LPGAT1 controls the stearate/palmitate ratio of phosphatidylethanolamine and phosphatidylcholine in sn-1 specific remodeling
Most mammalian phospholipids contain a saturated fatty acid at the sn-1 carbon atom and an unsaturated fatty acid at the sn-2 carbon atom of the glycerol backbone group. While the sn-2 linked chains undergo extensive remodeling by deacylation and reacylation (Lands cycle), it is not known how the composition of saturated fatty acids is controlled at the sn-1 position. Here, we demonstrate that lysophosphatidylglycerol acyltransferase 1 (LPGAT1) is an sn-1 specific acyltransferase that controls the stearate/palmitate ratio of phosphatidylethanolamine (PE) and phosphatidylcholine. Bacterially expressed murine LPGAT1 transferred saturated acyl-CoAs specifically into the sn-1 position of lysophosphatidylethanolamine (LPE) rather than lysophosphatidylglycerol and preferred stearoyl-CoA over palmitoyl-CoA as the substrate. In addition, genetic ablation of LPGAT1 in mice abolished 1-LPE:stearoyl-CoA acyltransferase activity and caused a shift from stearate to palmitate species in PE, dimethyl-PE, and phosphatidylcholine. Lysophosphatidylglycerol acyltransferase 1 KO mice were leaner and had a shorter life span than their littermate controls. Finally, we show that total lipid synthesis was reduced in isolated hepatocytes of LPGAT1 knockout mice. Thus, we conclude that LPGAT1 is an sn-1 specific LPE acyltransferase that controls the stearate/palmitate homeostasis of PE and the metabolites of the PE methylation pathway and that LPGAT1 plays a central role in the regulation of lipid biosynthesis with implications for body fat content and longevity.
A simple mechanistic explanation for Barth syndrome and cardiolipin remodeling
Barth syndrome is a multisystem disorder caused by an abnormal metabolism of the mitochondrial lipid cardiolipin. In this review, we discuss physical properties, biosynthesis, membrane assembly, and function of cardiolipin. We hypothesize that cardiolipin reduces packing stress in the inner mitochondrial membrane, which arises as a result of protein crowding. According to this hypothesis, patients with Barth syndrome are unable to meet peak energy demands because they fail to concentrate the proteins of oxidative phosphorylation to a high surface density in the inner mitochondrial membrane.
StaR-related lipid transfer-like domain-containing protein CLDP43 affects cardiolipin synthesis and mitochondrial function in Trypanosoma brucei
Cardiolipin is known to interact with bacterial and mitochondrial proteins and protein complexes. Unlike in Escherichia coli and Saccharomyces cerevisiae, the synthesis of cardiolipin is essential for growth of Trypanosoma brucei parasites in culture. Inhibition of cardiolipin production has been shown to result in major changes in the T. brucei proteome and energy metabolism, with CLDP43, a mitochondrial protein containing a StaR-related lipid transfer (START)-like domain, being depleted in a cardiolipin-dependent way. We now show that in T. brucei procyclic forms lacking CLDP43, cardiolipin metabolism and mitochondrial function are affected. Using quantitative and qualitative lipid analyses, we found that while steady-state levels of cardiolipin were elevated in CLDP43 knock-out parasites compared to parental cells, de novo formation of cardiolipin was down-regulated. In addition, depletion of CLDP43 resulted in partial loss of mitochondrial membrane potential and decreased ATP production via substrate level phosphorylation. Recombinant CLDP43 was found to bind cardiolipin and phosphatidic acid in lipid overlay experiments, suggesting that it may be involved in transport or synthesis of cardiolipin or its precursors in T. brucei.
Condensed Mitochondria Assemble Into the Acrosomal Matrix During Spermiogenesis
Mammalian spermatogenesis is associated with the transient appearance of condensed mitochondria, a singularity of germ cells with unknown function. Using proteomic analysis, respirometry, and electron microscopy with tomography, we studied the development of condensed mitochondria. Condensed mitochondria arose from orthodox mitochondria during meiosis by progressive contraction of the matrix space, which was accompanied by an initial expansion and a subsequent reduction of the surface area of the inner membrane. Compared to orthodox mitochondria, condensed mitochondria respired more actively, had a higher concentration of respiratory enzymes and supercomplexes, and contained more proteins involved in protein import and expression. After the completion of meiosis, the abundance of condensed mitochondria declined, which coincided with the onset of the biogenesis of acrosomes. Immuno-electron microscopy and the analysis of sub-cellular fractions suggested that condensed mitochondria or their fragments were translocated into the lumen of the acrosome. Thus, it seems condensed mitochondria are formed from orthodox mitochondria by extensive transformations in order to support the formation of the acrosomal matrix.
Cardiolipin remodeling enables protein crowding in the inner mitochondrial membrane
Mitochondrial cristae are extraordinarily crowded with proteins, which puts stress on the bilayer organization of lipids. We tested the hypothesis that the high concentration of proteins drives the tafazzin-catalyzed remodeling of fatty acids in cardiolipin, thereby reducing bilayer stress in the membrane. Specifically, we tested whether protein crowding induces cardiolipin remodeling and whether the lack of cardiolipin remodeling prevents the membrane from accumulating proteins. In vitro, the incorporation of large amounts of proteins into liposomes altered the outcome of the remodeling reaction. In yeast, the concentration of proteins involved in oxidative phosphorylation (OXPHOS) correlated with the cardiolipin composition. Genetic ablation of either remodeling or biosynthesis of cardiolipin caused a substantial drop in the surface density of OXPHOS proteins in the inner membrane of the mouse heart and Drosophila flight muscle mitochondria. Our data suggest that OXPHOS protein crowding induces cardiolipin remodelling and that remodeled cardiolipin supports the high concentration of these proteins in the inner mitochondrial membrane.
Structural basis for potassium transport in prokaryotes by KdpFABC
KdpFABC is an oligomeric K+ transport complex in prokaryotes that maintains ionic homeostasis under stress conditions. The complex comprises a channel-like subunit (KdpA) from the superfamily of K+ transporters and a pump-like subunit (KdpB) from the superfamily of P-type ATPases. Recent structural work has defined the architecture and generated contradictory hypotheses for the transport mechanism. Here, we use substrate analogs to stabilize four key intermediates in the reaction cycle and determine the corresponding structures by cryogenic electron microscopy. We find that KdpB undergoes conformational changes consistent with other representatives from the P-type superfamily, whereas KdpA, KdpC, and KdpF remain static. We observe a series of spherical densities that we assign as K+ or water and which define a pathway for K+ transport. This pathway runs through an intramembrane tunnel in KdpA and delivers ions to sites in the membrane domain of KdpB. Our structures suggest a mechanism where ATP hydrolysis is coupled to K+ transfer between alternative sites in KdpB, ultimately reaching a low-affinity site where a water-filled pathway allows release of K+ to the cytoplasm.
Enrichment of NPC1-deficient cells with the lipid LBPA stimulates autophagy, improves lysosomal function, and reduces cholesterol storage
Niemann-Pick C (NPC) is an autosomal recessive disorder characterized by mutations in the NPC1 or NPC2 genes encoding endo-lysosomal lipid transport proteins, leading to cholesterol accumulation and autophagy dysfunction. We have previously shown that enrichment of NPC1-deficient cells with the anionic lipid lysobisphosphatidic acid (LBPA; also called bis(monoacylglycerol)phosphate, BMP) via treatment with its precursor phosphatidylglycerol (PG) results in a dramatic decrease in cholesterol storage. However, the mechanisms underlying this reduction are unknown. In the present study, we showed using biochemical and imaging approaches in both NPC1-deficient cellular models and an NPC1 mouse model that PG incubation/LBPA enrichment significantly improved the compromised autophagic flux associated with NPC1 disease, providing a route for NPC1-independent endo-lysosomal cholesterol mobilization. PG/LBPA enrichment specifically enhanced the late stages of autophagy, and effects were mediated by activation of the lysosomal enzyme acid sphingomyelinase (ASM). PG incubation also led to robust and specific increases in LBPA species with polyunsaturated acyl chains, potentially increasing the propensity for membrane fusion events, which are critical for late-stage autophagy progression. Finally, we demonstrated that PG/LBPA treatment efficiently cleared cholesterol and toxic protein aggregates in Purkinje neurons of the NPC1I1061T mouse model. Collectively, these findings provide a mechanistic basis supporting cellular LBPA as a potential new target for therapeutic intervention in NPC disease.
Increased ROS-Mediated CaMKII Activation Contributes to Calcium Handling Abnormalities and Impaired Contraction in Barth Syndrome
Background: Mutations in tafazzin (TAZ), a gene required for biogenesis of cardiolipin, the signature phospholipid of the inner mitochondrial membrane, causes Barth syndrome (BTHS). Cardiomyopathy and risk of sudden cardiac death are prominent features of BTHS, but the mechanisms by which impaired cardiolipin biogenesis causes cardiac muscle weakness and arrhythmia are poorly understood. Methods: We performed in vivo electrophysiology to define arrhythmia vulnerability in cardiac specific TAZ knockout mice. Using cardiomyocytes derived from human induced pluripotent stem cells (iPSC-CMs) and cardiac specific TAZ knockout mice as model systems, we investigated the effect of TAZ inactivation on Ca2+ handling. Through genome editing and pharmacology, we defined a molecular link between TAZ mutation and abnormal Ca2+ handling and contractility. Results: A subset of mice with cardiac-specific TAZ inactivation developed arrhythmias including bidirectional ventricular tachycardia, atrial tachycardia, and complete atrioventricular block. Compared to WT, BTHS iPSC-CMs had increased diastolic Ca2+ and decreased Ca2+ transient amplitude. BTHS iPSC-CMs had higher levels of mitochondrial and cellular ROS than WT, which activated Ca2+/calmodulin-dependent protein kinase II (CaMKII). Activated CaMKII phosphorylated the cardiac ryanodine receptor (RYR2) on serine 2814, increasing Ca2+ leak through RYR2. Inhibition of this ROS-CaMKII-RYR2 pathway through pharmacological inhibitors or genome editing normalized aberrant Ca2+ handling in BTHS iPSC-CMs and improved their contractile function. Murine Taz knockout cardiomyocytes also exhibited elevated diastolic Ca2+ and decreased Ca2+ transient amplitude. These abnormalities were ameliorated by CaMKII or ROS inhibition. Conclusions: This study identified a molecular pathway that links TAZ mutation to abnormal Ca2+ handling and decreased cardiomyocyte contractility. This pathway may offer therapeutic opportunities to treat BTHS and potentially other diseases with elevated mitochondrial ROS production.
Cardiolipin deficiency in Barth syndrome is not associated with increasedÂ superoxide/H2 O2 production in heart and skeletal muscle mitochondria
Barth Syndrome (BTHS) is a rare X-linked genetic disorder caused by mutations in the gene encoding the transacylase tafazzin and characterized by loss of cardiolipin and severe cardiomyopathy. Mitochondrial oxidants have been implicated in the cardiomyopathy in BTHS. Eleven mitochondrial sites produce superoxide/H2 O2 at significant rates. Which of these sites generate oxidants at excessive rates in BTHS is unknown. Here, we measured the maximum capacity of superoxide/H2 O2 production from each site and the ex vivo rate of superoxide/H2 O2 production in the heart and skeletal muscle mitochondria of the tafazzin knockdown mice (tazkd) from 3 to 12 months of age. Despite reduced oxidative capacity, superoxide/H2 O2 production is indistinguishable between tazkd mice and wildtype littermates. These observations raise questions about the involvement of mitochondrial oxidants in BTHS pathology.
Protein crowding in the inner mitochondrial membrane
The inner membrane of mitochondria is known for its low lipid-to-protein ratio. Calculations based on the size and the concentration of the principal membrane components, suggest about half of the hydrophobic volume of the membrane is occupied by proteins. Such high degree of crowding is expected to strain the hydrophobic coupling between proteins and lipids unless stabilizing mechanisms are in place. Both protein supercomplexes and cardiolipin are likely to be critical for the integrity of the inner mitochondrial membrane because they reduce the energy penalty of crowding.