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Expression of human monolysocardiolipin acyltransferase-1 improves mitochondrial function in Barth syndrome lymphoblasts.

ABSTRACT: The mitochondrial polyglycerophospholipid cardiolipin (CL) is remodeled to obtain specific fatty acyl chains. This is predominantly accomplished by the transacylase enzyme tafazzin (TAZ). Barth syndrome (BTHS) patients with TAZ gene mutations exhibit impaired TAZ activity and loss in mitochondrial respiratory function. Previous studies identified monolysocardiolipin acyltransferase-1 (MLCL AT-1) as a mitochondrial enzyme capable of remodeling CL with fatty acid. In this study, we analyzed what relationship, if any, exists between TAZ and MLCL AT-1 with regard to CL remodeling and whether transfection of BTHS lymphoblasts with an MLCL AT-1 expression construct improves mitochondrial respiratory function. In healthy lymphoblasts, reduction in TAZ expression through TAZ RNAi transfection resulted in a compensatory increase in MLCL AT-1 mRNA, protein, and enzyme activity, but CL mass was unaltered. In contrast, BTHS lymphoblasts exhibited decreased TAZ gene and protein expression but in addition decreased MLCL AT-1 expression and CL mass. Transfection of BTHS lymphoblasts with MLCL AT-1 expression construct increased CL, improved mitochondrial basal respiration and protein leak, and decreased the proportion of cells producing superoxide but did not restore CL molecular species composition to control levels. In addition, BTHS lymphoblasts exhibited higher rates of glycolysis compared with healthy controls to compensate for reduced mitochondrial respiratory function. Mitochondrial supercomplex assembly was significantly impaired in BTHS lymphoblasts, and transfection of BTHS lymphoblasts with MLCL AT-1 expression construct did not restore supercomplex assembly. The results suggest that expression of MLCL AT-1 depends on functional TAZ in healthy cells. In addition, transfection of BTHS lymphoblasts with an MLCL AT-1 expression construct compensates, but not completely, for loss of mitochondrial respiratory function.

SUBMITTER: Mejia EM

PROVIDER: S-EPMC5961033 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Expression of human monolysocardiolipin acyltransferase-1 improves mitochondrial function in Barth syndrome lymphoblasts.

Mejia Edgard M EM Zegallai Hana H Bouchard Eric D ED Banerji Versha V Ravandi Amir A Hatch Grant M GM

The Journal of biological chemistry 20180321 20

The mitochondrial polyglycerophospholipid cardiolipin (CL) is remodeled to obtain specific fatty acyl chains. This is predominantly accomplished by the transacylase enzyme tafazzin (TAZ). Barth syndrome (BTHS) patients with <i>TAZ</i> gene mutations exhibit impaired TAZ activity and loss in mitochondrial respiratory function. Previous studies identified monolysocardiolipin acyltransferase-1 (MLCL AT-1) as a mitochondrial enzyme capable of remodeling CL with fatty acid. In this study, we analyzed ...[more]

PMID: 29563154

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Project description:Here we report the identification of a previously uncharacterized human protein as the human monolysocardiolipin acyltransferase-1 (MLCL AT-1). Pig liver mitochondria were treated with n-butyl alcohol followed by Q-Sepharose chromatography, preparative gel electrophoresis, cytidine diphosphate-1,2-diacyl-sn-glycerol-Sepharose chromatography, and finally monolysocardiolipin-adriamycin-agarose affinity chromatography. Elution with either monolysocardiolipin or linoleoyl coenzyme A revealed a major band at 74 kDa with high specific activity (2,300 pmol/min/mg) for the acylation of monolysocardiolipin to cardiolipin using [1-(14)C]linoleoyl coenzyme A as substrate. Matrix-assisted laser desorption ionization time-of-flight-mass spectrometry analysis followed by search of the Mascot protein data base revealed peptide matches consistent with a 59-kDa protein identified as unknown human protein (GenBank(TM) protein accession number AAX93141; nucleotide accession number AC011742.3). The purified human recombinant MLCL AT-1 protein utilized linoleoyl coenzyme A > oleoyl coenzyme A > palmitoyl coenzyme A for the specific acylation of monolysocardiolipin to cardiolipin. Expression of MLCL AT-1 in HeLa cells increased mitochondrial monolysocardiolipin acyltransferase activity and [1-(14)C]linoleic acid incorporated into cardiolipin, whereas RNA interference knockdown of MLCL AT-1 in HeLa cells resulted in reduction in enzyme activity and [1-(14)C]linoleic acid incorporated into cardiolipin. In contrast, expression of MLCL AT-1 in HeLa cells did not alter [1-(14)C]oleic or [1-(14)C]palmitate incorporation into cardiolipin indicating in vivo specificity for the remodeling of cardiolipin with linoleate. Finally, expression of MLCL AT-1 in Barth syndrome lymphoblasts, which exhibit cardiolipin levels 20% that of normal lymphoblasts, increased mitochondrial monolysocardiolipin acyltransferase activity, [1-(14)C]linoleic acid incorporation into cardiolipin, cardiolipin mass, and succinate dehydrogenase (mitochondrial complex II) activity compared with mock-transfected Barth syndrome lymphoblasts. The results identify MLCL AT-1 as a human mitochondrial monolysocardiolipin acyltransferase involved in the remodeling of cardiolipin.

Project description:BackgroundCardiomyopathy is a major clinical feature in Barth syndrome (BTHS), an X-linked mitochondrial lipid disorder caused by mutations in Tafazzin (TAZ), encoding a mitochondrial acyltransferase required for cardiolipin remodeling. Despite recent description of a mouse model of BTHS cardiomyopathy, an in-depth analysis of specific lipid abnormalities and mitochondrial form and function in an in vivo BTHS cardiomyopathy model is lacking.MethodsWe performed in-depth assessment of cardiac function, cardiolipin species profiles, and mitochondrial structure and function in our newly generated Taz cardiomyocyte-specific knockout mice and Cre-negative control mice (n≥3 per group).ResultsTaz cardiomyocyte-specific knockout mice recapitulate typical features of BTHS and mitochondrial cardiomyopathy. Fewer than 5% of cardiomyocyte-specific knockout mice exhibited lethality before 2 months of age, with significantly enlarged hearts. More than 80% of cardiomyocyte-specific knockout displayed ventricular dilation at 16 weeks of age and survived until 50 weeks of age. Full parameter analysis of cardiac cardiolipin profiles demonstrated lower total cardiolipin concentration, abnormal cardiolipin fatty acyl composition, and elevated monolysocardiolipin to cardiolipin ratios in Taz cardiomyocyte-specific knockout, relative to controls. Mitochondrial contact site and cristae organizing system and F1F0-ATP synthase complexes, required for cristae morphogenesis, were abnormal, resulting in onion-shaped mitochondria. Organization of high molecular weight respiratory chain supercomplexes was also impaired. In keeping with observed mitochondrial abnormalities, seahorse experiments demonstrated impaired mitochondrial respiration capacity.ConclusionsOur mouse model mirrors multiple physiological and biochemical aspects of BTHS cardiomyopathy. Our results give important insights into the underlying cause of BTHS cardiomyopathy and provide a framework for testing therapeutic approaches to BTHS cardiomyopathy, or other mitochondrial-related cardiomyopathies.

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Expression of human monolysocardiolipin acyltransferase-1 improves mitochondrial function in Barth syndrome lymphoblasts.

Publications

Expression of human monolysocardiolipin acyltransferase-1 improves mitochondrial function in Barth syndrome lymphoblasts.

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