Tafazzin Expression Research Articles

MAPK and Hippo signaling pathways crosstalk via the RAF-1/MST-2 interaction in malignant melanoma.

The aim of the present study was to expound on the interactions between the mitogen-activated protein kinase (MAPK) and Hippo pathway members, and to further elucidate the molecular mechanisms of melanoma tumorigenesis. Four melanoma cell lines (C32, HS695T, SK-MEL-28 and A375) were used in the present study. Western blotting was used to assess the expression levels of the MAPK and Hippo pathway effector proteins: rapidly accelerated fibrosarcoma-1 proto-oncogene, serine/threonine kinase (RAF-1); serine/threonine kinase3 (STK3; also known as MST-2); yes-associated protein (YAP); and tafazzin (TAZ). Immunoprecipitation was used to identify interactions between the effector proteins of the Hippo and MAPK pathways. RAF-1 was knocked down in melanoma cells using siRNA transfection, and cell proliferation, migration and invasion were determined by the MTT, wound-healing and Transwell invasion assays, respectively. Additionally, the cell cycle and apoptosis were analyzed by flow cytometry 48h after RAF-1 knockdown. We found that the expression levels of the four proteins were variable, and that the HS695T cells expressed the highest levels of RAF-1. Immunoprecipitation studies revealed that RAF-1 bound to MST-2 in melanoma cells. Knockdown of RAF-1 inhibited the expression of YAP and TAZ, but did not affect MST-2 expression. Additionally, RAF-1 knockdown in melanoma cells significantly inhibited cell proliferation, migration and invasion, and induced apoptosis in these cells. Collectively, our results indicate that the RAF-1/MST-2 interaction may be a novel link between the MAPK and Hippo pathways.

187. AAV Delivery of Tafazzin for the Correction of Mitochondrial Dysfunction in Barth Syndrome Using IPS Differentiated Cardiomyocytes

Barth Syndrome (BTHS) is caused by a single gene mutation affecting mitochondria and resulting in dilated cardiomyopathy, growth delay, neutropenia and severe exercise intolerance. This gene, Tafazzin (TAZ) located at Xq28, is responsible for cardiolipin (CL) remodeling in the inner mitochondrial membrane. Mutation/s in TAZ result in altered remodeling of CL and increasing Monolysocardiolpin (MLCL): CL ratios. This imbalance leads to abnormal mitochondrial cristae formation and decreased oxidative phosphorylation resulting in decreased ATP production and energy metabolism. Since BTHS is the result of a single gene defect, it is an ideal candidate for treatment by gene therapy. Therefore, we hypothesized that delivery of TAZ by adeno-associated virus (AAV) should increase TAZ gene expression and function, correcting the CL remodeling. Preliminary data, using fibroblasts directly differentiated to muscle resulted in increased gene expression of TAZ and a return to normal levels of CL. To further evaluate this gene therapy for effectiveness in the heart, we utilized patient derived induced pluripotent stem cells (IPSC) which were differentiated to cardiomyocytes. Upon confirmation of differentiation, control and diseased cells were treated with either AAV-TAZ or sham. TAZ gene and protein expression were measured as well as TAZ function through CL analysis. Muscle weakness and exercise intolerance have been described by patients, therefore to further stress the cardiomyocytes, cells were placed in hypoxia under nutrient limiting conditions and ATP production measured after 24 hours. Additional studies are being performed to evaluate mitochondrial activity as well as preclinical in vivo analysis.

The cellular and molecular mechanisms for neutropenia in Barth syndrome

Barth syndrome (BTHS), a rare, X-linked, recessive disease, is characterized by neutropenia and cardiomyopathy. BTHS is caused by loss-of-function mutations of the tafazzin (TAZ) gene. We developed a model of BTHS by transfecting human HL60 myeloid progenitor cells with TAZ-specific shRNAs. Results demonstrate a significant downregulation in TAZ expression, mimicking the effects of naturally occurring truncation mutations in TAZ. Flow cytometry analyses of cells with TAZ-specific, but not scrambled, shRNAs demonstrate nearly twofold increase in the proportion of annexin V-positive cells and significantly increased dissipation of mitochondrial membrane potential as determined by DIOC6 staining. Transfection of TAZ-specific shRNA had similar effects in U937 myeloid cells but not in lymphoid cell lines. Further studies in HL60 myeloid progenitor cells revealed aberrant release of cytochrome c from mitochondria and significantly elevated levels of activated caspase-3 in response to TAZ knockdown. Treatment with caspase-specific inhibitor zVAD-fmk resulted in substantially reduced apoptosis to near-normal levels. These data suggest that neutropenia in BTHS is attributable to increased dissipation of mitochondrial membrane potential, aberrant release of cytochrome c, activation of caspase-3, and accelerated apoptosis of myeloid progenitor cells, and that this defect can be partially restored in vitro by treatment with caspase-specific inhibitors.

The dynamics of cardiolipin synthesis post-mitochondrial fusion

Alteration in mitochondrial fusion may regulate mitochondrial metabolism. Since the phospholipid cardiolipin (CL) is required for function of the mitochondrial respiratory chain, we examined the dynamics of CL synthesis in growing Hela cells immediately after and 12 h post-fusion. Cells were transiently transfected with Mfn-2, to promote fusion, or Mfn-2 expressing an inactive GTPase for 24 h and de novo CL biosynthesis was examined immediately after or 12 h post-fusion. Western blot analysis confirmed elevated Mfn-2 expression and electron microscopic analysis revealed that Hela cell mitochondrial structure was normal immediately after and 12 h post-fusion. Cells expressing Mfn-2 exhibited reduced CL de novo biosynthesis from [1,3- 3H]glycerol immediately after fusion and this was due to a decrease in phosphatidylglycerol phosphate synthase (PGPS) activity and its mRNA expression. In contrast, 12 h post-mitochondrial fusion cells expressing Mfn-2 exhibited increased CL de novo biosynthesis from [1,3- 3H]glycerol and this was due to an increase in PGPS activity and its mRNA expression. Cells expressing Mfn-2 with an inactive GTPase activity did not exhibit alterations in CL de novo biosynthesis immediately after or 12 h post-fusion. The Mfn-2 mediated alterations in CL de novo biosynthesis were not accompanied by alterations in CL or monolysoCL mass. [1- 14C]Oleate incorporation into CL was elevated at 12 h post-fusion indicating increased CL resynthesis. The reason for the increased CL resynthesis was an increased mRNA expression of tafazzin, a mitochondrial CL resynthesis enzyme. Ceramide-induced expression of PGPS in Hela cells or in CHO cells did not alter expression of Mfn-2 indicating that Mfn-2 expression is independent of altered CL synthesis mediated by elevated PGPS. In addition, Mfn-2 expression was not altered in Hela cells expressing phospholipid scramblase-3 or a disrupted scramblase indicating that proper CL localization within mitochondria is not essential for Mfn-2 expression. The results suggest that immediately post-mitochondrial fusion CL de novo biosynthesis is “slowed down” and then 12 h post-fusion it is “upregulated”. The implications of this are discussed.

Tafazzin knockdown causes hypertrophy of neonatal ventricular myocytes

Mutation of the mitochondrial protein tafazzin causes dilated cardiomyopathy in Barth syndrome. We employed an adenovirus as a vector to transfer tafazzin small hairpin RNA (shRNA) into neonatal ventricular myocytes (NVMs) to investigate the effects of tafazzin knockdown. The tafazzin shRNA adenovirus consistently knocked down tafazzin mRNA and lowered cardiolipin while significantly decreasing the production of ATP by the mitochondria. The phosphorylation of AMP-activated protein kinase and mitochondrial density were both increased in tafazzin knockdown NVMs compared with scrambled shRNA controls. When we tested whether tafazzin knockdown causes hypertrophy in vitro, we found that the surface area of NVMs infected with tafazzin shRNA adenovirus was significantly increased, as were the protein synthesis and expression of the hypertrophic marker gene, brain natriuretic peptide. Taken together, our data support the concept that a decreased tafazzin expression causes cardiomyocyte hypertrophy in vitro.

Loss of Tafazzin (TAZ) Function and Accelerated Apoptosis of Human Bone Marrow Stem and Myeloid Progenitors in Barth Syndrome.

Abstract 549Barth syndrome (BTHS) is a severe X-linked stem cell disorder characterized by neutropenia, cardio- and skeletal myopathies, and growth retardation. Barth patients have a high rate of mortality due to progressive cardiomyopathy and/or overwhelming bacterial infections. Majority of Barth patients have mutations in the tafazzin (G4.5 or TAZ) gene that appear to truncate the tafazzin protein resulting in the loss of TAZ function. Based on protein homology, tafazzin is a phospholipid acyltransferase involved in remodeling cardiolipin (CL), the main lipid of the inner mitochondrial membrane. Therefore, BTHS patients exhibit reduced levels of total CL and accumulation of monolysocardiolipin. However, the function of TAZ protein and how these metabolic defects are triggered by TAZ mutations in BTHS remain largely unknown. The cellular or mouse models of this disorder are not availabel yet, and thus, the link between TAZ mutations and severe neutropenia in Barth syndrome remains elusive. Earlier study reported increased annexin V staining but absence of apoptosis in peripheral blood neutrophils. However, the patients' bone marrow stem and myeloid progenitor cells have not been examined. We hypothesized that TAZ mutations trigger accelerated apoptosis of bone marrow stem/progenitor cells, which in turn leads to reduced production of neutrophils in the bone marrow and severe neutropenia in Barth patients. To test this hypothesis, we used TAZ-specific shRNA to knock down the expression of the tafazzin gene in human myeloid progenitor HL60 cells and examined its effect on cell survival. Transfection of human myeloid progenitor cells with two different TAZ-specific but not control scrambled shRNA results in substantial down-regulation in the tafazzin expression level as determined by Western blot and confirmed by RT-PCR using TAZ and GAPDH-specific primers. Human myeloid progenitor cells with knocked down TAZ expression exhibit significantly elevated dissipation of mitochondrial membrane potential compared with control cells with scrambled shRNA as evidenced by flow cytometry analysis of DIOC6-labeled cells ((p<0.0009, n=3). A remarkably significant increase in proportion of apoptotic annexin-positive cells was also observed in response to knock-down of TAZ expression in myeloid progenitor cells compared with control cells with scrambled shRNA (p<0.0002, n=6). The observed increase in apoptosis in response to TAZ knock-down was caspase-3 dependent as evidenced by Western blot analysis. Treatment of the cells with caspase-specific inhibitor zVAD-fmk significantly improved cell survival characteristics to near normal level as determined by flow cytometry (p<0.02, n=4). Interestingly, knock-down of TAZ expression in human lymphoid cells failed to affect their cell survival or mitochondrial membrane potential, indicating that the loss of TAZ function exhibits lineage-specific effect. Analysis of bone marrow-derived CD34+ stem cells from a Barth patient positive for TAZ mutation cultured 24h in the presence of 10% autologous serum revealed nearly 3-fold increase in proportion of apoptotic annexin V positive CD34+ cells compared with the same cell subpopulation from a healthy volunteer (36% in Barth vs 13% in ctrl). More differentiated CD15+ neutrophil precursors also exhibit substantially increased rate of apoptosis compared with control (Barth 27% vs ctrl 17%). CD33+ myeloid-committed progenitor cells from Barth patient do not show increased annexin V binding compared with corresponding cell subpopulation from a healthy volunteer, but demonstrate approximately 2-fold increase in dissipation of mitochondrial membrane potential compared with control donor cells. Thus, these data demonstrate that tafazzin functions as an anti-apoptotic protein, the loss of function of the TAZ gene is cytotoxic to hematopoietic cells, and that severe neutropenia in patients with Barth syndrome is due to accelerated apoptosis of bone marrow myeloid progenitor cells. Our data also reveal that caspase-specific inhibitors may represent potentially therapeutic agents capable of restoring normal production of myeloid cells in Barth Syndrome. Disclosures:No relevant conflicts of interest to declare.

Drosophila Mitochondrial Membrane‐bound Tafazzin Protein Is A Transacylase

Barth syndrome is an X-linked mitochondria disorder with skeleton myopathy, cardiomyopathy and neutropenia, resulting from the tafazzin mutation. As demonstrated previously [Xu et al.: PNAS 103:11584, 2006], the mutation of full length Drosophila tafazzin has led to deficiency of cardiolipin and abnormality of phopholipids in the Drosophila animal model. We have further used recombinant baculovirus-Sf9 insect cell system to identify the biochemical reaction of a full length mitochondrial membrane-bound Drosophila tafazzin protein and related isoforms. Tafazzin expression induced a new enzymatic function in Sf9 cell mitochondria, namely 1-palmitoyl-2-[14C]-linoleoyl-phosphatidylcholine: monolysocardiolipin linoleoyltransferase (Xu et al.: JBC 2007). The highest rate was for the phosphatidylcholine-cardiolipin transacylation between various phospholipid-lysophospholipid pairs. Transacylation activities were about ten-fold higher for linoleoyl groups than for oleoyl groups, and negligible for arachidonoyl groups. The reverse reaction was also evident by transfering acyl groups from phospholipids to 1-[14C]-palmitoyl-2-lyso-phosphatidylcholine. The purified tafazzin protein in mitochodnria using GST-tag affinity column determined by Western blot and silver staining, catalyzed both forward and reverse transacylations between cardiolipin and phosphatidylcholine, but was unable to utilize acyl-CoA as substrates. The data show that Drosophila tafazzin is a CoA-independent, acyl-specific phospholipid transacylase with substrate preference for cardiolipin and phosphatidylcholine.

Barth syndrome associated with compound hemizygosity and heterozygosity of the TAZ and LDB3 genes

Barth syndrome is an X-linked recessive disorder caused by the tafazzin (TAZ) gene mutations and includes dilated cardiomyopathy (DCM) with left ventricular non-compaction, neutropenia, skeletal myopathy, abnormal mitochondria and 3-methylglutaconic aciduria. Dilated cardiomyopathy with left ventricular non-compaction transmitted as an autosomal dominant condition has also been associated with LIM domain-binding 3 (LDB3) gene defects. We describe a family in which the 12-year-old proband had left ventricular non-compaction and DCM. His mother had five miscarriages, two other sons who died in infancy, and a healthy son and daughter. The proband showed left ventricular non-compaction-DCM, skeletal myopathy, recurrent oral aphthous ulcers and cyclic neutropenia. The DCM progressively improved with age; medical therapy was discontinued at 5 years of age. At present, left ventricular function is normal and arrhythmias are absent. Magnetic resonance imaging documented left ventricular non-compaction. However, oral aphthous ulcers and cyclic neutropenia have recurred. In the proband we identified two novel mutations, one of maternal origin in the TAZ gene (p.[Glu202ValfsX15]) and one of paternal origin in the LDB3 gene (p.[Thr350Ile]). The mother, brother and father are healthy; although the latter two show prominent left ventricle trabeculation without dysfunction. Expression studies of TAZ and LDB3 genes were conducted in family members and controls. In the proband, brother and father, LDB3 expression was similar to control cases. TAZ and LDB3 expression progressively declined with age in control both blood and myocardial samples. However, an endomyocardial biopsy performed in the proband at 6 months of age, showed significantly lower TAZ and LDB3 expression than in age-matched myocardial controls. We believe that the clinical, genetic and expression data support the hypothesis that tafazzins are essential during fetal and early post-natal life.

The Enzymatic Function of Tafazzin

Tafazzin is a putative enzyme that is involved in cardiolipin metabolism, it may carry mutations responsible for Barth syndrome. To identify the biochemical reaction catalyzed by tafazzin, we expressed the full-length isoform of Drosophila melanogaster tafazzin in a baculovirus-Sf9 insect cell system. Tafazzin expression induced a new enzymatic function in Sf9 cell mitochondria, namely 1-palmitoyl-2-[14C]linoleoyl-phosphatidylcholine:monolysocardiolipin linoleoyltransferase. We also found evidence for the reverse reaction, because tafazzin expression caused transfer of acyl groups from phospholipids to 1-[14C]palmitoyl-2-lyso-phosphatidylcholine. An affinity-purified tafazzin construct, tagged with the maltose-binding protein, catalyzed both forward and reverse transacylations between cardiolipin and phosphatidylcholine, but was unable to utilize CoA or acyl-CoA as substrates. Whereas tafazzin supported transacylations between various phospholipid-lysophospholipid pairs, it showed the highest rate for the phosphatidylcholine-cardiolipin transacylation. Transacylation activities were about 10-fold higher for linoleoyl groups than for oleoyl groups, and they were negligible for arachidonoyl groups. The data show that Drosophila tafazzin is a CoA-independent, acyl-specific phospholipid transacylase with substrate preference for cardiolipin and phosphatidylcholine.