The mitochondrial quality control protein Yme1 is necessary to prevent defective mitophagy in a yeast model of Barth syndrome.

The Saccharomyces cerevisiae TAZ1 gene is an orthologue of human TAZ; both encode the protein tafazzin. Tafazzin is a transacylase that transfers acyl chains with unsaturated fatty acids from phospholipids to monolysocardiolipin to generate cardiolipin with unsaturated fatty acids. Mutations in human TAZ cause Barth syndrome, a fatal childhood cardiomyopathy biochemically characterized by reduced cardiolipin mass and increased monolysocardiolipin levels. To uncover cellular processes that require tafazzin to maintain cell health, we performed a synthetic genetic array screen using taz1Δ yeast cells to identify genes whose deletion aggravated its fitness. The synthetic genetic array screen uncovered several mitochondrial cellular processes that require tafazzin. Focusing on the i-AAA protease Yme1, a mitochondrial quality control protein that degrades misfolded proteins, we determined that in cells lacking both Yme1 and Taz1 function, there were substantive mitochondrial ultrastructural defects, ineffective superoxide scavenging, and a severe defect in mitophagy. We identify an important role for the mitochondrial protease Yme1 in the ability of cells that lack tafazzin function to maintain mitochondrial structural integrity and mitochondrial quality control and to undergo mitophagy.

A novel ß-adrenergic response element regulates both basal and agonist-induced expression of cyclin-dependent kinase 1 gene in cardiac fibroblasts.

Cardiac fibrosis, a known risk factor for heart disease, is typically caused by uncontrolled proliferation of fibroblasts and excessive deposition of extracellular matrix proteins in the myocardium. Cyclin-dependent kinase 1 (CDK1) is involved in the control of G2/M transit phase of the cell cycle. Here, we showed that isoproterenol (ISO)-induced cardiac fibrosis is associated with increased levels of CDK1 exclusively in fibroblasts in the adult mouse heart. Treatment of primary embryonic ventricular cell cultures with ISO (a nonselective ß-adrenergic receptor agonist) increased CDK1 protein expression in fibroblasts and promoted their cell cycle activity. Quantitative PCR analysis confirmed that ISO increases CDK1 transcription in a transient manner. Further, the ISO-responsive element was mapped to the proximal -100-bp sequence of the CDK1 promoter region using various 5'-flanking sequence deletion constructs. Sequence analysis of the -100-bp CDK1 minimal promoter region revealed two putative nuclear factor-Y (NF-Y) binding elements. Overexpression of the NF-YA subunit in primary ventricular cultures significantly increased the basal activation of the -100-bp CDK1 promoter construct but not the ISO-induced transcription of the minimal promoter construct. In contrast, dominant negative NF-YA expression decreased the basal activity of the minimal promoter construct and ISO treatment fully rescued the dominant negative effects. Furthermore, site-directed mutagenesis of the distal NF-Y binding site in the -100-bp CDK1 promoter region completely abolished both basal and ISO-induced promoter activation of the CDK1 gene. Collectively, our results raise an exciting possibility that targeting CDK1 or NF-Y in the diseased heart may inhibit fibrosis and subsequently confer cardioprotection.

NTE1-encoded phosphatidylcholine phospholipase b regulates transcription of phospholipid biosynthetic genes.

The Saccharomyces cerevisiae NTE1 gene encodes an evolutionarily conserved phospholipase B localized to the endoplasmic reticulum (ER) that degrades phosphatidylcholine (PC) generating glycerophosphocholine and free fatty acids. We show here that the activity of NTE1-encoded phospholipase B (Nte1p) prevents the attenuation of transcription of genes encoding enzymes involved in phospholipid synthesis in response to increased rates of PC synthesis by affecting the nuclear localization of the transcriptional repressor Opi1p. Nte1p activity becomes necessary for cells growing in inositol-free media under conditions of high rates of PC synthesis elicited by the presence of choline at 37 degrees C. The specific choline transporter encoded by the HNM1 gene is necessary for the burst of PC synthesis observed at 37 degrees C as follows: (i) Nte1p is dispensable in an hnm1Delta strain under these conditions, and (ii) there is a 3-fold increase in the rate of choline transport via the Hnm1p choline transporter upon a shift to 37 degrees C. Overexpression of NTE1 alleviated the inositol auxotrophy of a plethora of mutants, including scs2Delta, scs3Delta, ire1Delta, and hac1Delta among others. Overexpression of NTE1 sustained phospholipid synthesis gene transcription under conditions that normally repress transcription. This effect was also observed in a strain defective in the activation of free fatty acids for phosphatidic acid synthesis. No changes in the levels of phosphatidic acid were detected under conditions of altered expression of NTE1. Consistent with a synthetic impairment between challenged ER function and inositol deprivation, increased expression of NTE1 improved the growth of cells exposed to tunicamycin in the absence of inositol. We describe a new role for Nte1p toward membrane homeostasis regulating phospholipid synthesis gene transcription. We propose that Nte1p activity, by controlling PC abundance at the ER, affects lateral membrane packing and that this parameter, in turn, impacts the repressing transcriptional activity of Opi1p, the main regulator of phospholipid synthesis gene transcription.

Quantification of cardiac fibrosis by colour-subtractive computer-assisted image analysis.

1. Quantification of fibrosis is a key parameter in the assessment of the severity of cardiovascular disease and efficacy of future candidate therapies. Computer-assisted methods are frequently used to assess cardiac fibrosis in several experimental models. A brief survey indicated that there is a clear dearth of literature outlining detailed methodologies for computer-based assessment of cardiac fibrosis. The purpose of the present study was to provide a reliable method for a systematic assessment of cardiac fibrosis. 2. We induced cardiac fibrosis by isoproterenol (ISO) infusion in adult CD1 male mice and quantified fibrosis using a recently developed colour-subtractive computer-assisted image analysis (CS-CAIA) technique. Here, we provided a detailed description of our methodology to facilitate its wider use by other researchers. 3. We showed that the severity of ISO-induced cardiac fibrosis was similar in the apex, mid-ventricular ring and base of the adult CD1 mouse heart. In contrast with other species, such as rats and dogs, we found that uniform expression of beta(1)-adrenoceptors between different regions in CD1 mouse hearts correlated well with uniform induction of cardiac fibrosis. 4. A previous study found a negative correlation between levels of myocardial fibrosis and the degree of cardiac hypertrophy in ISO-treated Wistar rats. In contrast, we found a similar degree of cardiac fibrosis in our ISO-treated CD1 mice. 5. Our results suggest that CD1 mice are an ideal model system to study catecholamine-induced cardiac remodelling, as well as to screen candidate antifibrotic agents for future therapies.

Cardiolipin metabolism and its causal role in the etiology of the inherited cardiomyopathy Barth syndrome.

Cardiolipin (CL) is a phospholipid with many unique characteristics. CL is synthesized in the mitochondria and resides almost exclusively within the mitochondrial inner membrane. Unlike most phospholipids that have two fatty acyl chains, CL possesses four fatty acyl chains resulting in unique biophysical characteristics that impact several biological processes including membrane fission and fusion. In addition, several proteins directly bind CL including proteins within the electron transport chain, the ADP/ATP carrier, and proteins that mediate mitophagy. Tafazzin is an enzyme that remodels saturated fatty acyl chains within CL to unsaturated fatty acyl chains, loss of function mutations in the TAZ gene encoding tafazzin are causal for the inherited cardiomyopathy Barth syndrome. Cells from Barth syndrome patients as well as several models of Barth have reduced mitochondrial functions including impaired electron transport chain function and increased reactive oxygen species (ROS) production. Mitochondria in cells from Barth syndrome patients, as well as several model organism mimics of Barth syndrome, are large and lack cristae consistent with the recently described role of CL participating in the generation of mitochondrial membrane contact sites. Cells with an inactive TAZ gene have also been shown to have a decreased capacity to undergo mitophagy when faced with stresses such as increased ROS or decreased mitochondrial quality control. This review describes CL metabolism and how defects in CL metabolism cause Barth syndrome, the etiology of Barth syndrome, and known modifiers of Barth syndrome phenotypes some of which could be explored for their amelioration of Barth syndrome in higher organisms.

The Kap60-Kap95 karyopherin complex directly regulates phosphatidylcholine synthesis.

Phosphatidylcholine is the major phospholipid in eukaryotic cells. There are two main pathways for the synthesis of phosphatidylcholine: the CDP-choline pathway present in all eukaryotes and the phosphatidylethanolamine methylation pathway present in mammalian hepatocytes and some single celled eukaryotes, including the yeast Saccharomyces cerevisiae. In S. cerevisiae, the rate-determining step in the synthesis of phosphatidylcholine via the CDP-choline pathway is catalyzed by Pct1. Pct1 converts phosphocholine and CTP to CDP-choline and pyrophosphate. In this study, we determined that Pct1 is in the nucleoplasm and at endoplasmic reticulum and nuclear membranes. Pct1 directly interacts with the alpha-importin Kap60 via a bipartite basic region in Pct1, and this region of Pct1 was required for its entry into the nucleus. Pct1 also interacted with the beta-importin Kap95 in cell extracts, implying a model whereby Pct1 interacts with Kap60 and Kap95 with this tripartite complex transiting the nuclear pore. Exclusion of Pct1 from the nucleus by elimination of its nuclear localization signal or by decreasing Kap60 function did not affect the level of phosphatidylcholine synthesis. Diminution of Kap95 function resulted in almost complete ablation of phosphatidylcholine synthesis under conditions where Pct1 was extranuclear. The beta-importin Kap95 is a direct regulator membrane synthesis.