genes associated with
calcium-independent phospholipase A2 VIA
of the disease
Barth syndrome (BTHS) is an X-linked multisystemic disorder presenting with dilated cardiomyopathy, skeletal myopathy, cyclic neutropenia, and growth retardation. The disease is caused by mutations in the tafazzin gene that encodes a putative phospholipid acyltransferase. Accordingly, BTHS patients have phospholipid abnormalities, in particular, a reduced content and an altered composition of cardiolipin. Among the many cardiolipin species that can be formed by various fatty acid combinations, BTHS specifically affects the concentration of tetralinoleoyl-cardiolipin. This species is formed by remodeling of the four acyl groups of cardiolipin, suggesting that tafazzin is involved in the acyl group exchange. BTHS has generally been considered a mitochondrial disease because pathologic mitochondria have been found in tissue biopsies from BTHS patients and because cardiolipin is a specific mitochondrial lipid. However, the presence of multiple tafazzin transcripts raises the prospect that other cellular functions are affected as well (Xu, 2006 and references therein; see below).
Relevant studies of Barth syndrome
Xu, Y., Malhotra, A., Claypool, S.M., Ren, M. and Schlame, M. (2015). Tafazzins from Drosophila and mammalian cells assemble in large protein complexes with a short half-life. Mitochondrion 21: 27-32. PubMed ID: 25598000
The results confirm and expand previous observations made in yeast, in particular the association of tafazzin with heterogeneous protein complexes, and the requirement for cardiolipin to stabilize the largest of these complexes. The latter is consistent with the general role of cardiolipin in promoting supercomplex formation. Although some variations exist between yeast, flies, and mammals in terms of the assembly pattern of tafazzin, common to all is that tafazzin is recovered mostly in the 100–400 kDa zone of the Blue Native-PAGE gel while only a tiny portion migrates with supercomplexes. It was also confirmed that the smaller tafazzin complexes are not assembly intermediates of the larger ones (Xu, 2015).
Protein–protein interactions seem to be involved in the formation of tafazzin complexes in the 100–400 kDa weight range because they remain stable in the absence of cardiolipin and do not disintegrate upon treatment with phospholipase A2. However, the tafazzin–protein interactions are relatively non-specific, prompting complex formation not only in mitochondria but also in microsomes. Therefore, it could be possible that tafazzin could also interact with itself. Indeed, when tafazzin was over-expressed in insect cells, evidence for the presence of tafazzin dimers was found, but the overall distribution of the enzyme was shifted towards smaller complexes compared to wild-type Drosophila. These data suggest that the formation of tafazzin complexes in wild-type mitochondria is not the result of tafazzin oligomerization but requires the involvement of other proteins (Xu, 2015).
It was found that the half-life of tafazzin is only a few hours in H9c2 and HEK 293 cells, which is much shorter than the half-life of other mitochondrial proteins such as VDAC, ATP synthase, and the respiratory complexes. It is known that mitochondrial proteins exhibit different rates of degradation and that protein degradation can function as a control mechanism. The short half-life of tafazzin is interesting in light of a recent study showing that tafazzin nearly vanishes from yeast cells in which the MICOS complex has been altered by Aim24 deletion combined with Mic22/Mic26 mutations. Thus, the state of the MICOS complex, a large protein assembly that resides near cristae junctions and bridges the outer and the inner membrane, has an impact on the steady-state concentration of tafazzin (Xu, 2015).
Tafazzin degrades rapidly, regardless of the protein complex it is associated with, which indicates it is highly susceptible to proteases. It has been shown that the degradation of mutated tafazzins is catalyzed by iAAA, but it remains unclear at this point which degradation system is involved in maintaining the steady-state level of wild-type tafazzin. Continuous clearance of tafazzin from the mitochondria ensures that its steady-state concentration can be altered rapidly (Xu, 2015).
In summary, data from this study suggest that tafazzin is incorporated into multiple protein complexes but resides there only for a relatively short period of time. During its lifetime, tafazzin exchanges acyl groups between membrane phospholipids, a reaction that is thought to support membrane dynamics or crista assembly by lowering the energy of curvature formation. Recent evidence has implicated a protein complex formed by prohibitins and DNAJC19 in cardiolipin remodeling, which suggests that tafazzin may operate in the vicinity of this complex. The short-lived nature of tafazzin leads to the speculation that tafazzin participates in transient protein assemblies that are formed as intermediates in the biogenesis of mitochondrial membranes (Xu, 2015).
Malhotra, A., Edelman-Novemsky, I., Xu, Y., Plesken, H., Ma, J., Schlame, M. and Ren, M. (2009). Role of calcium-independent phospholipase A2 in the pathogenesis of Barth syndrome. Proc Natl Acad Sci U S A 106: 2337-2341. PubMed ID: 19164547
It was found that tafazzin deficiency in Drosophila, which alters CL homeostasis and reduces CL levels, also disrupts spermatid individualization during spermatogenesis, resulting in male sterility, and that this male-sterile phenotype can be suppressed by inactivation of the CL-degrading enzyme iPLA2-VIA, which partially restores CL homeostasis in double-mutant flies. These observations suggest that CL content, or at least the MLCL/CL ratio, plays a critical role in Drosophila spermatid individualization. It has been recently shown that the final stage of spermatid differentiation in Drosophila involves an apoptosis-like mechanism, in which the cytochrome c-dependent caspase activation is required for the elimination of excess cytoplasm. Cardiolipin has been shown to play important roles in mitochondria-dependent apoptosis and a recent report demonstrats that CL deficiency increases cells' resistance to apoptosis. Therefore, CL deficiency in Drosophila testes may prevent the syncytial spermatids from initiating the apoptosis-like mechanism required for normal spermatid individualization. (Malhotra, 2009).
The cardinal characteristics of Barth syndrome are cardioskeletal myopathy, exercise intolerance, neutropenia, abnormal mitochondria, and altered CL metabolism. Because in eukaryotes CL is localized exclusively in mitochondria and is required for optimal mitochondrial function, it has been generally assumed that the defective CL metabolism causes the pathophysiology of Barth syndrome. This study tested this hypothesis by genetically manipulating CL metabolism in the Drosophila model. It was found that partial restoration of CL homeostasis through genetic inactivation of iPLA2-VIA suppresses the male-sterile phenotype of tafazzin-deficient flies; this provides the direct evidence that altered CL metabolism is a major contributing factor in Barth syndrome (Malhotra, 2009).
The most abundant CL molecular species from various organisms and tissues contain only 1 or 2 types of fatty acids. In many mammalian tissues, the predominant fatty acyl moiety in CL is linoleic acid (C18:2). For example, 80% of CL molecules in heart and skeletal muscle are tetralinoleoyl CL. However, the role of CL molecular species in vivo remains speculative. The characteristic fatty acyl composition of CL in vivo is achieved through tafazzin-dependent remodeling of nascent CL. However, tafazzin deficiency, such as in Barth syndrome, results not only in abnormal CL acyl composition, but also in CL depletion and monolyso-CL accumulation. Thus, it is unclear which aspect of the CL metabolic disorder contributes to the pathogenesis of Barth syndrome. The finding in this study that the male-sterile phenotype of tafazzin-deficient flies can be suppressed by genetic inactivation of iPLA2-VIA, which prevents CL depletion and monolyso-CL accumulation without correcting the abnormal CL acyl composition, suggests that the abnormal levels of CL and/or monolyso-CL are important pathogenetic factors. Because a cardiolipin synthase mutant of yeast exhibits abnormal mitochondrial function, it is likely that the low CL content is critical in the molecular mechanism of Barth syndrome. Nevertheless, because Barth syndrome is a multisystem disorder, involvement of monolyso-CL accumulation and abnormal CL acyl composition may also play a role in certain tissues and organs (Malhotra, 2009).
The mature acyl chain composition of CL is achieved through a remodeling process, which requires the action of tafazzin. It has been previously shown that tafazzin catalyzes phospholipid-lysophospholipid transacylation that involves both deacylation of a phospholipid such as CL and reacylation of a monolyso-phospholipid, such as monolyso-CL. Unlike the CoA-dependent deacylation-reacylation cycle (Lands cycle), in which a nascent phospholipid is deacylated by a phospholipase A to yield a free fatty acid and a lysophospholipid that is then reacylated by an acyl-CoA-dependent acyltransferase, transacylation does not require acyl-CoA, and proceeds directly by transferring a fatty acyl chain from a phospholipid to a lysophospholipid; no phospholipase is involved and no free fatty acid is generated in the process. It was found that although the calcium-independent phospholipase A2, iPLA2-VIA, is not required for CL remodeling, in the absence of tafazzin, i.e., in the Barth syndrome model, the enzyme plays a major role in the depletion of CL and the accumulation of monolyso-CL (Malhotra, 2009).
The finding that the phenotypic features of tafazzin deficiency can be suppressed by inhibiting iPLA2-VIA activity identifies this enzyme as a potential target for therapeutic intervention in Barth syndrome. Indeed, it was found that treatment of cultured lymphoblasts from Barth patients with the iPLA2 inhibitor BEL partially restores CL homeostasis. The calcium-independent iPLA2-VIA has been implicated in a variety of biological processes, including phospholipid remodeling, arachidonic acid release, apoptosis, and store-operated calcium entry. In addition, iPLA2-VIA knockout mice develop age-dependent neurological impairment and mutations in the iPLA2-VIA gene have been identified in patients with infantile neuroaxonal dystrophy and neurodegeneration with iron accumulation in the brain. Therefore, a therapeutic approach to Barth syndrome based on the inhibition of iPLA2-VIA is likely to require either careful titration of the phospholipase inhibitor, or even its tissue-specific targeting (Malhotra, 2009).Xu, Y., Condell, M., Plesken, H., Edelman-Novemsky, I., Ma, J., Ren, M. and Schlame, M. (2006). A Drosophila model of Barth syndrome. Proc Natl Acad Sci U S A 103: 11584-11588. PubMed ID: 16855048
Like BTHS patients, tafazzin-deficient Drosophila mutants are unable to form the dominant molecular species of cardiolipin. However, the dominant molecular species are not identical in humans and in Drosophila, suggesting that tafazzin plays a general role in cardiolipin remodeling, rather than a specific role in the formation of any particular cardiolipin species. In both humans and Drosophila, tafazzin deficiency leads to (i) a diversification of the cardiolipin pattern, i.e., formation of multiple species at the expense of the dominant species, and (ii) a decline of the total cardiolipin content (Xu, 2006).
This specific form of cardiolipin deficiency is associated with abnormalities of the cristae membranes, which bear some resemblance to abnormalities caused by age, hyperoxia, or certain mutations. In all of these examples, an atypical pattern of cristae architecture emerges, albeit with variations in shape and form. For instance, in tafazzin-deficient flight muscle, characteristic stacks of hyperdense, closely apposed membranes, were found which were deposited inside the mitochondria. A very similar morphology has been observed in heart biopsies from BTHS patients. According to electron tomographic studies, these hyperdense structures may represent collapsed cristae with obliterated intracrista space (Xu, 2006).
Physiologic consequences of tafazzin deficiency are only beginning to emerge from clinical and experimental studies. In cell cultures, poor coupling between respiration and phosphorylation has been observed, but a general breakdown of energy metabolism is unlikely to be the pathogenetic mechanism of BTHS, because the symptoms are often subtle and confined to selected organ systems. Deletion of full-length tafazzin from Drosophila does not alter lifespan or heart rate, and the mutants appear to be in good overall health. However, the tafazzin mutants may become vulnerable once they are removed from the optimal culture environment and exposed to wildlife conditions. This proposition may be tested by lifespan studies under conditions of stress (Xu, 2006).
Tafazzin deletion causes motor weakness, strongly supporting the specific role of this protein in muscle function. Muscle mitochondria may be susceptible to tafazzin deletion because they require a high concentration of cardiolipin and because they may not tolerate deviations from the normal cardiolipin composition. For instance, mammalian skeletal muscle contains cardiolipin with a very specific molecular composition, mainly consisting of tetralinoleoyl species. The requirement for specific cardiolipins may be related to the structural organization, high density, or tight association with myofibrils, which is characteristic of muscle mitochondria. The selective effect on muscle function makes Drosophila an appropriate animal model of BTHS, the further exploration of which should yield insights into the mechanism by which tafazzin mutations cause myopathy (Xu, 2006).
Gawrisch, K. (2012). Tafazzin senses curvature. Nat Chem Biol 8: 811-812. PubMed ID: 22987008
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Date revised: 4 August 2015
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