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Inactivation of mitochondrial aspartate aminotransferase contributes to the respiratory deficit of yeast frataxin-deficient cells Dominika Sliwa, Julien Dairou, Jean-Michel Camadro, Renata Santos To cite this version: Dominika Sliwa, Julien Dairou, Jean-Michel Camadro, Renata Santos. Inactivation of mitochondrial aspartate aminotransferase contributes to the respiratory deficit of yeast frataxin-deficient cells. Bio- chemical Journal, Portland Press, 2012, 441 (3), pp.945 - 953. 10.1042/BJ20111574. hal-02328543 HAL Id: hal-02328543 https://hal.archives-ouvertes.fr/hal-02328543 Submitted on 23 Oct 2019 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. www.biochemj.org Biochem. J. (2012) 441, 945–953 (Printed in Great Britain) doi:10.1042/BJ20111574 945 Inactivation of mitochondrial aspartate aminotransferase contributes to the respiratory deficit of yeast frataxin-deficient cells Dominika SLIWA*, Julien DAIROU†, Jean-Michel CAMADRO* and Renata SANTOS*1 *Institut Jacques Monod, CNRS-Universite´ Paris Diderot, Sorbonne Paris Cite,´ 15 rue Hel´ ene` Brion, 75205 Paris cedex 13, France, and †Unite´ BFA (EAC 4413), UniversiteParis´ Diderot, Sorbonne Paris Cite-CNRS,´ 4 rue Marie Andree´ Lagroua Weill Halle, 75205 Paris Cedex 13, France Friedreich’s ataxia is a hereditary neurodegenerative disease deacetylated in yfh1 mitochondria suggesting that inactivation caused by reduced expression of mitochondrial frataxin. Frataxin could be due to this post-translational modification. Mutants deficiency causes impairment in respiratory capacity, disruption deficient in iron–sulfur cluster assembly or lacking mitochondrial of iron homoeostasis and hypersensitivity to oxidants. Although DNA also showed decreased activity of Aat1, suggesting that the redox properties of NAD (NAD + and NADH) are essential Aat1 inactivation was a secondary phenotype in yfh1 cells. for energy metabolism, only few results are available concerning Interestingly, deletion of the AAT1 gene in a wild-type strain homoeostasis of these nucleotides in frataxin-deficient cells. In the caused respiratory deficiency and disruption of iron homoeostasis present study, we show that the malate–aspartate NADH shuttle without any sensitivity to oxidative stress. Our results show that is impaired in Saccharomyces cerevisiae frataxin-deficient cells secondary inactivation of Aat1 contributes to the amplification of (yfh1) due to decreased activity of cytosolic and mitochondrial the respiratory defect observed in yfh1 cells. Further implication isoforms of malate dehydrogenase and to complete inactivation of mitochondrial protein deacetylation in the physiology of of the mitochondrial aspartate aminotransferase (Aat1). A frataxin-deficient cells is anticipated. considerable decrease in the amount of mitochondrial acetylated proteins was observed in the yfh1 mutant compared with Key words: frataxin, Friedreich’s ataxia, malate–aspartate NADH wild-type. Aat1 is acetylated in wild-type mitochondria and shuttle, mitochondrion, protein acetylation, yeast. INTRODUCTION significantly increased in the heart of transgenic mice ubiquitously overexpressing frataxin compared with control animals, FA (Friedreich’s ataxia) is the most common autosomal recessive indicating enhanced mitochondrial energy conversion and inherited ataxia. It is a slowly progressive neurodegenerative antioxidant defence capacity with increasing frataxin levels [8]. disease that affects the central and peripheral nervous system. Besides being an enzymatic oxidoreductase cofactor, NAD + Biochemical Journal Other organs can be affected in FA; 60% of patients develop is the substrate of enzymes that catalyse protein modification cardiomyopathy and almost 40% of patients present diabetes or reactions. In particular, the SIRT (sirtuin)-mediated protein glucose intolerance [1]. The most common mutation observed in deacetylation and the ADP-ribosylation, catalysed by PARP FA patients is an unstable hyperexpansion of a GAA trinucleotide [poly(ADP-ribose) polymerase] family, regulate gene expression, repeat in the first intron of the FXN gene that leads to the formation DNA repair, aging, calcium signalling, generation of ROS of a non-usual B-DNA structure and heterochromatin conforma- (reactive oxygen species) and cell death [9,10]. In yeast, NAD + tion that causes transcriptional silencing [2,3]. The consequence biosynthesis involves a de novo pathway from tryptophan and is decreased expression of the encoded protein, frataxin [4]. several salvage pathways from various precursors, such as Frataxin is a small mitochondrial matrix protein that participates nicotinic acid mononucleotide and nicotinamide riboside [11]. in iron–sulfur cluster assembly [5]. Frataxin deficiency induces NAD + biosynthesis appears to occur outside the mitochondria a decrease in the activities of iron–sulfur cluster enzymes and in because all enzymes are localized in the cytosol and the nucleus, respiratory capacity, perturbation of cellular iron homoeostasis with the exception of Bna4, which is a mitochondrial kynurenine and hypersensitivity to oxidants [6]. Therefore major phenotypes 3-mono-oxygenase required for de novo NAD + synthesis from of frataxin deficiency are related to energy metabolism and kynurenine. This implies that NAD + must be imported into the antioxidant response. Although pyridine nucleotides NAD mitochondria. Two mitochondrial NAD + transporters, Ndt1 and (NAD + and NADH) and NADP (NADP + and NADPH) are Ndt2, perform this function [12]. In addition, reducing equivalents electron carriers essential for energy production and defence from NADH can be exchanged between the cytosol and the against oxidative stress, little is known about the regulation of mitochondria by NADH shuttles, such as the glycerol 3-phosphate their pools in frataxin-deficient cells. We showed previously shuttle, the ethanol–acetaldehyde shuttle and the malate–aspartate that the NADPH/NADP + ratio was considerably lower in the shuttle [13,14]. The glycerol 3-phosphate shuttle only oxidizes yeast Saccharomyces cerevisiae frataxin-deficient mutant (yfh1) cytosolic NADH transferring the electrons directly to the compared with wild-type, which, in addition to the disturbed respiratory chain. This shuttle has two components, the cytosolic glutathione redox status, contributed to the severe oxidative stress glycerol-3-phosphate dehydrogenase (Gpd1/2) and the inner condition observed in yfh1 cells [7]. Another study showed that mitochondrial membrane glycerol-3-phosphate dehydrogenase the levels of reduced glutathione, ATP, NADPH and NADH were (Gut2). The ethanol–acetaldehyde shuttle is composed of Abbreviations used: 2D, two-dimensional; DTT, dithiothreitol; FA, Friedreich’s ataxia; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; HA, haemagglutinin; LDH, lactate dehydrogenase; PHO, phosphate-responsive signalling; PL, pyridoxal; PLP, pyridoxal 5-phosphate; PM, pyridoxamine; PN, pyridoxine; ROS, reactive oxygen species; SIRT, sirtuin; TCA, tricarboxylic acid. 1 To whom correspondence should be addressed (email [email protected]). c The Authors Journal compilation c 2012 Biochemical Society 946 D. Sliwa and others mitochondrial alcohol dehydrogenase (Adh3) that converts bromide, 1/100 diluted in the same medium and treated for ethanol into acetaldehyde, producing NADH and the cytosolic another 24 h. Isolated clones were verified by genetic crossing isoforms (Adh1/2) that perform the inverse reaction and synthes- with ρ0 tester strains and plating on YPGly medium (1% yeast ize NAD + in the cytosol. Acetaldehyde and ethanol freely diffuse extract, 2% Bacto peptone and 2% glycerol). Unless stated through mitochondrial membranes. The yeast malate–aspartate all experiments were carried out in YPD medium (1% yeast ◦ shuttle components include mitochondrial and cytosolic malate extract, 2% Bacto peptone and 2% D-glucose) at 30 C. All dehydrogenase (Mdh1/2) and aspartate aminotransferase (Aat1/2) media were supplemented with 200 μg/ml adenine. For growth and the mitochondrial aspartate/glutamate exchanger Agc1 [15]. under anaerobic conditions, the medium was supplemented with In yeast, sugar metabolism and biomass production (synthesis of 30 mg/l ergosterol and 2 ml/l Tween 80 (Jacomex anaerobic amino acids, nucleic acids and reduced lipids) result in NADH glove box; oxygen concentration below 5 p.p.m.). generation in the cytosol [13,14]. For the most part of cytosolic NADH, reoxidation to NAD + takes place in the mitochondria by the electron transport chain [14]. Under conditions where respir- Cell fractionation ation is repressed, glycerol production provides NAD + regener- Mitochondrial and extra-mitochondrial fractions were prepared ation in the cytosol [14]. Therefore NADH shuttles are required from stationary-phase cultured cells (D600 ∼4) in YPD medium. for maintaining the NAD + /NADH ratios in the mitochondrial and Cell pellets were resuspended in ice-cold 50 mM potassium cytosolic NAD pools. The biological importance of this function phosphate buffer, pH 7.4, and 0.6 M
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  • GOT2 Antibody Cat

    GOT2 Antibody Cat

    GOT2 Antibody Cat. No.: 25-873 GOT2 Antibody Specifications HOST SPECIES: Rabbit SPECIES REACTIVITY: Human, Mouse, Rat Antibody produced in rabbits immunized with a synthetic peptide corresponding a region IMMUNOGEN: of human GOT2. TESTED APPLICATIONS: ELISA, WB GOT2 antibody can be used for detection of GOT2 by ELISA at 1:312500. GOT2 antibody APPLICATIONS: can be used for detection of GOT2 by western blot at 1 μg/mL, and HRP conjugated secondary antibody should be diluted 1:50,000 - 100,000. POSITIVE CONTROL: 1) 293T Cell Lysate PREDICTED MOLECULAR 45 kDa WEIGHT: Properties PURIFICATION: Antibody is purified by peptide affinity chromatography method. CLONALITY: Polyclonal CONJUGATE: Unconjugated PHYSICAL STATE: Liquid September 29, 2021 1 https://www.prosci-inc.com/got2-antibody-25-873.html Purified antibody supplied in 1x PBS buffer with 0.09% (w/v) sodium azide and 2% BUFFER: sucrose. CONCENTRATION: batch dependent For short periods of storage (days) store at 4˚C. For longer periods of storage, store GOT2 STORAGE CONDITIONS: antibody at -20˚C. As with any antibody avoid repeat freeze-thaw cycles. Additional Info OFFICIAL SYMBOL: GOT2 ALTERNATE NAMES: GOT2, FLJ40994, kat4, kativ, mitaat, KAT4, KATIV, mitAAT ACCESSION NO.: NP_002071 PROTEIN GI NO.: 73486658 GENE ID: 2806 USER NOTE: Optimal dilutions for each application to be determined by the researcher. Background and References Glutamic-oxaloacetic transaminase is a pyridoxal phosphate-dependent enzyme which exists in cytoplasmic and inner-membrane mitochondrial forms, GOT1 and GOT2, respectively. GOT plays a role in amino acid metabolism and the urea and tricarboxylic acid cycles. The two enzymes are homodimeric and show close homology.Glutamic- oxaloacetic transaminase is a pyridoxal phosphate-dependent enzyme which exists in BACKGROUND: cytoplasmic and inner-membrane mitochondrial forms, GOT1 and GOT2, respectively.