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.
The Function of Tafazzin, a Mitochondrial Phospholipid-Lysophospholipid Acyltransferase
Tafazzin is a mitochondrial enzyme that exchanges fatty acids between phospholipids by phospholipid-lysophospholipid transacylation. The reaction alters the molecular species composition and, as a result, the physical properties of lipids. In vivo, the most important substrate of tafazzin is the mitochondria-specific lipid cardiolipin. Tafazzin mutations cause the human disease Barth syndrome, which presents with cardiomyopathy, skeletal muscle weakness, fatigue, and other symptoms, probably all related to mitochondrial dysfunction. The reason why mitochondria require tafazzin is still not known but recent evidence suggests that tafazzin may lower the energy cost associated with protein crowding in the inner mitochondrial membrane.
AAV Gene Therapy Prevents and Reverses Heart Failure in A Murine Knockout Model of Barth Syndrome
Rationale: Barth syndrome (BTHS) is an X-linked cardiac and skeletal myopathy caused by mutation of the gene Tafazzin (TAZ). Currently there is no targeted treatment for BTHS. Lack of a proper genetic animal model that recapitulates the features of BTHS has hindered understanding of disease pathogenesis and therapeutic development. Objective: We characterized murine germline (TAZ-KO) and cardiac specific (TAZ-CKO) Taz knockout models and tested the efficacy of AAV-mediated TAZ gene replacement therapy. Methods and Results: TAZ-KO caused embryonic and neonatal lethality, impaired growth, dilated cardiomyopathy, and skeletal myopathy. TAZ-KO mice that survived the neonatal period developed progressive, severe cardiac dysfunction and fibrosis. Cardiomyocyte specific inactivation of floxed Taz in CMs using Myh6-Cre caused progressive dilated cardiomyopathy without fetal or perinatal loss. Using both constitutive and conditional knockout models, we tested the efficacy and durability of Taz replacement by AAV gene therapy. Neonatal AAV-TAZ rescued neonatal death, cardiac dysfunction, and fibrosis in TAZ-KO mice, and both prevented and reversed established cardiac dysfunction in TAZ-KO and TAZ-CKO models. However, both neonatal and adult therapies required high CM transduction (~70%) for durable efficacy. Conclusions: TAZ-KO and TAZ-CKO mice recapitulate many of the key clinical features of BTHS. AAV-mediated gene replacement is efficacious when a sufficient fraction of CMs are transduced.
A Bayesian Analysis to Determine the Prevalence of Barth Syndrome in the Pediatric Population
OBJECTIVE:To determine the prevalence of Barth syndrome in the pediatric population. STUDY DESIGN/METHODS:Data were collected from the Barth Syndrome Foundation Registry and relevant literature. With the advent of genetic testing and whole-exome sequencing, a multipronged Bayesian analysis was used to estimate the prevalence of Barth syndrome based on published data on the incidence and prevalence of cardiomyopathy and neutropenia, and the respective subpopulations of patients with Barth syndrome indicated in these publications. RESULTS:Based on 7 published studies of cardiomyopathy and 2 published studies of neutropenia, the estimated prevalence of Barth syndrome is approximately 1 case per million male population. This contrasts with 99 cases in the Barth Syndrome Foundation Registry, 58 of which indicate a US location, and only 230-250 cases known worldwide. CONCLUSIONS:It appears that Barth syndrome is greatly underdiagnosed. There is a need for better education and awareness of this rare disease to move toward early diagnosis and treatment.
Lipidome-wide 13C flux analysis: a novel tool to estimate the turnover of lipids in organisms and cultures
Lipid metabolism plays an important role in the regulation of cellular homeostasis. However, since it is difficult to measure the actual rates of synthesis and degradation of individual lipid species, lipid compositions are used often as a surrogate to evaluate lipid metabolism even though they provide only static snapshots of the lipodome. Here, we designed a simple method to determine the turnover rate of phospholipid and acylglycerol species based on the incorporation of 13C6-glucose combined with LC-MS/MS. We labeled adult Drosophila melanogaster with 13C6-glucose that incorporates into the entire lipidome, derived kinetic parameters from mass spectra, and studied effects of deletion of CG6718, the fly homologue of the calcium-independent phospholipase A2Î², on lipid metabolism. Although 13C6-glucose gave rise to a complex pattern of 13C incorporation, we were able to identify discrete isotopomers in which 13C atoms were confined to the glycerol group. With these isotopomers, we calculated turnover rate constants, half-life times, and fluxes of the glycerol backbone of multiple lipid species. To perform these calculations, we estimated the fraction of labeled molecules in glycerol-3-phosphate, the lipid precursor, by mass isotopomer distribution analysis of the spectra of phosphatidylglycerol. When we applied this method to D. melanogaster, we found a range of lipid half-lives from 2 to 200 days, demonstrated tissue-specific fluxes of individual lipid species, and identified a novel function of CG6718 in triacylglycerol metabolism. This method provides fluxomics-type data with significant potential to improve the understanding of complex lipid regulation in a variety of research models.
Analysis of phospholipid synthesis in mitochondria
Mitochondria and their associated membranes actively participate in biosynthesis, trafficking, and degradation of cellular phospholipids. Two crucial lipid biosynthetic activities of mitochondria include (i) the decarboxylation of phosphatidylserine to phosphatidylethanolamine and (ii) the de novo synthesis of cardiolipin. Here we describe protocols to measure these two activities, applying isotope-labeled or exogenous substrates in combination with thin-layer chromatography or mass spectrometry.