RESUMO
Phospholipids are important biomolecules for the study of lipidomics, signal transduction, biodiesel, and synthetic biology; however, it is difficult to synthesize and analyze phospholipids in a defined in vitro condition. Here, we present a protocol for in vitro production and quantification of phospholipids. We describe steps for preparing a cell-free system consisting of fatty acid synthesis and a gene expression system that synthesizes acyltransferases on liposomes. The whole reaction can be completed within a day and the products are quantified by liquid chromatography-mass spectrometry. For complete details on the use and execution of this protocol, please refer to Eto et al.1.
Assuntos
Sistema Livre de Células , Ácidos Graxos , Fosfolipídeos , Fosfolipídeos/metabolismo , Fosfolipídeos/biossíntese , Ácidos Graxos/metabolismo , Ácidos Graxos/biossíntese , Sistema Livre de Células/metabolismo , Expressão Gênica/genética , Lipossomos/metabolismo , Lipossomos/química , Cromatografia Líquida/métodos , Aciltransferases/genética , Aciltransferases/metabolismo , Espectrometria de Massas/métodosRESUMO
In cells, phospholipids contain acyl chains of variable lengths and saturation, features that affect their functions. Their de novo synthesis in the endoplasmic reticulum takes place via the cytidine diphosphate diacylglycerol (CDP-DAG) and Kennedy pathways, which are conserved in eukaryotes. PA is a key intermediate for all phospholipids (PI, PIPs, PS, PE, PC, PG and CL). The de novo synthesis of PA occurs by acylation of glycerophosphate leading to the synthesis of 1-acyl lysoPA and subsequent acylation of 1-acyl lysoPA at the sn-2 position. Using membranes from Escherichia coli overexpressing MLG1, we showed that the yeast gene MLG1 encodes an acyltransferase, leading specifically to the synthesis of PA from 1-acyl lysoPA. Moreover, after their de novo synthesis, phospholipids can be remodelled by acyl exchange with one and/or two acyl chains exchanged at the sn-1 and/or sn-2 position. Based on shotgun lipidomics of the reference and mlg1Δ strains, as well as biochemical assays for acyltransferase activities, we identified an additional remodelling activity for Mlg1p, namely, incorporation of palmitic acid into the sn-1 position of PS and PE. By using confocal microscopy and subcellular fractionation, we also found that this acyltransferase is located in ER membranes associated with mitochondria, a finding that highlights the importance of these organelles in the global cellular metabolism of lipids.
Assuntos
Aciltransferases , Retículo Endoplasmático , Mitocôndrias , Fosfolipídeos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Retículo Endoplasmático/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Aciltransferases/metabolismo , Aciltransferases/genética , Fosfolipídeos/metabolismo , Fosfolipídeos/biossíntese , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Mitocôndrias/metabolismo , Mitocôndrias/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Membranas Intracelulares/metabolismoRESUMO
In Saccharomyces cerevisiae, the transcriptional repressor Opi1 regulates the expression of genes involved in phospholipid synthesis responding to the abundance of the phospholipid precursor phosphatidic acid at the endoplasmic reticulum. We report here the identification of the conserved leucine zipper (LZ) domain of Opi1 as a hot spot for gain of function mutations and the characterization of the strongest variant identified, Opi1N150D. LZ modeling posits asparagine 150 embedded on the hydrophobic surface of the zipper and specifying dynamic parallel homodimerization by allowing electrostatic bonding across the hydrophobic dimerization interface. Opi1 variants carrying any of the other three ionic residues at amino acid 150 were also repressing. Genetic analyses showed that Opi1N150D variant is dominant, and its phenotype is attenuated when loss of function mutations identified in the other two conserved domains are present in cis. We build on the notion that membrane binding facilitates LZ dimerization to antagonize an intramolecular interaction of the zipper necessary for repression. Dissecting Opi1 protein in three polypeptides containing each conserved region, we performed in vitro analyses to explore interdomain interactions. An Opi11-190 probe interacted with Opi1291-404, the C terminus that bears the activator interacting domain (AID). LZ or AID loss of function mutations attenuated the interaction of the probes but was unaffected by the N150D mutation. We propose a model for Opi1 signal transduction whereby synergy between membrane-binding events and LZ dimerization antagonizes intramolecular LZ-AID interaction and transcriptional repression.
Assuntos
Zíper de Leucina , Fosfolipídeos , Proteínas Repressoras , Proteínas de Saccharomyces cerevisiae , Fosfolipídeos/biossíntese , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Multimerização ProteicaRESUMO
Binding of general transcription factors TFIID and TFIIA to basal promoters is rate-limiting for transcriptional initiation of eukaryotic protein-coding genes. Consequently, activator proteins interacting with subunits of TFIID and/or TFIIA can drastically increase the rate of initiation events. Yeast transcriptional activator Ino2 interacts with several Taf subunits of TFIID, among them the multifunctional Taf1 protein. In contrast to mammalian Taf1, yeast Taf1 lacks bromodomains which are instead encoded by separate proteins Bdf1 and Bdf2. In this work, we show that Bdf1 not only binds to acetylated histone H4 but can also be recruited by Ino2 and unrelated activators such as Gal4, Rap1, Leu3 and Flo8. An activator-binding domain was mapped in the N-terminus of Bdf1. Subunits Toa1 and Toa2 of yeast TFIIA directly contact sequences of basal promoters and TFIID subunit TBP but may also mediate the influence of activators. Indeed, Ino2 efficiently binds to two separate structural domains of Toa1, specifically with its N-terminal four-helix bundle structure required for dimerization with Toa2 and its C-terminal ß-barrel domain contacting TBP and sequences of the TATA element. These findings complete the functional analysis of yeast general transcription factors Bdf1 and Toa1 and identify them as targets of activator proteins.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Proteínas que Contêm Bromodomínio , Fosfolipídeos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Fator de Transcrição TFIIA , Fatores de Transcrição , Fosfolipídeos/biossíntese , Fosfolipídeos/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteína de Ligação a TATA-Box/genética , Proteína de Ligação a TATA-Box/metabolismo , Fator de Transcrição TFIIA/genética , Fator de Transcrição TFIIA/metabolismo , Fator de Transcrição TFIID/genética , Fator de Transcrição TFIID/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas que Contêm Bromodomínio/genética , Proteínas que Contêm Bromodomínio/metabolismoRESUMO
BACKGROUND & AIMS: Hepatocyte growth and proliferation depends on membrane phospholipid biosynthesis. Short-chain fatty acids (SCFAs) generated by bacterial fermentation, delivered through the gut-liver axis, significantly contribute to lipid biosynthesis. We therefore hypothesized that dysbiotic insults like antibiotic treatment not only affect gut microbiota, but also impair hepatic lipid synthesis and liver regeneration. METHODS: Stable isotope labeling and 70% partial hepatectomy (PHx) was carried out in C57Bl/6J wild-type mice, in mice treated with broad-spectrum antibiotics, in germ-free mice and mice colonized with minimal microbiota. The microbiome was analyzed by 16S rRNA gene sequencing and microbial culture. Gut content, liver, blood and primary hepatocyte organoids were tested by mass spectrometry-based lipidomics, quantitative reverse-transcription PCR (qRT-PCR), immunoblot and immunohistochemistry for expression of proliferative and lipogenic markers. Matched biopsies from hyperplastic and hypoplastic liver tissue of patients subjected to surgical intervention to induce hyperplasia were analyzed by qRT-PCR for lipogenic enzymes. RESULTS: Three days of antibiotic treatment induced persistent dysbiosis with significantly decreased beta-diversity and richness, but a massive increase of Proteobacteria, accompanied by decreased colonic SCFAs. After PHx, antibiotic-treated mice showed delayed liver regeneration, increased mortality, impaired hepatocyte proliferation and decreased hepatic phospholipid synthesis. Expression of the lipogenic enzyme SCD1 was upregulated after PHx but delayed by antibiotic treatment. Germ-free mice essentially recapitulated the phenotype of antibiotic treatment. Phospholipid biosynthesis, hepatocyte proliferation, liver regeneration and survival were rescued in gnotobiotic mice colonized with a minimal SCFA-producing microbial community. SCFAs induced the growth of murine hepatocyte organoids and hepatic SCD1 expression in mice. Further, SCD1 was required for proliferation of human hepatoma cells and was associated with liver regeneration in human patients. CONCLUSION: Gut microbiota are pivotal for hepatic membrane phospholipid biosynthesis and liver regeneration. IMPACT AND IMPLICATIONS: Gut microbiota affect hepatic lipid metabolism through the gut-liver axis, but the underlying mechanisms are poorly understood. Perturbations of the gut microbiome, e.g. by antibiotics, impair the production of bacterial metabolites, which normally serve as building blocks for membrane lipids in liver cells. As a consequence, liver regeneration and survival after liver surgery is severely impaired. Even though this study is preclinical, its results might allow physicians in the future to improve patient outcomes after liver surgery, by modulation of gut microbiota or their metabolites.
Assuntos
Membrana Celular , Microbioma Gastrointestinal , Hepatócitos , Regeneração Hepática , Fosfolipídeos , Animais , Humanos , Camundongos , Antibacterianos/farmacologia , Microbioma Gastrointestinal/genética , Microbioma Gastrointestinal/fisiologia , Hiperplasia/metabolismo , Hiperplasia/patologia , Fígado/patologia , Regeneração Hepática/fisiologia , Camundongos Endogâmicos C57BL , Fosfolipídeos/biossíntese , Fosfolipídeos/metabolismo , RNA Ribossômico 16S , Hepatócitos/metabolismo , Membrana Celular/metabolismoRESUMO
Under certain cellular conditions, functional proteins undergo misfolding, leading to a transition into oligomers which precede the formation of amyloid fibrils. Misfolding proteins are associated with neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. While the importance of lipid membranes in misfolding and disease aetiology is broadly accepted, the influence of lipid membranes during therapeutic design has been largely overlooked. This study utilized a biophysical approach to provide mechanistic insights into the effects of two lipid membrane systems (anionic and zwitterionic) on the inhibition of amyloid-ß 40 and α-synuclein amyloid formation at the monomer, oligomer and fibril level. Large unilamellar vesicles (LUVs) were shown to increase fibrillization and largely decrease the effectiveness of two well-known polyphenol fibril inhibitors, (-)-epigallocatechin gallate (EGCG) and resveratrol; however, use of immunoblotting and ion mobility mass spectrometry revealed this occurs through varying mechanisms. Oligomeric populations in particular were differentially affected by LUVs in the presence of resveratrol, an elongation phase inhibitor, compared to EGCG, a nucleation targeted inhibitor. Ion mobility mass spectrometry showed EGCG interacts with or induces more compact forms of monomeric protein typical of off-pathway structures; however, binding is reduced in the presence of LUVs, likely due to partitioning in the membrane environment. Competing effects of the lipids and inhibitor, along with reduced inhibitor binding in the presence of LUVs, provide a mechanistic understanding of decreased inhibitor efficacy in a lipid environment. Together, this study highlights that amyloid inhibitor design may be misguided if effects of lipid membrane composition and architecture are not considered during development.
Assuntos
Precursor de Proteína beta-Amiloide/genética , Amiloide/genética , Doença de Parkinson/genética , alfa-Sinucleína/genética , Amiloide/efeitos dos fármacos , Amiloide/ultraestrutura , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/ultraestrutura , Proteínas Amiloidogênicas/antagonistas & inibidores , Proteínas Amiloidogênicas/genética , Catequina/análogos & derivados , Catequina/farmacologia , Humanos , Bicamadas Lipídicas/metabolismo , Lipídeos de Membrana/genética , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/patologia , Fosfolipídeos/biossíntese , Fosfolipídeos/genética , Polifenóis/farmacologia , alfa-Sinucleína/ultraestruturaRESUMO
The cell envelope is essential for viability in all domains of life. It retains enzymes and substrates within a confined space while providing a protective barrier to the external environment. Destabilising the envelope of bacterial pathogens is a common strategy employed by antimicrobial treatment. However, even in one of the best studied organisms, Escherichia coli, there remain gaps in our understanding of how the synthesis of the successive layers of the cell envelope are coordinated during growth and cell division. Here, we used a whole-genome phenotypic screen to identify mutants with a defective cell envelope. We report that loss of yhcB, a conserved gene of unknown function, results in loss of envelope stability, increased cell permeability and dysregulated control of cell size. Using whole genome transposon mutagenesis strategies, we report the comprehensive genetic interaction network of yhcB, revealing all genes with a synthetic negative and a synthetic positive relationship. These genes include those previously reported to have a role in cell envelope biogenesis. Surprisingly, we identified genes previously annotated as essential that became non-essential in a ΔyhcB background. Subsequent analyses suggest that YhcB functions at the junction of several envelope biosynthetic pathways coordinating the spatiotemporal growth of the cell, highlighting YhcB as an as yet unexplored antimicrobial target.
Assuntos
Parede Celular/genética , Proteínas de Escherichia coli/genética , Lipopolissacarídeos/genética , Oxirredutases/genética , Peptidoglicano/genética , Divisão Celular/genética , Membrana Celular/genética , Membrana Celular/microbiologia , Parede Celular/microbiologia , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Lipopolissacarídeos/biossíntese , Mutagênese , Fosfolipídeos/biossíntese , Fosfolipídeos/genéticaRESUMO
The cold-water gorgonian coral Paragorgia arborea is considered as a foundation species of deep-sea ecosystems in the northern Atlantic and Pacific oceans. To advance lipidomic studies of deep-sea corals, molecular species compositions of diacylglycerol ethers (DAGE), which are specific storage lipids of corals, and structural glycerophospholipids (GPL) including ethanolamine, choline, inositol and serine GPL (PE, PC, PI, and PS, respectively) were analyzed in P. arborea by HPLC and tandem mass spectrometry. In DAGE molecules, alkyl groups (16:0, 14:0, and 18:1), polyunsaturated fatty acids (PUFA), and monounsaturated FA are mainly substituted the glycerol moiety at position sn-1, sn-2, and sn-3, respectively. The ether form (1-O-alkyl-2-acyl) predominates in PE and PC, while PI is comprised of the 1,2-diacyl form. Both ether and diacyl forms were observed in PS. At position sn-2, C20 PUFA are mainly attached to PC, but C24 PUFA, soft coral chemotaxonomic markers, concentrate in PS, PI, and PE. A comparison of non-polar parts of molecules has shown that DAGE, ether PE, and ether PC can originate from one set of 1-O-alkyl-2-acyl-sn-glycerols. Ether PE may be converted to ether PS by the base-exchange reaction. A diacylglycerol unit generated from phosphatidic acid can be a precursor for diacyl PS, PC, and PI. Thus, a lipidomic approach has confirmed the difference in biosynthetic origins between ether and diacyl lipids of deep-sea gorgonians.
Assuntos
Antozoários/metabolismo , Diglicerídeos/biossíntese , Lipidômica , Fosfolipídeos/biossíntese , Animais , Éteres , Ácidos Graxos/metabolismo , Metabolismo dos Lipídeos , Lipidômica/métodosRESUMO
Here, we present the main features of human acid sphingomyelinase (ASM), its biosynthesis, processing and intracellular trafficking, its structure, its broad substrate specificity, and the proposed mode of action at the surface of the phospholipid substrate carrying intraendolysosomal luminal vesicles. In addition, we discuss the complex regulation of its phospholipid cleaving activity by membrane lipids and lipid-binding proteins. The majority of the literature implies that ASM hydrolyses solely sphingomyelin to generate ceramide and ignores its ability to degrade further substrates. Indeed, more than twenty different phospholipids are cleaved by ASM in vitro, including some minor but functionally important phospholipids such as the growth factor ceramide-1-phosphate and the unique lysosomal lysolipid bis(monoacylglycero)phosphate. The inherited ASM deficiency, Niemann-Pick disease type A and B, impairs mainly, but not only, cellular sphingomyelin catabolism, causing a progressive sphingomyelin accumulation, which furthermore triggers a secondary accumulation of lipids (cholesterol, glucosylceramide, GM2) by inhibiting their turnover in late endosomes and lysosomes. However, ASM appears to be involved in a variety of major cellular functions with a regulatory significance for an increasing number of metabolic disorders. The biochemical characteristics of ASM, their potential effect on cellular lipid turnover, as well as a potential impact on physiological processes will be discussed.
Assuntos
Fosfolipídeos/biossíntese , Esfingomielina Fosfodiesterase/biossíntese , Esfingomielina Fosfodiesterase/metabolismo , Transporte Biológico , Ceramidas/metabolismo , Colesterol/metabolismo , Endossomos/metabolismo , Humanos , Lisossomos/metabolismo , Lipídeos de Membrana/metabolismo , Doença de Niemann-Pick Tipo A/metabolismo , Fosfolipídeos/metabolismo , Esfingomielina Fosfodiesterase/fisiologia , Esfingomielinas/metabolismo , Fosfolipases Tipo C/metabolismo , Fosfolipases Tipo C/fisiologiaRESUMO
Prokineticin-2 (Prok2) is an important secreted protein likely involved in the pathogenesis of several acute and chronic neurological diseases through currently unidentified regulatory mechanisms. The initial mechanical injury of neurons by traumatic brain injury triggers multiple secondary responses including various cell death programs. One of these is ferroptosis, which is associated with dysregulation of iron and thiols and culminates in fatal lipid peroxidation. Here, we explore the regulatory role of Prok2 in neuronal ferroptosis in vitro and in vivo. We show that Prok2 prevents neuronal cell death by suppressing the biosynthesis of lipid peroxidation substrates, arachidonic acid-phospholipids, via accelerated F-box only protein 10 (Fbxo10)-driven ubiquitination, degradation of long-chain-fatty-acid-CoA ligase 4 (Acsl4), and inhibition of lipid peroxidation. Mice injected with adeno-associated virus-Prok2 before controlled cortical impact injury show reduced neuronal degeneration and improved motor and cognitive functions, which could be inhibited by Fbxo10 knockdown. Our study shows that Prok2 mediates neuronal cell deaths in traumatic brain injury via ferroptosis.
Assuntos
Lesões Encefálicas Traumáticas/patologia , Córtex Cerebral/patologia , Ferroptose , Hormônios Gastrointestinais/metabolismo , Neuropeptídeos/metabolismo , Adulto , Idoso , Animais , Lesões Encefálicas Traumáticas/cirurgia , Células Cultivadas , Córtex Cerebral/citologia , Coenzima A Ligases/metabolismo , Modelos Animais de Doenças , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Feminino , Hormônios Gastrointestinais/genética , Técnicas de Silenciamento de Genes , Humanos , Peroxidação de Lipídeos , Masculino , Camundongos , Pessoa de Meia-Idade , Mitocôndrias/patologia , Neurônios/citologia , Neurônios/patologia , Neuropeptídeos/genética , Fosfolipídeos/biossíntese , Cultura Primária de Células , Proteólise , UbiquitinaçãoRESUMO
Choline phospholipids (PLs) such as phosphatidylcholine (PC) and 1-alkyl-2-acyl-sn-glycerophosphocholine are important components for cell membranes and also serve as a source of several lipid mediators. These lipids are biosynthesized in mammals in the final step of the CDP-choline pathway by the choline phosphotransferases choline phosphotransferase 1 (CPT1) and choline/ethanolamine phosphotransferase 1 (CEPT1). However, the contributions of these enzymes to the de novo biosynthesis of lipids remain unknown. Here, we established and characterized CPT1- and CEPT1-deficient human embryonic kidney 293 cells. Immunohistochemical analyses revealed that CPT1 localizes to the trans-Golgi network and CEPT1 to the endoplasmic reticulum. Enzyme assays and metabolic labeling with radiolabeled choline demonstrated that loss of CEPT1 dramatically decreases choline PL biosynthesis. Quantitative PCR and reintroduction of CPT1 and CEPT1 revealed that the specific activity of CEPT1 was much higher than that of CPT1. LC-MS/MS analysis of newly synthesized lipid molecular species from deuterium-labeled choline also showed that these enzymes have similar preference for the synthesis of PC molecular species, but that CPT1 had higher preference for 1-alkyl-2-acyl-sn-glycerophosphocholine with PUFA than did CEPT1. The endogenous level of PC was not reduced by the loss of these enzymes. However, several 1-alkyl-2-acyl-sn-glycerophosphocholine molecular species were reduced in CPT1-deficient cells and increased in CEPT1-deficient cells when cultured in 0.1% FBS medium. These results suggest that CEPT1 accounts for most choline PL biosynthesis activity, and that both enzymes are responsible for the production of different lipid molecular species in distinct organelles.
Assuntos
Colina/biossíntese , Diacilglicerol Colinofosfotransferase/metabolismo , Fosfolipídeos/biossíntese , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Células Cultivadas , Células HEK293 , HumanosRESUMO
The structural challenges faced by eukaryotic cells through the cell cycle are key for understanding cell viability and proliferation. We tested the hypothesis that the biosynthesis of structural lipids is linked to the cell cycle. If true, this would suggest that the cell's structure is important for progress through and perhaps even control of the cell cycle. Lipidomics (31P NMR and MS), proteomics (Western immunoblotting) and transcriptomics (RT-qPCR) techniques were used to profile the lipid fraction and characterise aspects of its metabolism at seven stages of the cell cycle of the model eukaryote, Desmodesmus quadricauda. We found considerable, transient increases in the abundance of phosphatidylethanolamine during the G1 phase (+35%, ethanolamine phosphate cytidylyltransferase increased 2·5×) and phosphatidylglycerol (+100%, phosphatidylglycerol synthase increased 22×) over the G1/pre-replication phase boundary. The relative abundance of phosphatidylcholine fell by ~35% during the G1. N-Methyl transferases for the conversion of phosphatidylethanolamine into phosphatidylcholine were not found in the de novo transcriptome profile, though a choline phosphate transferase was found, suggesting that the Kennedy pathway is the principal route for the synthesis of PC. The fatty acid profiles of the four most abundant lipids suggested that these lipids were not generally converted between one another. This study shows for the first time that there are considerable changes in the biosynthesis of the three most abundant phospholipid classes in the normal cell cycle of D. quadricauda, by margins large enough to elicit changes to the physical properties of membranes.
Assuntos
Ciclo Celular , Células Eucarióticas/citologia , Células Eucarióticas/metabolismo , Fosfolipídeos/biossíntese , Colina/metabolismo , Metabolismo dos LipídeosRESUMO
The de novo formation of lipid membranes from minimal reactive precursors is a major goal in synthetic cell research. In nature, the synthesis of membrane phospholipids is orchestrated by numerous enzymes, including fatty acid synthases and membrane-bound acyltransferases. However, these enzymatic pathways are difficult to fully reproduce in vitro. As such, the reconstitution of phospholipid membrane synthesis from simple metabolic building blocks remains a challenge. Here, we describe a chemoenzymatic strategy for lipid membrane generation that utilizes a soluble bacterial fatty acid synthase (cgFAS I) to synthesize palmitoyl-CoA in situ from acetyl-CoA and malonyl-CoA. The fatty acid derivative spontaneously reacts with a cysteine-modified lysophospholipid by native chemical ligation (NCL), affording a noncanonical amidophospholipid that self-assembles into micron-sized membrane-bound vesicles. To our knowledge, this is the first example of reconstituting phospholipid membrane formation directly from acetyl-CoA and malonyl-CoA precursors. Our results demonstrate that combining the specificity and efficiency of a type I fatty acid synthase with a highly selective bioconjugation reaction provides a biomimetic route for the de novo formation of membrane-bound vesicles.
Assuntos
Ácido Graxo Sintase Tipo I/metabolismo , Fosfolipídeos/biossíntese , Ácido Graxo Sintase Tipo I/química , Estrutura Molecular , Fosfolipídeos/químicaRESUMO
Bacterial membranes are primarily composed of phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and cardiolipin (CL). In the canonical PE biosynthesis pathway, phosphatidylserine (PS) is decarboxylated by the Psd enzyme. CL formation typically depends on CL synthases (Cls) using two PG molecules as substrates. Only few bacteria produce phosphatidylcholine (PC), the hallmark of eukaryotic membranes. Most of these bacteria use phospholipid N-methyltransferases to successively methylate PE to PC and/or a PC synthase (Pcs) to catalyze the condensation of choline and CDP-diacylglycerol (CDP-DAG) to PC. In this study, we show that membranes of Pseudomonas species able to interact with eukaryotes contain PE, PG, CL and PC. More specifically, we report on PC formation and a poorly characterized CL biosynthetic pathway in the plant pathogen P. syringae pv. tomato. It encodes a Pcs enzyme responsible for choline-dependent PC biosynthesis. CL formation is catalyzed by a promiscuous phospholipase D (PLD)-type enzyme (PSPTO_0095) that we characterized in vivo and in vitro. Like typical bacterial CL biosynthesis enzymes, it uses PE and PG for CL production. This enzyme is also able to convert PE and glycerol to PG, which is then combined with another PE molecule to synthesize CL. In addition, the enzyme is capable of converting ethanolamine or methylated derivatives into the corresponding phospholipids such as PE both in P. syringae and in E. coli. It can also hydrolyze CDP-DAG to yield phosphatidic acid (PA). Our study adds an example of a promiscuous Cls enzyme able to synthesize a suite of products according to the available substrates.
Assuntos
Fosfolipídeos/biossíntese , Plantas/microbiologia , Pseudomonas syringae/enzimologia , Pseudomonas syringae/fisiologia , Especificidade por SubstratoRESUMO
Contacts between organelles create microdomains that play major roles in regulating key intracellular activities and signaling pathways, but whether they also regulate systemic functions remains unknown. Here, we report the ultrastructural organization and dynamics of the inter-organellar contact established by sheets of curved rough endoplasmic reticulum closely wrapped around the mitochondria (wrappER). To elucidate the in vivo function of this contact, mouse liver fractions enriched in wrappER-associated mitochondria are analyzed by transcriptomics, proteomics, and lipidomics. The biochemical signature of the wrappER points to a role in the biogenesis of very-low-density lipoproteins (VLDL). Altering wrappER-mitochondria contacts curtails VLDL secretion and increases hepatic fatty acids, lipid droplets, and neutral lipid content. Conversely, acute liver-specific ablation of Mttp, the most upstream regulator of VLDL biogenesis, recapitulates this hepatic dyslipidemia phenotype and promotes remodeling of the wrappER-mitochondria contact. The discovery that liver wrappER-mitochondria contacts participate in VLDL biology suggests an involvement of inter-organelle contacts in systemic lipid homeostasis.
Assuntos
Retículo Endoplasmático/metabolismo , Homeostase , Lipídeos/química , Fígado/metabolismo , Mitocôndrias/metabolismo , Animais , Retículo Endoplasmático/ultraestrutura , Enterócitos/metabolismo , Inativação Gênica , Hepatócitos/metabolismo , Imageamento Tridimensional , Intestino Delgado/citologia , Lipoproteínas VLDL/biossíntese , Masculino , Metabolômica , Camundongos Endogâmicos C57BL , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/metabolismo , Fosfolipídeos/biossíntese , Proteínas/metabolismoRESUMO
Over 80 human diseases have been attributed to defects in complex lipid metabolism. A majority of them have been reported recently in the setting of rapid advances in genomic technology and their increased use in clinical settings. Lipids are ubiquitous in human biology and play roles in many cellular and intercellular processes. While inborn errors in lipid metabolism can affect every organ system with many examples of genetic heterogeneity and pleiotropy, the clinical manifestations of many of these disorders can be explained based on the disruption of the metabolic pathway involved. In this review, we will discuss the physiological function of major pathways in complex lipid metabolism, including nonlysosomal sphingolipid metabolism, acylceramide metabolism, de novo phospholipid synthesis, phospholipid remodeling, phosphatidylinositol metabolism, mitochondrial cardiolipin synthesis and remodeling, and ether lipid metabolism as well as common clinical phenotypes associated with each.
Assuntos
Metabolismo dos Lipídeos , Lipídeos/química , Redes e Vias Metabólicas/fisiologia , Cardiolipinas/biossíntese , Cardiolipinas/química , Homeostase , Humanos , Lipídeos/biossíntese , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Fenótipo , Fosfatidilinositóis/biossíntese , Fosfatidilinositóis/química , Fosfolipídeos/biossíntese , Fosfolipídeos/química , Esfingolipídeos/biossíntese , Esfingolipídeos/químicaRESUMO
Fatty acid biosynthesis (FASII) enzymes are considered valid targets for antimicrobial drug development against the human pathogen Staphylococcus aureus However, incorporation of host fatty acids confers FASII antibiotic adaptation that compromises prospective treatments. S. aureus adapts to FASII inhibitors by first entering a nonreplicative latency period, followed by outgrowth. Here, we used transcriptional fusions and direct metabolite measurements to investigate the factors that dictate the duration of latency prior to outgrowth. We show that stringent response induction leads to repression of FASII and phospholipid synthesis genes. (p)ppGpp induction inhibits synthesis of malonyl-CoA, a molecule that derepresses FapR, a key regulator of FASII and phospholipid synthesis. Anti-FASII treatment also triggers transient expression of (p)ppGpp-regulated genes during the anti-FASII latency phase, with concomitant repression of FapR regulon expression. These effects are reversed upon outgrowth. GTP depletion, a known consequence of the stringent response, also occurs during FASII latency, and is proposed as the common signal linking these responses. We next showed that anti-FASII treatment shifts malonyl-CoA distribution between its interactants FapR and FabD, toward FapR, increasing expression of the phospholipid synthesis genes plsX and plsC during outgrowth. We conclude that components of the stringent response dictate malonyl-CoA availability in S. aureus FASII regulation, and contribute to latency prior to anti-FASII-adapted outgrowth. A combinatory approach, coupling a (p)ppGpp inducer and an anti-FASII, blocks S. aureus outgrowth, opening perspectives for bi-therapy treatment.IMPORTANCEStaphylococcus aureus is a major human bacterial pathogen for which new inhibitors are urgently needed. Antibiotic development has centered on the fatty acid synthesis (FASII) pathway, which provides the building blocks for bacterial membrane phospholipids. However, S. aureus overcomes FASII inhibition and adapts to anti-FASII by using exogenous fatty acids that are abundant in host environments. This adaptation mechanism comprises a transient latency period followed by bacterial outgrowth. Here, we use metabolite sensors and promoter reporters to show that responses to stringent conditions and to FASII inhibition intersect, in that both involve GTP and malonyl-CoA. These two signaling molecules contribute to modulating the duration of latency prior to S. aureus adaptation outgrowth. We exploit these novel findings to propose a bi-therapy treatment against staphylococcal infections.
Assuntos
Antibacterianos/farmacologia , Ácidos Graxos/antagonistas & inibidores , Guanosina Pentafosfato/fisiologia , Guanosina Trifosfato/fisiologia , Malonil Coenzima A/fisiologia , Staphylococcus aureus/efeitos dos fármacos , Adaptação Fisiológica/efeitos dos fármacos , Ácidos Graxos/biossíntese , Humanos , Malonil Coenzima A/análise , Mupirocina/farmacologia , Fosfolipídeos/biossíntese , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/fisiologiaRESUMO
Enteroviruses are among the most common human infectious agents. While infections are often mild, the severe neuropathogenesis associated with recent outbreaks of emerging non-polio enteroviruses, such as EV-A71 and EV-D68, highlights their continuing threat to public health. In recent years, our understanding of how non-polio enteroviruses co-opt cellular pathways has greatly increased, revealing intricate host-virus relationships. In this review, we focus on newly identified mechanisms by which enteroviruses hijack the cellular machinery to promote their replication and spread, and address their potential for the development of host-directed therapeutics. Specifically, we discuss newly identified cellular receptors and their contribution to neurotropism and spread, host factors required for viral entry and replication, and recent insights into lipid acquisition and replication organelle biogenesis. The comprehensive knowledge of common cellular pathways required by enteroviruses could expose vulnerabilities amenable for host-directed therapeutics against a broad spectrum of enteroviruses. Since this will likely include newly arising strains, it will better prepare us for future epidemics. Moreover, identifying host proteins specific to neurovirulent strains may allow us to better understand factors contributing to the neurotropism of these viruses.
Assuntos
Viroses do Sistema Nervoso Central/virologia , Sistema Nervoso Central/virologia , Infecções por Enterovirus/virologia , Enterovirus/patogenicidade , Tropismo Viral , Animais , Autofagia , Enterovirus/genética , Enterovirus/fisiologia , Genoma Viral , Interações Hospedeiro-Patógeno , Humanos , Sítios Internos de Entrada Ribossomal , Fosfolipídeos/biossíntese , Biossíntese de Proteínas , RNA Viral/biossíntese , Receptores Virais/metabolismo , Compartimentos de Replicação Viral/fisiologia , Compartimentos de Replicação Viral/ultraestrutura , Internalização do Vírus , Replicação ViralRESUMO
OBJECTIVE: T cell activation triggers metabolic reprogramming to meet increased demands for energy and metabolites required for cellular proliferation. Ethanolamine phospholipid synthesis has emerged as a regulator of metabolic shifts in stem cells and cancer cells, which led us to investigate its potential role during T cell activation. METHODS: As selenoprotein I (SELENOI) is an enzyme participating in two metabolic pathways for the synthesis of phosphatidylethanolamine (PE) and plasmenyl PE, we generated SELENOI-deficient mouse models to determine loss-of-function effects on metabolic reprogramming during T cell activation. Ex vivo and in vivo assays were carried out along with metabolomic, transcriptomic, and protein analyses to determine the role of SELENOI and the ethanolamine phospholipids synthesized by this enzyme in cell signaling and metabolic pathways that promote T cell activation and proliferation. RESULTS: SELENOI knockout (KO) in mouse T cells led to reduced de novo synthesis of PE and plasmenyl PE during activation and impaired proliferation. SELENOI KO did not affect T cell receptor signaling, but reduced activation of the metabolic sensor AMPK. AMPK was inhibited by high [ATP], consistent with results showing SELENOI KO causing ATP accumulation, along with disrupted metabolic pathways and reduced glycosylphosphatidylinositol (GPI) anchor synthesis/attachment CONCLUSIONS: T cell activation upregulates SELENOI-dependent PE and plasmenyl PE synthesis as a key component of metabolic reprogramming and proliferation.
Assuntos
Etanolamina/metabolismo , Fosfolipídeos/biossíntese , Selenoproteínas/metabolismo , Linfócitos T/metabolismo , Animais , Proliferação de Células , Etanolaminas/metabolismo , Feminino , Glicólise , Glicosilfosfatidilinositóis/metabolismo , Lipogênese/genética , Lipogênese/fisiologia , Masculino , Redes e Vias Metabólicas , Metabolômica , Camundongos , Camundongos Knockout , Fosfatidiletanolaminas/metabolismo , Selenoproteínas/deficiência , Selenoproteínas/genéticaRESUMO
As a promising novel marine fish model for future research on marine ecotoxicology as well as an animal model of human disease, the genome information of yellowstripe goby (Mugilogobius chulae) remains unknown. Here we report the first annotated chromosome-level reference genome assembly for yellowstripe goby. A 20.67-cM sex determination region was discovered on chromosome 5 and seven potential sex-determining genes were identified. Based on combined genome and transcriptome data, we identified three key lipid metabolic pathways for high-fat accumulation in the liver of yellowstripe goby. The changes in the expression patterns of MGLL and CPT1 at different development stage of the liver, and the expansion of the ABCA1 gene, innate immune gene TLR23, and TRIM family genes may help in balancing high-fat storage in hepatocytes and steatohepatitis. These results may provide insights into understanding the molecular mechanisms of sex determination and high-fat storage in the liver of marine fishes.