RESUMO
The interphase nuclear envelope (NE) is extensively remodeled during nuclear pore complex (NPC) insertion. How this remodeling occurs and why it requires Torsin ATPases, which also regulate lipid metabolism, remains poorly understood. Here, we show that Drosophila Torsin (dTorsin) affects lipid metabolism via the NEP1R1-CTDNEP1 phosphatase and the Lipin phosphatidic acid (PA) phosphatase. This includes that Torsins remove NEP1R1-CTDNEP1 from the NE in fly and mouse cells, leading to subsequent Lipin exclusion from the nucleus. NEP1R1-CTDNEP1 downregulation also restores nuclear pore membrane fusion in post-mitotic dTorsinKO fat body cells. However, dTorsin-associated nuclear pore defects do not correlate with lipidomic abnormalities and are not resolved by silencing of Lipin. Further testing confirmed that membrane fusion continues in cells with hyperactivated Lipin. It also led to the surprising finding that excessive PA metabolism inhibits recruitment of the inner ring complex Nup35 subunit, resulting in elongated channel-like structures in place of mature nuclear pores. We conclude that the NEP1R1-CTDNEP1 phosphatase affects interphase NPC biogenesis by lipid-dependent and lipid-independent mechanisms, explaining some of the pleiotropic effects of Torsins.
Assuntos
Proteínas de Drosophila/metabolismo , Poro Nuclear/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster , Corpo Adiposo/citologia , Corpo Adiposo/metabolismo , Metabolismo dos Lipídeos , Fusão de Membrana , Fosfoproteínas Fosfatases/genéticaRESUMO
Emerging clinical data show that three ceramide molecules, Cer d18:1/16:0, Cer d18:1/24:1, and Cer d18:1/24:0, are biomarkers of a fatal outcome in patients with cardiovascular disease. This finding raises basic questions about their metabolic origin, their contribution to disease pathogenesis, and the utility of targeting the underlying enzymatic machinery for treatment of cardiometabolic disorders. Here, we outline the development of a potent N-acetylgalactosamine-conjugated antisense oligonucleotide engineered to silence ceramide synthase 2 specifically in hepatocytes in vivo. We demonstrate that this compound reduces the ceramide synthase 2 mRNA level and that this translates into efficient lowering of protein expression and activity as well as Cer d18:1/24:1 and Cer d18:1/24:0 levels in liver. Intriguingly, we discover that the hepatocyte-specific antisense oligonucleotide also triggers a parallel modulation of blood plasma ceramides, revealing that the biomarkers predictive of cardiovascular death are governed by ceramide biosynthesis in hepatocytes. Our work showcases a generic therapeutic framework for targeting components of the ceramide enzymatic machinery to disentangle their roles in disease causality and to explore their utility for treatment of cardiometabolic disorders.
Assuntos
Doenças Cardiovasculares , Oligonucleotídeos Antissenso , Oxirredutases , Biomarcadores , Doenças Cardiovasculares/genética , Ceramidas , Inativação Gênica , Hepatócitos , Humanos , Oligonucleotídeos Antissenso/genética , Oxirredutases/antagonistas & inibidores , PlasmaRESUMO
TOR1A/TorsinA mutations cause two incurable diseases: a recessive congenital syndrome that can be lethal, and a dominantly-inherited childhood-onset dystonia (DYT-TOR1A). TorsinA has been linked to phosphatidic acid lipid metabolism in Drosophila melanogaster. Here we evaluate the role of phosphatidic acid phosphatase (PAP) enzymes in TOR1A diseases using induced pluripotent stem cell-derived neurons from patients, and mouse models of recessive Tor1a disease. We find that Lipin PAP enzyme activity is abnormally elevated in human DYT-TOR1A dystonia patient cells and in the brains of four different Tor1a mouse models. Its severity also correlated with the dosage of Tor1a/TOR1A mutation. We assessed the role of excess Lipin activity in the neurological dysfunction of Tor1a disease mouse models by interbreeding these with Lpin1 knock-out mice. Genetic reduction of Lpin1 improved the survival of recessive Tor1a disease-model mice, alongside suppressing neurodegeneration, motor dysfunction, and nuclear membrane pathology. These data establish that TOR1A disease mutations cause abnormal phosphatidic acid metabolism, and suggest that approaches that suppress Lipin PAP enzyme activity could be therapeutically useful for TOR1A diseases.
Assuntos
Chaperonas Moleculares/metabolismo , Fosfatidato Fosfatase/metabolismo , Animais , Encéfalo/patologia , Modelos Animais de Doenças , Distonia/genética , Distonia/metabolismo , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Chaperonas Moleculares/genética , Mutação , Neurônios/metabolismo , Fosfatidato Fosfatase/genética , Fosfatidato Fosfatase/fisiologiaRESUMO
The acyl-CoA binding protein (ACBP) plays a key role in chaperoning long-chain acyl-CoAs into lipid metabolic processes and acts as an important regulatory hub in mammalian physiology. This is highlighted by the recent finding that mice devoid of ACBP suffer from a compromised epidermal barrier and delayed weaning, the physiological process where newborns transit from a fat-based milk diet to a carbohydrate-rich diet. To gain insights into how ACBP impinges on weaning and the concomitant remodeling of whole-body lipid metabolism we performed a comparative lipidomics analysis charting the absolute abundance of 613 lipid molecules in liver, muscle and plasma from weaning and adult Acbp knockout and wild type mice. Our results reveal that ACBP deficiency affects primarily lipid metabolism of liver and plasma during weaning. Specifically, we show that ACBP deficient mice have elevated levels of hepatic cholesteryl esters, and that lipids featuring an 18:1 fatty acid moiety are increased in Acbp depleted mice across all tissues investigated. Our results also show that the perturbation of systemic lipid metabolism in Acbp knockout mice is transient and becomes normalized and similar to that of wild type as mice grow older. These findings demonstrate that ACBP serves crucial functions in maintaining lipid metabolic homeostasis in mice during weaning.
Assuntos
Inibidor da Ligação a Diazepam/deficiência , Metabolismo dos Lipídeos/fisiologia , Animais , Ésteres do Colesterol/metabolismo , Ácidos Graxos/metabolismo , Lipídeos/fisiologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos KnockoutRESUMO
Neuronal activity causes use-dependent decline in protein function. However, it is unclear how this is coupled to local quality control mechanisms. We show in Drosophila that the endocytic protein Endophilin-A (EndoA) connects activity-induced calcium influx to synaptic autophagy and neuronal survival in a Parkinson disease-relevant fashion. Mutations in the disordered loop, including a Parkinson disease-risk mutation, render EndoA insensitive to neuronal stimulation and affect protein dynamics: when EndoA is more flexible, its mobility in membrane nanodomains increases, making it available for autophagosome formation. Conversely, when EndoA is more rigid, its mobility reduces, blocking stimulation-induced autophagy. Balanced stimulation-induced autophagy is required for dopagminergic neuron survival, and a variant in the human ENDOA1 disordered loop conferring risk to Parkinson disease also blocks nanodomain protein mobility and autophagy both in vivo and in human-induced dopaminergic neurons. Thus, we reveal a mechanism that neurons use to connect neuronal activity to local autophagy and that is critical for neuronal survival.
Assuntos
Doença de Parkinson , Animais , Humanos , Autofagia/genética , Cálcio/metabolismo , Neurônios Dopaminérgicos/metabolismo , Drosophila/metabolismo , Mutação/genética , Doença de Parkinson/genética , Doença de Parkinson/metabolismoRESUMO
Reliable, cost-effective, and gold-standard absolute quantification of non-esterified cholesterol in human plasma is of paramount importance in clinical lipidomics and for the monitoring of metabolic health. Here, we compared the performance of three mass spectrometric approaches available for direct detection and quantification of cholesterol in extracts of human plasma. These approaches are high resolution full scan Fourier transform mass spectrometry (FTMS) analysis, parallel reaction monitoring (PRM), and novel multiplexed MS/MS (MSX) technology, where fragments from selected precursor ions are detected simultaneously. Evaluating the performance of these approaches in terms of dynamic quantification range, linearity, and analytical precision showed that the MSX-based approach is superior to that of the FTMS and PRM-based approaches. To further show the efficacy of this approach, we devised a simple routine for extensive plasma lipidome characterization using only 8 µL of plasma, using a new commercially available ready-to-spike-in mixture with 14 synthetic lipid standards, and executing a single 6 min sample injection with combined MSX analysis for cholesterol quantification and FTMS analysis for quantification of sterol esters, glycerolipids, glycerophospholipids, and sphingolipids. Using this simple routine afforded reproducible and absolute quantification of 200 lipid species encompassing 13 lipid classes in human plasma samples. Notably, the analysis time of this procedure can be shortened for high throughput-oriented clinical lipidomics studies or extended with more advanced MSALL technology (Almeida R. et al., J. Am. Soc. Mass Spectrom. 26, 133-148 [1]) to support in-depth structural elucidation of lipid molecules. Graphical Abstract á .
Assuntos
Análise Química do Sangue/métodos , Colesterol/sangue , Lipídeos/sangue , Espectrometria de Massas/métodos , Animais , Feminino , Análise de Fourier , Humanos , Masculino , Camundongos Endogâmicos C57BL , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem/métodosRESUMO
Total fatty acid analysis is a routine method in many areas, including lipotyping of individuals in personalized medicine, analysis of foodstuffs, and optimization of oil production in biotechnology. This analysis is commonly done by converting fatty acyl (FA) chains of intact lipids into FA methyl esters (FAMEs) and monitoring these by gas-chromatography (GC)-based methods, typically requiring at least 15 min of analysis per sample. Here, we describe a novel method that supports fast, precise and accurate absolute quantification of total FA levels in human plasma and serum samples. The method uses acid-catalyzed transesterification with 18O-enriched H2O (i.e., H218O) to convert FA chains into 18O-labeled free fatty acids. The resulting "mass-tagged" FA analytes can be specifically monitored with improved signal-to-background by 1 min of high resolution Fourier transform mass spectrometry (FTMS) on an Orbitrap-based mass spectrometer. By benchmarking to National Institute of Standards and Technology (NIST) certified standard reference materials we show that the performance of our method is comparable, and at times superior, to that of gold-standard GC-based methods. In addition, we demonstrate that the method supports the accurate quantification of FA differences in samples obtained in dietary intervention studies and also affords specific monitoring of ingested stable isotope-labeled fatty acids (13C16-palmitate) in normoinsulinemic and hyperinsulinemic human subjects. Overall, our novel high-throughput method is generic and suitable for many application areas, spanning basic research to personalized medicine, and is particularly useful for laboratories equipped with high resolution mass spectrometers, but lacking access to GC-based instrumentation.
Assuntos
Ácidos Graxos/sangue , Espectrometria de Massas , Cromatografia Líquida de Alta Pressão , Ésteres/química , Ácidos Graxos/química , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Marcação por Isótopo , Isótopos de Oxigênio/química , Água/químicaRESUMO
Advances in mass spectrometry-based lipidomics have in recent years prompted efforts to standardize the annotation of the vast number of lipid molecules that can be detected in biological systems. These efforts have focused on cataloguing, naming and drawing chemical structures of intact lipid molecules, but have provided no guidelines for annotation of lipid fragment ions detected using tandem and multi-stage mass spectrometry, albeit these fragment ions are mandatory for structural elucidation and high confidence lipid identification, especially in high throughput lipidomics workflows. Here we propose a nomenclature for the annotation of lipid fragment ions, describe its implementation and present a freely available web application, termed ALEX123 lipid calculator, that can be used to query a comprehensive database featuring curated lipid fragmentation information for more than 430,000 potential lipid molecules from 47 lipid classes covering five lipid categories. We note that the nomenclature is generic, extendable to stable isotope-labeled lipid molecules and applicable to automated annotation of fragment ions detected by most contemporary lipidomics platforms, including LC-MS/MS-based routines.
Assuntos
Bases de Dados de Compostos Químicos , Íons/química , Lipídeos/química , Algoritmos , Íons/classificação , Íons/isolamento & purificação , Marcação por Isótopo , Lipídeos/classificação , Lipídeos/isolamento & purificação , Estrutura Molecular , Espectrometria de Massas em TandemRESUMO
Long-chain fatty acyl-CoA esters are key intermediates in numerous lipid metabolic pathways, and recognized as important cellular signaling molecules. The intracellular flux and regulatory properties of acyl-CoA esters have been proposed to be coordinated by acyl-CoA-binding domain containing proteins (ACBDs). The ACBDs, which comprise a highly conserved multigene family of intracellular lipid-binding proteins, are found in all eukaryotes and ubiquitously expressed in all metazoan tissues, with distinct expression patterns for individual ACBDs. The ACBDs are involved in numerous intracellular processes including fatty acid-, glycerolipid- and glycerophospholipid biosynthesis, ß-oxidation, cellular differentiation and proliferation as well as in the regulation of numerous enzyme activities. Little is known about the specific roles of the ACBDs in the regulation of these processes, however, recent studies have gained further insights into their in vivo functions and provided further evidence for ACBD-specific functions in cellular signaling and lipid metabolic pathways. This review summarizes the structural and functional properties of the various ACBDs, with special emphasis on the function of ACBD1, commonly known as ACBP.
Assuntos
Acil Coenzima A/fisiologia , Inibidor da Ligação a Diazepam/fisiologia , Metabolismo dos Lipídeos , Sequência de Aminoácidos , Animais , Vias Biossintéticas , Proteínas de Transporte/metabolismo , Ésteres , Humanos , Dados de Sequência Molecular , Transdução de SinaisRESUMO
For more than ten years the nematode Caenorhabditis elegans has proven to be a valuable model for studies of the host response to various bacterial and fungal pathogens. When exposed to a pathogenic organism, a clear response is elicited in the nematode, which is characterized by specific alterations on the transcriptional and translational levels. Early on, researchers took advantage of the possibility to conduct large-scale investigations of the C. elegans immune response. Multiple studies demonstrated that C. elegans does indeed mount a protective response against invading pathogens, thus rendering this small nematode a very useful and simple host model for the study of innate immunity and host-pathogen interactions. Here, we provide an overview of key aspects of innate immunity in C. elegans revealed by recent whole-genome transcriptomics and proteomics studies of the global response of C. elegans to various bacterial and fungal pathogens.