Bodipy, Cy3 and Cy5 hydrazides. Using this approach, Western blot and immunodetection are no longer needed, shortening the procedure and increasing accuracy. Combination of Cy3 and Cy5 hydrazides allows multiplexing analyses in a single two-dimensional gel. Derivatization with Bodipy hydrazide allows easy matching of the spots of interest and those obtained by general fluorescent protein staining methods, which facilitates excising target proteins from the gels and identifying them. This method is effective for detecting protein carbonylation in samples of proteins submitted to metal-catalyzed oxidation "in vitro" and assessing the effect of hydrogen peroxide and chronological aging on protein oxidative damage in yeast cells.