Cardiolipin Remodeling Enzyme Research Articles

ETMR-20. CARDIOLIPIN ACYL CHAIN MODULATION AS A NOVEL MITOCHONDRIAL-FOCUSED THERAPEUTIC TARGET IN ETMR

Abstract Embryonal tumor with multilayered rosettes (ETMR) is a highly aggressive CNS neoplasm that occurs almost exclusively in infants and is associated with an extremely poor prognosis. Dysregulated mitochondrial bioenergetics and dynamics have been associated with the initiation and progression of diverse cancers and studies have linked metabolic rewiring to chemotherapy resistance. Cardiolipins are mitochondrial-specific lipids that reside in the mitochondrial membrane and their fatty acid composition has been extensively shown to regulate mitochondrial function and dynamics. Despite the known functional significance of cardiolipin structure, their role in mitochondrial regulation of brain tumors remains ill-defined. Using mass spectrometry imaging, we identified a shift to shorter acyl chain cardiolipins within the rapidly proliferating embryonal tumor cells in patient samples and patient-derived cell lines grown as 3D tumorspheres. Western blot analysis of the enzymes involved in cardiolipin synthesis and remodeling identified a significant increase in the expression of the cardiolipin remodeling enzyme, LCLAT1/ALCAT1. Orthogonal imaging techniques including immunohistochemistry, transmission electron microscopy and super-resolution microscopy correlated shorter acyl chain remodeling of cardiolipin with fragmented mitochondria (increased fission) and aberrant cristae structure. Further studies identified increased expression of the fission protein Drp1, decreased expression of respiratory chain enzymes and altered respiration in the embryonal tumor cells. Ongoing studies will utilize multiple methods to modulate cardiolipin acyl chain structure and use MS and MSI to correlate cardiolipin fatty acid structure to mitochondrial function and growth inhibition in the embryonal tumor cells.

Insights into complex I assembly: Function of NDUFAF1 and a link with cardiolipin remodeling.

Respiratory complex I is a~1-MDa proton pump in mitochondria. Its structure has been revealed in great detail, but the structural basis of its assembly, in humans involving at least 15 assembly factors, is essentially unknown. We determined cryo-electron microscopy structures of assembly intermediates associated with assembly factor NDUFAF1 in a yeast model system. Subunits ND2 and NDUFC2 together with assembly factors NDUFAF1 and CIA84 form the nucleation point of the NDUFAF1-dependent assembly pathway. Unexpectedly, the cardiolipin remodeling enzyme tafazzin is an integral component of this core complex. In a later intermediate, all 12 subunits of the proximal proton pump module have assembled. NDUFAF1 locks the central ND3 subunit in an assembly-competent conformation, and major rearrangements of central subunits are required for complex I maturation.

Tafazzin deficiency attenuates anti-cluster of differentiation 40 and interleukin-4 activation of mouse B lymphocytes.

Barth syndrome (BTHS) is a rare X-linked genetic disease caused by mutations in TAFAZZIN. The tafazzin (Taz) protein is a cardiolipin remodeling enzyme required for maintaining mitochondrial function. Patients with BTHS exhibit impaired mitochondrial respiratory chain and metabolic function and are susceptible to serious infections. B lymphocytes (B cells) play a vital role in humoral immunity required to eradicate circulating antigens from pathogens. Intact mitochondrial respiration is required for proper B-cell function. We investigated whether Taz deficiency in mouse B cells altered their response to activation by anti-cluster of differentiation 40 (anti-CD40) + interleukin-4 (IL-4). B cells were isolated from 3-4-month-old wild type (WT) or tafazzin knockdown (TazKD) mice and were stimulated with anti-CD40 + IL-4 for 24h and cellular bioenergetics, surface marker expression, proliferation, antibody production, and proteasome and immunoproteasome activities determined. TazKD B cells exhibited reduced mRNA expression of Taz, lowered levels of cardiolipin, and impairment in both oxidative phosphorylation and glycolysis compared to WT B cells. In addition, anti-CD40 + IL-4 stimulated TazKD B cells expressed lower levels of the immunogenic surface markers, cluster of differentiation 86 (CD86) and cluster of differentiation 69 (CD69), exhibited a lower proliferation rate, reduced production of immunoglobulin M and immunoglobulin G, and reduced proteasome and immunoproteasome proteolytic activities compared to WT B cells stimulated with anti-CD40 + IL-4. The results indicate that Taz is required to support T-cell-dependent signaling activation of mouse B cells.

Does ALCAT1, a cardiolipin-remodeling enzyme, alter the metabolic landscape of HeLa cells?

Does ALCAT1, a cardiolipin-remodeling enzyme, alter the metabolic landscape of HeLa cells?

N-oleoylethanolamide treatment of lymphoblasts deficient in Tafazzin improves cell growth and mitochondrial morphology and dynamics.

Barth syndrome (BTHS) is caused by mutations in the TAZ gene encoding the cardiolipin remodeling enzyme, Tafazzin. The study objective was to quantitatively examine growth characteristics and mitochondrial morphology of transformed lymphoblast cell lines derived from five patients with BTHS relative to five healthy controls, as well as the therapeutic potential of oleoylethanolamide (OEA) and linoleoylethanolamide (LEA). These bioactive lipids both activate PPARα, which may be therapeutic. BTHS lymphoblasts grew more slowly than controls, suggesting lymphopenia merits clinical investigation. Treatment of BTHS lymphoblasts with OEA, but not LEA, significantly restored mitochondrial membrane potential, as well as colony growth in all BTHS lymphoblast lines, although a full growth rescue was not achieved. Quantification analysis of electron micrographs from three BTHS and healthy lymphoblast donors indicated similar numbers of mitochondria per cell, but lower average cristae length per mitochondrion, and higher mitochondrial density. Additionally, BTHS lymphoblasts had larger mitochondria, and a higher percentage of abnormally large mitochondria (> 1 μm2) than healthy controls. Notably, OEA treatment significantly restored mitochondrial size, without affecting density or cristae lengths. Cardiolipin total content, relative linoleic acid content and monolysocardiolipin:cardiolipin ratios were not improved by OEA, indicating that effects on growth, and mitochondrial morphology and function, occurred without resolving this deficit. However, immunoblotting showed higher levels of OPA1, a biomarker for mitochondrial fusion, in BTHS lymphoblasts, which was attenuated by OEA treatment, implicating altered mitochondrial dynamics in the pathology and treatment of BTHS.

Tafazzin deficiency impairs mitochondrial metabolism and function of lipopolysaccharide activated B lymphocytes in mice.

B lymphocytes are responsible for humoral immunity and play a key role in the immune response. Optimal mitochondrial function is required to support B cell activity during activation. We examined how deficiency of tafazzin, a cardiolipin remodeling enzyme required for mitochondrial function, alters the metabolic activity of B cells and their response to activation by lipopolysaccharide in mice. B cells were isolated from 3-month-old wild type or tafazzin knockdown mice and incubated for up to 72h with lipopolysaccharide and cell proliferation, expression of cell surface markers, secretion of antibodies and chemokines, proteasome and immunoproteasome activities, and metabolic function determined. In addition, proteomic analysis was performed to identify altered levels of proteins involved in survival, immunogenic, proteasomal and mitochondrial processes. Compared to wild type lipopolysaccharide activated B cells, lipopolysaccharide activated tafazzin knockdown B cells exhibited significantly reduced proliferation, lowered expression of cluster of differentiation 86 and cluster of differentiation 69 surface markers, reduced secretion of immunoglobulin M antibody, reduced secretion of keratinocytes-derived chemokine and macrophage-inflammatory protein-2, reduced proteasome and immunoproteasome activities, and reduced mitochondrial respiration and glycolysis. Proteomic analysis revealed significant alterations in key protein targets that regulate cell survival, immunogenicity, proteasomal processing and mitochondrial function consistent with the findings of the above functional studies. The results indicate that the cardiolipin transacylase enzyme tafazzin plays a key role in regulating mouse B cell function and metabolic activity during activation through modulation of mitochondrial function.

Berberine elevates cardiolipin in heart of offspring from mouse dams with high fat diet-induced gestational diabetes mellitus

Berberine (BBR) is an isoquinoline alkaloid from plants known to improve cardiac mitochondrial function in gestational diabetes mellitus (GDM) offspring but the mechanism is poorly understood. We examined the role of the mitochondrial phospholipid cardiolipin (CL) in mediating this cardiac improvement. C57BL/6 female mice were fed either a Lean-inducing low-fat diet or a GDM-inducing high-fat diet for 6 weeks prior to breeding. Lean and GDM-exposed male offspring were randomly assigned a low-fat, high-fat, or high-fat diet containing BBR at weaning for 12 weeks. The content of CL was elevated in the heart of GDM offspring fed a high fat diet containing BBR. The increase in total cardiac CL was due to significant increases in the most abundant and functionally important CL species, tetralinoleoyl-CL and this correlated with an increase in the expression of the CL remodeling enzyme tafazzin. Additionally, BBR treatment increased expression of cardiac enzymes involved in fatty acid uptake and oxidation and electron transport chain subunits in high fat diet fed GDM offspring. Thus, dietary BBR protection from cardiac dysfunction in GDM exposed offspring involves improvement in mitochondrial function mediated through increased synthesis of CL.

Changes of cardiolipin involve in the occurrence and development of mitochondrial dysfunction associated with non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) is one of the most common liver diseases, which is often accompanied by mitochondrial dysfunction. Cardiolipin is a special lipid of mitochondrial inner membrane and any change of cardiolipin may cause series of mitochondria-relevant diseases. Changes in cardiolipin content, fatty acid side chain composition after cardiolipin remodeling, and the content of cardiolipin remodeling enzymes play an important role in the occurrence and development of NAFLD-related mitochondrial dysfunction.

Tafazzin (TAZ) Regulates the Differentiation of AML Cells By Reducing Levels of the Phospholipid Phosphatidylethanolamine

AML cells have unique mitochondrial characteristics with an increased reliance on mitochondrial metabolism and oxidative phosphorylation. To identify new biological vulnerabilities in the mitochondria of AML, we conducted a CRISPR knockout screen. CAS9-overexpressing human OCI-AML2 leukemia cells were transduced with a library of 91,320 sgRNAs in barcoded lentiviral vectors targeting 17,237 nuclear-encoded genes. Cells were harvested, genomic DNA was isolated, and the relative abundance of sgRNAs were determined by sequencing barcodes 14 days after puromycin selection. We focused on the sgRNAs targeting the 1050 mitochondrial proteins to identify targets in the mitochondrial proteome whose knockout reduced AML growth and viability.The cardiolipin remodeling enzyme tafazzin (TAZ) was among the top 1% of mitochondrial hits. Using individual sgRNA, we confirmed that knockout of TAZ reduced the growth of CAS9-OCI-AML2 cells by >70%, thus validating the findings from our screen. We also knocked down TAZ with 2 independent shRNA and demonstrated reductions in growth and viability of a panel of AML cells: OCI-AML2 (>80%), TEX (>50%), K562 (>50%), and U937 (>40%). Moreover, TAZ knockdown significantly reduced the engraftment of TEX leukemia cells in vivo by 80%, indicating that TAZ-knockdown reduces AML growth in vivo and can target leukemia initiating cells. In contrast, knockdown of TAZ in mouse models did not impair normal hematopoiesis nor reduced the abundance of hematopoietic stem cells, although more subtle defects in the hematopoietic stem cells might explain transient episodes of neutropenia seen in Barth's syndrome, a congenital condition associated with X-linked TAZ mutations.TAZ is responsible for the majority of Cardiolipin (CL) remodeling under physiological conditions. As expected the knockdown of TAZ in both AML and normal mouse hematopoietic cells increased the substrate (monlysocardiolipin) to product (CL) ratio of TAZ. CL is required for the proper localization, and efficient function of, respiratory chain enzymes. However, in AML cells, knockdown of TAZ did not alter respiratory chain complex activity, basal oxygen consumption, or respiratory chain reserve capacity.Recent studies have shown that mitochondrial pathways can regulate cell fate and differentiation independent of their effects on oxidative phosphorylation. Therefore, we examined changes in AML cell differentiation after TAZ knockdown. Knockdown of TAZ promoted the differentiation of AML cells as evidenced by increased non-specific esterase staining and increased CD11b expression on the cell surface.In breast cancer cells decreasing phosphatidylethanolamine (PE) levels, induced the differentiation of these cells. As TAZ regulates phospholipid remodeling, therefore we measured levels of PE and phosphatidylserine (PS) after TAZ-knockdown by spot densitometry. Interestingly, knockdown of TAZ in OCI-AML2 cells decreased PE and increased PS lipid levels. To determine whether alterations in PE and PS phospholipids are functionally important for differentiation of AML cells, we treated AML cells with MMV007285, an inhibitor of the phosphatidylserine decarboxylase (PISD), an enzyme that converts PS to PE. MMV007285 mimicked the effects of TAZ-knockdown and increased differentiation of OCI-AML2 and 8227 AML cells.In summary, the cardiolipin remodeling enzyme TAZ regulates the differentiation of AML cells by controlling levels of PS and PE, thereby highlighting a new mechanism by which phospholipids and mitochondrial enzymes regulate AML cell fate and differentiation. Moreover, PISD inhibition may be a novel therapeutic strategy to selectively promote the differentiation of AML. DisclosuresNo relevant conflicts of interest to declare.

The Arabidopsis thaliana lysophospholipid acyltransferase At1g78690p acylates lysocardiolipins

The Arabidopsis thaliana lysophospholipid acyltransferase At1g78690 acylates a variety of lysophospholipids such as lyso phosphatidylglycerol, lyso phosphatidylethanolamine and lyso phosphatidylserine. Despite di-acylate phosphatidylglycerol being a substrate, overexpression of At1g78690 in Escherichia coli leads to the accumulation of acyl-PG. Here we show that cardiolipin also accumulates in cells overexpressing At1g78690. To help try and explain this observation, we show, using a liquid chromatography mass spectrometry (LC-MS) based assay, that At1g78690 utilizes both mono- and di-lyso cardiolipin as an acyl acceptor. Because At1g78690 shares high homology (∼40%) with the cardiolipin remodeling enzyme tafazzin, we also tested whether At1g78690 was able to catalyze a tafazzin-like transacylation reaction. Di-linoleoyl phosphatidylcholine was used as the acyl donor and mono-lyso cardiolipin was used as the acyl acceptor in a reaction and the reaction was monitored by LC-MS. No transfer of the linoleoyl chains was detected in an At1g78690 dependent manner suggesting that, despite the strong homology, these enzymes catalyze unique reactions.

Lysocardiolipin acyltransferase regulates TGF-β mediated lung fibroblast differentiation

Lysocardiolipin acyltransferase (LYCAT), a cardiolipin remodeling enzyme, plays a key role in mitochondrial function and vascular development. We previously reported that reduced LYCAT mRNA levels in peripheral blood mononuclear cells correlated with poor pulmonary function outcomes and decreased survival in IPF patients. Further LYCAT overexpression reduced lung fibrosis, and LYCAT knockdown accentuated experimental pulmonary fibrosis. NADPH Oxidase 4 (NOX4) expression and oxidative stress are known to contribute to lung fibroblast differentiation and progression of fibrosis. In this study, we investigated the role of LYCAT in TGF-β mediated differentiation of human lung fibroblasts to myofibroblasts, and whether this occurred through mitochondrial superoxide and NOX4 mediated hydrogen peroxide (H2O2) generation. Our data indicated that LYCAT expression was up-regulated in primary lung fibroblasts isolated from IPF patients and bleomycin-challenged mice, compared to controls. In vitro, siRNA-mediated SMAD3 depletion inhibited TGF-β stimulated LYCAT expression in human lung fibroblasts. ChIP immunoprecipitation assay revealed TGF-β stimulated SMAD2/3 binding to the endogenous LYCAT promoter, and mutation of the SMAD2/3 binding sites (−179/−183 and −540/−544) reduced TGF-β-stimulated LYCAT promoter activity. Overexpression of LYCAT attenuated TGF-β-induced mitochondrial and intracellular oxidative stress, NOX4 expression and differentiation of human lung fibroblasts. Further, pretreatment with Mito-TEMPO, a mitochondrial superoxide scavenger, blocked TGF-β-induced mitochondrial superoxide, NOX4 expression and differentiation of human lung fibroblasts. Treatment of human lung fibroblast with NOX1/NOX4 inhibitor, GKT137831, also attenuated TGF-β induced fibroblast differentiation and mitochondrial oxidative stress. Collectively, these results suggest that LYCAT is a negative regulator of TGF-β-induced lung fibroblast differentiation by modulation of mitochondrial superoxide and NOX4 dependent H2O2 generation, and this may serve as a potential therapeutic target for human lung fibrosis.

LPS impairs oxygen utilization in epithelia by triggering degradation of the mitochondrial enzyme Alcat1.

Cardiolipin (also known as PDL6) is an indispensable lipid required for mitochondrial respiration that is generated through de novo synthesis and remodeling. Here, the cardiolipin remodeling enzyme, acyl-CoA:lysocardiolipin-acyltransferase-1 (Alcat1; SwissProt ID, Q6UWP7) is destabilized in epithelia by lipopolysaccharide (LPS) impairing mitochondrial function. Exposure to LPS selectively decreased levels of carbon 20 (C20)-containing cardiolipin molecular species, whereas the content of C18 or C16 species was not significantly altered, consistent with decreased levels of Alcat1. Alcat1 is a labile protein that is lysosomally degraded by the ubiquitin E3 ligase Skp-Cullin-F-box containing the Fbxo28 subunit (SCF-Fbxo28) that targets Alcat1 for monoubiquitylation at residue K183. Interestingly, K183 is also an acetylation-acceptor site, and acetylation conferred stability to the enzyme. Histone deacetylase 2 (HDAC2) interacted with Alcat1, and expression of a plasmid encoding HDAC2 or treatment of cells with LPS deacetylated and destabilized Alcat1, whereas treatment of cells with a pan-HDAC inhibitor increased Alcat1 levels. Alcat1 degradation was partially abrogated in LPS-treated cells that had been silenced for HDAC2 or treated with MLN4924, an inhibitor of Cullin-RING E3 ubiquitin ligases. Thus, LPS increases HDAC2-mediated Alcat1 deacetylation and facilitates SCF-Fbxo28-mediated disposal of Alcat1, thus impairing mitochondrial integrity.

The mitochondrial cardiolipin remodeling enzyme lysocardiolipin acyltransferase is a novel target in pulmonary fibrosis.

Lysocardiolipin acyltransferase (LYCAT), a cardiolipin-remodeling enzyme regulating the 18:2 linoleic acid pattern of mammalian mitochondrial cardiolipin, is necessary for maintaining normal mitochondrial function and vascular development. We hypothesized that modulation of LYCAT expression in lung epithelium regulates development of pulmonary fibrosis. To define a role for LYCAT in human and murine models of pulmonary fibrosis. We analyzed the correlation of LYCAT expression in peripheral blood mononuclear cells (PBMCs) with the outcomes of pulmonary functions and overall survival, and used the murine models to establish the role of LYCAT in fibrogenesis. We studied the LYCAT action on cardiolipin remodeling, mitochondrial reactive oxygen species generation, and apoptosis of alveolar epithelial cells under bleomycin challenge. LYCAT expression was significantly altered in PBMCs and lung tissues from patients with idiopathic pulmonary fibrosis (IPF), which was confirmed in two preclinical murine models of IPF, bleomycin- and radiation-induced pulmonary fibrosis. LYCAT mRNA expression in PBMCs directly and significantly correlated with carbon monoxide diffusion capacity, pulmonary function outcomes, and overall survival. In both bleomycin- and radiation-induced pulmonary fibrosis murine models, hLYCAT overexpression reduced several indices of lung fibrosis, whereas down-regulation of native LYCAT expression by siRNA accentuated fibrogenesis. In vitro studies demonstrated that LYCAT modulated bleomycin-induced cardiolipin remodeling, mitochondrial membrane potential, reactive oxygen species generation, and apoptosis of alveolar epithelial cells, potential mechanisms of LYCAT-mediated lung protection. This study is the first to identify modulation of LYCAT expression in fibrotic lungs and offers a novel therapeutic approach for ameliorating lung inflammation and pulmonary fibrosis.

Cardiolipin biosynthesis and remodeling enzymes are altered during development of heart failure

Cardiolipin (CL) is responsible for modulation of activities of various enzymes involved in oxidative phosphorylation. Although energy production decreases in heart failure (HF), regulation of cardiolipin during HF development is unknown. Enzymes involved in cardiac cardiolipin synthesis and remodeling were studied in spontaneously hypertensive HF (SHHF) rats, explanted hearts from human HF patients, and nonfailing Sprague Dawley (SD) rats. The biosynthetic enzymes cytidinediphosphatediacylglycerol synthetase (CDS), phosphatidylglycerolphosphate synthase (PGPS) and cardiolipin synthase (CLS) were investigated. Mitochondrial CDS activity and CDS-1 mRNA increased in HF whereas CDS-2 mRNA in SHHF and humans, not in SD rats, decreased. PGPS activity, but not mRNA, increased in SHHF. CLS activity and mRNA decreased in SHHF, but mRNA was not significantly altered in humans. Cardiolipin remodeling enzymes, monolysocardiolipin acyltransferase (MLCL AT) and tafazzin, showed variable changes during HF. MLCL AT activity increased in SHHF. Tafazzin mRNA decreased in SHHF and human HF, but not in SD rats. The gene expression of acyl-CoA: lysocardiolipin acyltransferase-1, an endoplasmic reticulum MLCL AT, remained unaltered in SHHF rats. The results provide mechanisms whereby both cardiolipin biosynthesis and remodeling are altered during HF. Increases in CDS-1, PGPS, and MLCL AT suggest compensatory mechanisms during the development of HF. Human and SD data imply that similar trends may occur in human HF, but not during nonpathological aging, consistent with previous cardiolipin studies.

The microsomal cardiolipin remodeling enzyme acyl-CoA lysocardiolipin acyltransferase is an acyltransferase of multiple anionic lysophospholipids

Phospholipids are subjected to remodeling through the Lands cycle to attain appropriate FA compositions. In recent years, at least two families of lysophospholipid acyltransferases have been identified. Acyl-CoA lysocardiolipin acyltransferase 1 (ALCAT1) was initially identified as a microsomal lysocardiolipin acyltransferase. However, the physiological relevance of how this enzyme is involved in cardiolipin remodeling has not been elucidated. We report in this study that ALCAT1 possesses acyltransferase activities toward lysophosphatidylinositol (LPI) and lysophosphatidylglycerol (LPG). Membrane preparations from human embryonic kidney 293 (HEK293) cells overexpressing human ALCAT1 demonstrated significant increases in LPI acyltransferase (LPIAT) and LPG acyltransferase (LPGAT) activities using a variety of fatty acyl-CoAs. The enzyme affinities toward LPI and LPG were determined through kinetic studies suggesting that the LPI binding affinity to ALCAT1 depends on fatty acyl-CoA. Reduced expression of ALCAT1 in Hela cells resulted in significant reductions of LPIAT and LPGAT activities, but not ALCAT activity. Through structural and functional studies, we have identified critical amino acids D168 and L169 within ALCAT1 that are potentially involved in lysophospholipid substrate binding. Our studies provide the molecular basis for future investigations of the physiological function of ALCAT1 and offer evidence of critical amino acids involved in substrate binding for the family of glycerolipid acyltransferases.

Lycat and cloche at the Switch Between Blood Vessel Growth and Differentiation?

See related article, pages 1057–1064 The formation of the cardiovascular system starts in the mouse embryo at approximately embryonic day (E)7.0 to E7.5. The first blood vessels in the extraembryonic membranes, the major intraembryonic vessels, and the heart form by vasculogenesis, the in situ differentiation of mesodermal cells that give rise to “blood-islands.” The latter are composed of hemangioblasts, the common precursors of endothelial and blood cells. Hemangioblasts situated in the lumen of the blood islands will further differentiate into hematocytoblasts, the precursors of all 3 lineages of blood cells. In contrast, hemangioblasts lining the walls of the blood islands will give rise to angioblasts that form endothelial cells.1 Migrating angioblasts from the proximal lateral mesoderm assemble symmetrically at the lateral sides of the embryo to establish 2 preendocardial tubes. They fuse to give rise to the primordial heart.2 While vasculogenesis is still proceeding, the uniform blood islands begin to remodel to a network of large and small vessels by the process of angiogenesis, preferentially intussusceptive microvascular growth.3,4 Gene expression and targeting studies have identified vascular endothelial growth factor and its 2 receptors, KDR/flk-1 and flt-1, as critical for the formation and early remodeling of the blood islands. Vascular endothelial growth factor is produced by endodermal and mesodermal cells at the onset of hemangioblast formation, whereas its receptors are expressed in the future endothelial cells lining the blood islands.5 Flk-1−/− embryos lack blood islands throughout the embryo and yolk sac.6 In flt-1−/− embryos, blood islands do not properly remodel but form large blood channels.7 Inactivation of a single vascular endothelial growth factor allele caused multiple embryonic malformations including the heart, rudimentary dorsal aortae, and a reduced number of blood cells.8,9 All deletions were lethal between days E8.5 and E11 to …

A Novel Cardiolipin-remodeling Pathway Revealed by a Gene Encoding an Endoplasmic Reticulum-associated Acyl-CoA:Lysocardiolipin Acyltransferase (ALCAT1) in Mouse

Cardiolipin is a major membrane polyglycerophospholipid that is required for the reconstituted activity of a number of key mitochondrial enzymes involved in energy metabolism. Cardiolipin is subjected to remodeling subsequent to its de novo biosynthesis to attain appropriate acyl composition for its biological functions. Yet, the enzyme(s) involved in the remodeling process have not been identified. We report here the identification and characterization of a murine gene that encodes an acyl-CoA:lysocardiolipin acyltransferase 1 (ALCAT1). Expression of the ALCAT1 cDNA in either insect or mammalian cells led to a significant increase in acyl-CoA:monolysocardiolipin acyltransferase and acyl-CoA: dilysocardiolipin acyltransferase activities that exhibited a dependence upon ALCAT1 enzyme levels. The recombinant ALCAT1 enzyme recognizes both monolysocardiolipin and dilysocardiolipin as substrates with a preference for linoleoyl-CoA and oleoyl-CoA as acyl donors. In contrast, no significant increases in acyltransferase activities by the recombinant ALCAT1 were detected against either glycerol-3-phosphate or a variety of other lysophospholipids as substrates, including lysophosphatidylcholine, lysophosphatidylethanolamine, and lysophosphatidylserine. Immunocytohistochemical analysis showed that the ALCAT1 enzyme is localized in the endoplasmic reticulum, which is supported by a significant ALCAT activity in isolated liver and heart microsomes. Northern blot analysis indicates that the mouse ALCAT1 is widely distributed, with the highest expression in heart and liver. In support of a role for ALCAT1 in maintaining heart function, the ALCAT1 gene is conserved among different species of vertebrates, but not in non-atrium organisms. ALCAT1 represents the first identified cardiolipin-remodeling enzyme from any living organism; its identification implies a novel role for the endoplasmic reticulum in cardiolipin metabolism.