Marisa W. Friederich

Fatty liver is associated with reduced SIRT3 activity and mitochondrial protein hyperacetylation.

Acetylation has recently emerged as an important mechanism for controlling a broad array of proteins mediating cellular adaptation to metabolic fuels. Acetylation is governed, in part, by SIRTs (sirtuins), class III NAD(+)-dependent deacetylases that regulate lipid and glucose metabolism in liver during fasting and aging. However, the role of acetylation or SIRTs in pathogenic hepatic fuel metabolism under nutrient excess is unknown. In the present study, we isolated acetylated proteins from total liver proteome and observed 193 preferentially acetylated proteins in mice fed on an HFD (high-fat diet) compared with controls, including 11 proteins not previously identified in acetylation studies. Exposure to the HFD led to hyperacetylation of proteins involved in gluconeogenesis, mitochondrial oxidative metabolism, methionine metabolism, liver injury and the ER (endoplasmic reticulum) stress response. Livers of mice fed on the HFD had reduced SIRT3 activity, a 3-fold decrease in hepatic NAD(+) levels and increased mitochondrial protein oxidation. In contrast, neither SIRT1 nor histone acetyltransferase activities were altered, implicating SIRT3 as a dominant factor contributing to the observed phenotype. In Sirt3⁻(/)⁻ mice, exposure to the HFD further increased the acetylation status of liver proteins and reduced the activity of respiratory complexes III and IV. This is the first study to identify acetylation patterns in liver proteins of HFD-fed mice. Our results suggest that SIRT3 is an integral regulator of mitochondrial function and its depletion results in hyperacetylation of critical mitochondrial proteins that protect against hepatic lipotoxicity under conditions of nutrient excess.

Clinical and biochemical characterization of four patients with mutations in ECHS1

BackgroundShort-chain enoyl-CoA hydratase (SCEH, encoded by ECHS1) catalyzes hydration of 2-trans-enoyl-CoAs to 3(S)-hydroxy-acyl-CoAs. SCEH has a broad substrate specificity and is believed to play an important role in mitochondrial fatty acid oxidation and in the metabolism of branched-chain amino acids. Recently, the first patients with SCEH deficiency have been reported revealing only a defect in valine catabolism. We investigated the role of SCEH in fatty acid and branched-chain amino acid metabolism in four newly identified patients. In addition, because of the Leigh-like presentation, we studied enzymes involved in bioenergetics.MethodsMetabolite, enzymatic, protein and genetic analyses were performed in four patients, including two siblings. Palmitate loading studies in fibroblasts were performed to study mitochondrial β-oxidation. In addition, enoyl-CoA hydratase activity was measured with crotonyl-CoA, methacrylyl-CoA, tiglyl-CoA and 3-methylcrotonyl-CoA both in fibroblasts and liver to further study the role of SCEH in different metabolic pathways. Analyses of pyruvate dehydrogenase and respiratory chain complexes were performed in multiple tissues of two patients.ResultsAll patients were either homozygous or compound heterozygous for mutations in the ECHS1 gene, had markedly reduced SCEH enzymatic activity and protein level in fibroblasts. All patients presented with lactic acidosis. The first two patients presented with vacuolating leukoencephalopathy and basal ganglia abnormalities. The third patient showed a slow neurodegenerative condition with global brain atrophy and the fourth patient showed Leigh-like lesions with a single episode of metabolic acidosis. Clinical picture and metabolite analysis were not consistent with a mitochondrial fatty acid oxidation disorder, which was supported by the normal palmitate loading test in fibroblasts. Patient fibroblasts displayed deficient hydratase activity with different substrates tested. Pyruvate dehydrogenase activity was markedly reduced in particular in muscle from the most severely affected patients, which was caused by reduced expression of E2 protein, whereas E2 mRNA was increased.ConclusionsDespite its activity towards substrates from different metabolic pathways, SCEH appears to be only crucial in valine metabolism, but not in isoleucine metabolism, and only of limited importance for mitochondrial fatty acid oxidation. In severely affected patients SCEH deficiency can cause a secondary pyruvate dehydrogenase deficiency contributing to the clinical presentation.

Mutations in the mitochondrial cysteinyl-tRNA synthase gene, CARS2, lead to a severe epileptic encephalopathy and complex movement disorder

Background Mitochondrial disease is often suspected in cases of severe epileptic encephalopathy especially when a complex movement disorder, liver involvement and progressive developmental regression are present. Although mutations in either mitochondrial DNA or POLG are often present, other nuclear defects in mitochondrial DNA replication and protein translation have been associated with a severe epileptic encephalopathy. Methods and results We identified a proband with an epileptic encephalopathy, complex movement disorder and a combined mitochondrial respiratory chain enzyme deficiency. The child presented with neurological regression, complex movement disorder and intractable seizures. A combined deficiency of mitochondrial complexes I, III and IV was noted in liver tissue, along with increased mitochondrial DNA content in skeletal muscle. Incomplete assembly of complex V, using blue native polyacrylamide gel electrophoretic analysis and complex I, using western blotting, suggested a disorder of mitochondrial transcription or translation. Exome sequencing identified compound heterozygous mutations in CARS2, a mitochondrial aminoacyl-tRNA synthetase. Both mutations affect highly conserved amino acids located within the functional ligase domain of the cysteinyl-tRNA synthase. A specific decrease in the amount of charged mt-tRNACys was detected in patient fibroblasts compared with controls. Retroviral transfection of the wild-type CARS2 into patient skin fibroblasts led to the correction of the incomplete assembly of complex V, providing functional evidence for the role of CARS2 mutations in disease aetiology. Conclusions Our findings indicate that mutations in CARS2 result in a mitochondrial translational defect as seen in individuals with mitochondrial epileptic encephalopathy.

Mitochondrial energy failure in HSD10 disease is due to defective mtDNA transcript processing

Muscle, heart and liver were analyzed in a male subject who succumbed to HSD10 disease. Respiratory chain enzyme analysis and BN-PAGE showed reduced activities and assembly of complexes I, III, IV, and V. The mRNAs of all RNase P subunits were preserved in heart and overexpressed in muscle, but MRPP2 protein was severely decreased. RNase P upregulation correlated with increased expression of mitochondrial biogenesis factors and preserved mitochondrial enzymes in muscle, but not in heart where this compensatory mechanism was incomplete. We demonstrate elevated amounts of unprocessed pre-tRNAs and mRNA transcripts encoding mitochondrial subunits indicating deficient RNase P activity. This study provides evidence of abnormal mitochondrial RNA processing causing mitochondrial energy failure in HSD10 disease.

A new mutation in MT-ND1 m.3928G>C p.V208L causes Leigh disease with infantile spasms

New mutations in mitochondrial DNA encoded genes of complex I are rarely reported. An infant developed Leigh disease with infantile spasms. Complex I enzyme activity was deficient and response to increasing coenzyme Q concentrations was reduced. Complex I assembly was intact. A new mutation in MT-ND1 m.3928G>C p.V208L, affecting a conserved amino acid in a critical domain, part of the coenzyme Q binding pocket, was present at high heteroplasmy. The unaffected mother did not carry measurable mutant mitochondrial DNA, but concern remained for gonadal mosaicism. Prenatal testing was possible for a subsequent sibling. The ND1 p.V208L mutation causes Leigh disease.

Mutations in the accessory subunit NDUFB10 result in isolated complex I deficiency and illustrate the critical role of intermembrane space import for complex I holoenzyme assembly

An infant presented with fatal infantile lactic acidosis and cardiomyopathy, and was found to have profoundly decreased activity of respiratory chain complex I in muscle, heart and liver. Exome sequencing revealed compound heterozygous mutations in NDUFB10, which encodes an accessory subunit located within the PD part of complex I. One mutation resulted in a premature stop codon and absent protein, while the second mutation replaced the highly conserved cysteine 107 with a serine residue. Protein expression of NDUFB10 was decreased in muscle and heart, and less so in the liver and fibroblasts, resulting in the perturbed assembly of the holoenzyme at the 830 kDa stage. NDUFB10 was identified together with three other complex I subunits as a substrate of the intermembrane space oxidoreductase CHCHD4 (also known as Mia40). We found that during its mitochondrial import and maturation NDUFB10 transiently interacts with CHCHD4 and acquires disulfide bonds. The mutation of cysteine residue 107 in NDUFB10 impaired oxidation and efficient mitochondrial accumulation of the protein and resulted in degradation of non-imported precursors. Our findings indicate that mutations in NDUFB10 are a novel cause of complex I deficiency associated with a late stage assembly defect and emphasize the role of intermembrane space proteins for the efficient assembly of complex I.

Potentially Diagnostic Electron Paramagnetic Resonance Spectra Elucidate the Underlying Mechanism of Mitochondrial Dysfunction in the Deoxyguanosine Kinase Deficient Rat Model of a Genetic Mitochondrial DNA Depletion Syndrome

A novel rat model for a well-characterized human mitochondrial disease, mitochondrial DNA depletion syndrome with associated deoxyguanosine kinase (DGUOK) deficiency, is described. The rat model recapitulates the pathologic and biochemical signatures of the human disease. The application of electron paramagnetic (spin) resonance (EPR) spectroscopy to the identification and characterization of respiratory chain abnormalities in the mitochondria from freshly frozen tissue of the mitochondrial disease model rat is introduced. EPR is shown to be a sensitive technique for detecting mitochondrial functional abnormalities in situ and, here, is particularly useful in characterizing the redox state changes and oxidative stress that can result from depressed expression and/or diminished specific activity of the distinct respiratory chain complexes. As EPR requires no sample preparation or non-physiological reagents, it provides information on the status of the mitochondrion as it was in the functioning state. On its own, this information is of use in identifying respiratory chain dysfunction; in conjunction with other techniques, the information from EPR shows how the respiratory chain is affected at the molecular level by the dysfunction. It is proposed that EPR has a role in mechanistic pathophysiological studies of mitochondrial disease and could be used to study the impact of new treatment modalities or as an additional diagnostic tool.

New insights into the phenotype of FARS2 deficiency.

Mutations in FARS2 are known to cause dysfunction of mitochondrial translation due to deficient aminoacylation of the mitochondrial phenylalanine tRNA. Here, we report three novel mutations in FARS2 found in two patients in a compound heterozygous state. The missense mutation c.1082C > T (p.Pro361Leu) was detected in both patients. The mutations c.461C > T (p.Ala154Val) and c.521_523delTGG (p.Val174del) were each detected in one patient. We report abnormal in vitro aminoacylation assays as a functional validation of the molecular genetic findings. Based on the phenotypic data of previously reported subjects and the two subjects reported here, we conclude that FARS2 deficiency can be associated with two phenotypes: (i) an epileptic phenotype, and (ii) a spastic paraplegia phenotype.

Activation of a cryptic splice site in the mitochondrial elongation factor GFM1 causes combined OXPHOS deficiency

We report the clinical, biochemical, and molecular findings in two brothers with encephalopathy and multi-systemic disease. Abnormal transferrin glycoforms were suggestive of a type I congenital disorder of glycosylation (CDG). While exome sequencing was negative for CDG related candidate genes, the testing revealed compound heterozygous mutations in the mitochondrial elongation factor G gene (GFM1). One of the mutations had been reported previously while the second, novel variant was found deep in intron 6, activating a cryptic splice site. Functional studies demonstrated decreased GFM1 protein levels, suggested disrupted assembly of mitochondrial complexes III and V and decreased activities of mitochondrial complexes I and IV, all indicating combined OXPHOS deficiency.

Pathogenic variants in glutamyl-tRNAGln amidotransferase subunits cause a lethal mitochondrial cardiomyopathy disorder

Mitochondrial protein synthesis requires charging a mitochondrial tRNA with its amino acid. Here, the authors describe pathogenic variants in the GatCAB protein complex genes required for the generation of glutaminyl-mt-tRNAGln, that impairs mitochondrial translation and presents with cardiomyopathy.AbstractMitochondrial protein synthesis requires charging mt-tRNAs with their cognate amino acids by mitochondrial aminoacyl-tRNA synthetases, with the exception of glutaminyl mt-tRNA (mt-tRNAGln). mt-tRNAGln is indirectly charged by a transamidation reaction involving the GatCAB aminoacyl-tRNA amidotransferase complex. Defects involving the mitochondrial protein synthesis machinery cause a broad spectrum of disorders, with often fatal outcome. Here, we describe nine patients from five families with genetic defects in a GatCAB complex subunit, including QRSL1, GATB, and GATC, each showing a lethal metabolic cardiomyopathy syndrome. Functional studies reveal combined respiratory chain enzyme deficiencies and mitochondrial dysfunction. Aminoacylation of mt-tRNAGln and mitochondrial protein translation are deficient in patients’ fibroblasts cultured in the absence of glutamine but restore in high glutamine. Lentiviral rescue experiments and modeling in S. cerevisiae homologs confirm pathogenicity. Our study completes a decade of investigations on mitochondrial aminoacylation disorders, starting with DARS2 and ending with the GatCAB complex.