RESUMO
BACKGROUND: Allergens can cross the epithelial barrier to enter the body but how this cellular passage affects protein structures and the downstream interactions with the immune system are still open questions. OBJECTIVE: We show the molecular details and the effects of three non-specific lipid transfer proteins (nsLTPs; Mal d 3, Cor a 8 and Pru p 3) upon epithelial cell uptake and transport. METHODS: We used fluorescent imaging, flow cytometry, proteomic and lipidomic screenings to identify the mechanism involved in nsLTP cellular uptake and signaling on selected epithelial and transgenic cell lines. RESULTS: NsLTPs are transported across the epithelium without affecting cell membrane stability or viability and allergen uptake was largely impaired by inhibition of clathrin-mediated endocytosis (CME). Analysis of the lipidome associated with nsLTPs showed a wide variety of lipid ligands predicted to bind inside the allergen hydrophobic cavity. Importantly, the internalization of nsLTPs was contingent upon these ligands in the protein complex.nsLTPs were found to initiate cellular signaling via TLR2 but not the CD1d receptor, despite neither being essential for nsLTP endocytosis. We also provide evidence that the three allergens induced intracellular stress signaling through activation of the NOD2 pathway. CONCLUSIONS: Our work consolidates the current model on nsLTP-epithelial cell interplay and adds molecular details about cell transport and signaling. Additionally, we have developed a versatile toolbox to extend these investigations to other allergens and cell types.
RESUMO
Eukaryotic ribosome synthesis involves more than 200 assembly factors, which promote ribosomal RNA (rRNA) processing, modification and folding, and assembly of ribosomal proteins. The formation and maturation of the earliest pre-60S particles requires structural remodeling by the Npa1 complex, but is otherwise still poorly understood. Here, we introduce Rbp95 (Ycr016w), a constituent of early pre-60S particles, as a novel ribosome assembly factor. We show that Rbp95 is both genetically and physically linked to most Npa1 complex members and to ribosomal protein Rpl3. We demonstrate that Rbp95 is an RNA-binding protein containing two independent RNA-interacting domains. In vivo, Rbp95 associates with helix H95 in the 3' region of the 25S rRNA, in close proximity to the binding sites of Npa1 and Rpl3. Additionally, Rbp95 interacts with several snoRNAs. The absence of Rbp95 results in alterations in the protein composition of early pre-60S particles. Moreover, combined mutation of Rbp95 and Npa1 complex members leads to a delay in the maturation of early pre-60S particles. We propose that Rbp95 acts together with the Npa1 complex during early pre-60S maturation, potentially by promoting pre-rRNA folding events within pre-60S particles.
Assuntos
Proteínas Nucleares/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos , Proteínas de Saccharomyces cerevisiae/metabolismo , Precursores de RNA/metabolismo , RNA Ribossômico/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Saccharomyces cerevisiae/genéticaRESUMO
Sterols exert a profound influence on numerous cellular processes, playing a crucial role in both health and disease. However, comprehending the effects of sterol dysfunction on cellular physiology is challenging. Consequently, numerous processes affected by impaired sterol biosynthesis still elude our complete understanding. In this study, we made use of yeast strains that produce cholesterol instead of ergosterol and investigated the cellular response mechanisms on the transcriptome as well as the lipid level. The exchange of ergosterol for cholesterol caused the downregulation of phosphatidylethanolamine and phosphatidylserine and upregulation of phosphatidylinositol and phosphatidylcholine biosynthesis. Additionally, a shift towards polyunsaturated fatty acids was observed. While the sphingolipid levels dropped, the total amounts of sterols and triacylglycerol increased, which resulted in 1.7-fold enlarged lipid droplets in cholesterol-producing yeast cells. In addition to internal storage, cholesterol and its precursors were excreted into the culture supernatant, most likely by the action of ABC transporters Snq2, Pdr12 and Pdr15. Overall, our results demonstrate that, similarly to mammalian cells, the production of non-native sterols and sterol precursors causes lipotoxicity in K. phaffii, mainly due to upregulated sterol biosynthesis, and they highlight the different survival and stress response mechanisms on multiple, integrative levels.
Assuntos
Fitosteróis , Esteróis , Animais , Humanos , Saccharomyces cerevisiae , Ergosterol , Colesterol , MamíferosRESUMO
BACKGROUND: Lipid homeostasis is an evolutionarily conserved process that is crucial for energy production, storage and consumption. Drosophila larvae feed continuously to achieve the roughly 200-fold increase in size and accumulate sufficient reserves to provide all energy and nutrients necessary for the development of the adult fly. The mechanisms controlling this metabolic program are poorly understood. RESULTS: Herein we identified a highly conserved gene, orsai (osi), as a key player in lipid metabolism in Drosophila. Lack of osi function in the larval fat body, the regulatory hub of lipid homeostasis, reduces lipid reserves and energy output, evidenced by decreased ATP production and increased ROS levels. Metabolic defects due to reduced Orsai (Osi) in time trigger defective food-seeking behavior and lethality. Further, we demonstrate that downregulation of Lipase 3, a fat body-specific lipase involved in lipid catabolism in response to starvation, rescues the reduced lipid droplet size associated with defective orsai. Finally, we show that osi-related phenotypes are rescued through the expression of its human ortholog ETFRF1/LYRm5, known to modulate the entry of ß-oxidation products into the electron transport chain; moreover, knocking down electron transport flavoproteins EtfQ0 and walrus/ETFA rescues osi-related phenotypes, further supporting this mode of action. CONCLUSIONS: These findings suggest that Osi may act in concert with the ETF complex to coordinate lipid homeostasis in the fat body in response to stage-specific demands, supporting cellular functions that in turn result in an adaptive behavioral response.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Metabolismo dos Lipídeos , Animais , Humanos , Trifosfato de Adenosina/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Corpo Adiposo/metabolismo , Flavoproteínas/metabolismo , Larva , Lipase/genética , Lipase/metabolismo , Metabolismo dos Lipídeos/genética , Lipídeos , Espécies Reativas de Oxigênio/metabolismoRESUMO
We report on the response of asymmetric lipid membranes composed of palmitoyl oleoyl phosphatidylethanolamine and palmitoyl oleoyl phosphatidylglycerol, to interactions with the frog peptides L18W-PGLa and magainin 2 (MG2a), as well as the lactoferricin derivative LF11-215. In particular we determined the peptide-induced lipid flip-flop, as well as membrane partitioning of L18W-PGLa and LF11-215, and vesicle dye-leakage induced by L18W-PGLa. The ability of L18W-PGLa and MG2a to translocate through the membrane appears to correlate with the observed lipid flip-flop, which occurred at the fastest rate for L18W-PGLa. The higher structural flexibility of LF11-215 in turn allows this peptide to insert into the bilayers without detectable changes of membrane asymmetry. The increased vulnerability of asymmetric membranes to L18W-PGLa in terms of permeability, appears to be a consequence of tension differences between the compositionally distinct leaflets, but not due to increased peptide partitioning.
Assuntos
Peptídeos Antimicrobianos , Bicamadas Lipídicas , Membrana Celular , MagaininasRESUMO
We achieve automated and reliable annotation of lipid species and their molecular structures in high-throughput data from chromatography-coupled tandem mass spectrometry using decision rule sets embedded in Lipid Data Analyzer (LDA; http://genome.tugraz.at/lda2). Using various low- and high-resolution mass spectrometry instruments with several collision energies, we proved the method's platform independence. We propose that the software's reliability, flexibility, and ability to identify novel lipid molecular species may now render current state-of-the-art lipid libraries obsolete.
Assuntos
Cromatografia Líquida/métodos , Lipídeos/análise , Lipídeos/química , Espectrometria de Massas em Tandem/métodos , Algoritmos , Animais , Fígado/química , Camundongos , Estrutura Molecular , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
The formation of ribosomal subunits is a highly dynamic process that is initiated in the nucleus and involves more than 200 trans-acting factors, some of which accompany the pre-ribosomes into the cytoplasm and have to be recycled into the nucleus. The inhibitor diazaborine prevents cytoplasmic release and recycling of shuttling pre-60S maturation factors by inhibiting the AAA-ATPase Drg1. The failure to recycle these proteins results in their depletion in the nucleolus and halts the pathway at an early maturation step. Here, we made use of the fast onset of inhibition by diazaborine to chase the maturation path in real-time from 27SA2 pre-rRNA containing pre-ribosomes localized in the nucleolus up to nearly mature 60S subunits shortly after their export into the cytoplasm. This allows for the first time to put protein assembly and disassembly reactions as well as pre-rRNA processing into a chronological context unraveling temporal and functional linkages during ribosome maturation.
Assuntos
Nucléolo Celular/metabolismo , Citoplasma/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/metabolismo , Transporte Biológico/efeitos dos fármacos , Compostos de Boro/farmacologia , Fluorescência , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/química , Subunidades Ribossômicas Maiores de Eucariotos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/metabolismo , Imagem com Lapso de Tempo/métodosRESUMO
Sepsis is a major cause of mortality in critically ill patients and associated with cardiac dysfunction, a complication linked to immunological and metabolic aberrations. Cardiac neutrophil infiltration and subsequent release of myeloperoxidase (MPO) leads to the formation of the oxidant hypochlorous acid (HOCl) that is able to chemically modify plasmalogens (ether-phospholipids) abundantly present in the heart. This reaction gives rise to the formation of reactive lipid species including aldehydes and chlorinated fatty acids. During the present study, we tested whether endotoxemia increases MPO-dependent lipid oxidation/modification in the mouse heart. In hearts of lipopolysaccharide-injected mice, we observed significantly higher infiltration of MPO-positive cells, increased fatty acid content, and formation of 2-chlorohexadecanal (2-ClHDA), an MPO-derived plasmalogen modification product. Using murine HL-1 cardiomyocytes as in vitro model, we show that exogenously added HOCl attacks the cellular plasmalogen pool and gives rise to the formation of 2-ClHDA. Addition of 2-ClHDA to HL-1 cardiomyocytes resulted in conversion to 2-chlorohexadecanoic acid and 2-chlorohexadecanol, indicating fatty aldehyde dehydrogenase-mediated redox metabolism. However, a recovery of only 40% indicated the formation of non-extractable (protein) adducts. To identify protein targets, we used a clickable alkynyl analog, 2-chlorohexadec-15-yn-1-al (2-ClHDyA). After Huisgen 1,3-dipolar cycloaddition of 5-tetramethylrhodamine azide (N3-TAMRA) and two dimensional-gel electrophoresis (2D-GE), we were able to identify 51 proteins that form adducts with 2-ClHDyA. Gene ontology enrichment analyses revealed an overrepresentation of heat shock and chaperone, energy metabolism, and cytoskeletal proteins as major targets. Our observations in a murine endotoxemia model demonstrate formation of HOCl-modified lipids in the heart, while pathway analysis in vitro revealed that the chlorinated aldehyde targets specific protein subsets, which are central to cardiac function.
Assuntos
Aldeídos/metabolismo , Endotoxemia/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Peroxidase/metabolismo , Animais , Biomarcadores , Química Click , Endotoxemia/etiologia , Ácidos Graxos/metabolismo , Ácido Hipocloroso/metabolismo , Lipopolissacarídeos/administração & dosagem , Camundongos , Proteoma , Proteômica/métodos , Espécies Reativas de Oxigênio/metabolismoRESUMO
During inflammation, activated leukocytes release cytotoxic mediators that compromise blood-brain barrier (BBB) function. Under inflammatory conditions, myeloperoxidase (MPO) is critically involved in inflicting BBB damage. We used genetic and pharmacological approaches to investigate whether MPO induces aberrant lipid homeostasis at the BBB in a murine endotoxemia model. To corroborate findings in a human system we studied the impact of sera from sepsis and non-sepsis patients on brain endothelial cells (hCMEC/D3). In response to endotoxin, the fatty acid, ceramide, and sphingomyelin content of isolated mouse brain capillaries dropped and barrier dysfunction occurred. In mice, genetic deficiency or pharmacological inhibition of MPO abolished these alterations. Studies in metabolic cages revealed increased physical activity and less pronounced sickness behavior of MPO-/- compared to wild-type mice in response to sepsis. In hCMEC/D3 cells, exogenous tumor necrosis factor α (TNFα) potently regulated gene expression of pro-inflammatory cytokines and a set of genes involved in sphingolipid (SL) homeostasis. Notably, treatment of hCMEC/D3 cells with sera from septic patients reduced cellular ceramide concentrations and induced barrier and mitochondrial dysfunction. In summary, our in vivo and in vitro data revealed that inflammatory mediators including MPO, TNFα induce dysfunctional SL homeostasis in brain endothelial cells. Genetic and pharmacological inhibition of MPO attenuated endotoxin-induced alterations in SL homeostasis in vivo, highlighting the potential role of MPO as drug target to treat inflammation-induced brain dysfunction.
Assuntos
Encéfalo/irrigação sanguínea , Células Endoteliais/metabolismo , Peroxidase/metabolismo , Sepse/metabolismo , Esfingolipídeos/metabolismo , Animais , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Capilares/metabolismo , Capilares/patologia , Linhagem Celular , Células Cultivadas , Células Endoteliais/citologia , Células Endoteliais/patologia , Homeostase , Humanos , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Sepse/patologiaRESUMO
S-Adenosyl-l-homocysteine hydrolase (AdoHcy hydrolase; Sah1 in yeast/AHCY in mammals) degrades AdoHcy, a by-product and strong product inhibitor of S-adenosyl-l-methionine (AdoMet)-dependent methylation reactions, to adenosine and homocysteine (Hcy). This reaction is reversible, so any elevation of Hcy levels, such as in hyperhomocysteinemia (HHcy), drives the formation of AdoHcy, with detrimental consequences for cellular methylation reactions. HHcy, a pathological condition linked to cardiovascular and neurological disorders, as well as fatty liver among others, is associated with a deregulation of lipid metabolism. Here, we developed a yeast model of HHcy to identify mechanisms that dysregulate lipid metabolism. Hcy supplementation to wildtype cells up-regulated cellular fatty acid and triacylglycerol content and induced a shift in fatty acid composition, similar to changes observed in mutants lacking Sah1. Expression of the irreversible bacterial pathway for AdoHcy degradation in yeast allowed us to dissect the impact of AdoHcy accumulation on lipid metabolism from the impact of elevated Hcy. Expression of this pathway fully suppressed the growth deficit of sah1 mutants as well as the deregulation of lipid metabolism in both the sah1 mutant and Hcy-exposed wildtype, showing that AdoHcy accumulation mediates the deregulation of lipid metabolism in response to elevated Hcy in yeast. Furthermore, Hcy supplementation in yeast led to increased resistance to cerulenin, an inhibitor of fatty acid synthase, as well as to a concomitant decline of condensing enzymes involved in very long-chain fatty acid synthesis, in line with the observed shift in fatty acid content and composition.
Assuntos
Adenosil-Homocisteinase/metabolismo , Ácidos Graxos/metabolismo , Metabolismo dos Lipídeos , S-Adenosil-Homocisteína/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosil-Homocisteinase/genética , Ácidos Graxos/genética , Hiper-Homocisteinemia/genética , Hiper-Homocisteinemia/metabolismo , Modelos Biológicos , Mutação , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
We measured the effect of intrinsic lipid curvature, J0, on structural properties of asymmetric vesicles made of palmitoyl-oleoyl-phosphatidylethanolamine (POPE; J0<0) and palmitoyl-oleoyl-phosphatidylcholine (POPC; J0â¼0). Electron microscopy and dynamic light scattering were used to determine vesicle size and morphology, and x-ray and neutron scattering, combined with calorimetric experiments and solution NMR, yielded insights into leaflet-specific lipid packing and melting processes. Below the lipid melting temperature we observed strong interleaflet coupling in asymmetric vesicles with POPE inner bilayer leaflets and outer leaflets enriched in POPC. This lipid arrangement manifested itself by lipids melting cooperatively in both leaflets, and a rearrangement of lipid packing in both monolayers. On the other hand, no coupling was observed in vesicles with POPC inner bilayer leaflets and outer leaflets enriched in POPE. In this case, the leaflets melted independently and did not affect each other's acyl chain packing. Furthermore, we found no evidence for transbilayer structural coupling above the melting temperature of either sample preparation. Our results are consistent with the energetically preferred location of POPE residing in the inner leaflet, where it also resides in natural membranes, most likely causing the coupling of both leaflets. The loss of this coupling in the fluid bilayers is most likely the result of entropic contributions.
Assuntos
Bicamadas Lipídicas/química , Fenômenos Mecânicos , Fosfatidilcolinas/química , Fosfatidiletanolaminas/químicaRESUMO
Adipose triglyceride lipase (ATGL) initiates intracellular triglyceride (TG) catabolism. In humans, ATGL deficiency causes neutral lipid storage disease with myopathy (NLSDM) characterized by a systemic TG accumulation. Mice with a genetic deletion of ATGL (AKO) also accumulate TG in many tissues. However, neither NLSDM patients nor AKO mice are exceedingly obese. This phenotype is unexpected considering the importance of the enzyme for TG catabolism in white adipose tissue (WAT). In this study, we identified the counteracting mechanisms that prevent excessive obesity in the absence of ATGL. We used "healthy" AKO mice expressing ATGL exclusively in cardiomyocytes (AKO/cTg) to circumvent the cardiomyopathy and premature lethality observed in AKO mice. AKO/cTg mice were protected from high-fat diet (HFD)-induced obesity despite complete ATGL deficiency in WAT and normal adipocyte differentiation. AKO/cTg mice were highly insulin sensitive under hyperinsulinemic-euglycemic clamp conditions, eliminating insulin insensitivity as a possible protective mechanism. Instead, reduced food intake and altered signaling by peroxisome proliferator-activated receptor-gamma (PPAR-γ) and sterol regulatory element binding protein-1c in WAT accounted for the phenotype. These adaptations led to reduced lipid synthesis and storage in WAT of HFD-fed AKO/cTg mice. Treatment with the PPAR-γ agonist rosiglitazone reversed the phenotype. These results argue for the existence of an adaptive interdependence between lipolysis and lipid synthesis. Pharmacological inhibition of ATGL may prove useful to prevent HFD-induced obesity and insulin resistance.
Assuntos
Adaptação Fisiológica , Dieta Hiperlipídica , Comportamento Alimentar , Lipase/fisiologia , Lipólise , Obesidade/prevenção & controle , Animais , Lipase/genética , Camundongos , Camundongos Knockout , Obesidade/metabolismo , PPAR gama/genética , PPAR gama/metabolismo , FenótipoRESUMO
The importance of peroxisomes for adipocyte function is poorly understood. Herein, we provide insights into the critical role of peroxin 16 (PEX16)-mediated peroxisome biogenesis in adipocyte development and lipid metabolism. Pex16 is highly expressed in adipose tissues and upregulated during adipogenesis of murine and human cells. We demonstrate that Pex16 is a target gene of the adipogenesis "master-regulator" PPARγ. Stable silencing of Pex16 in 3T3-L1 cells strongly reduced the number of peroxisomes while mitochondrial number was unaffected. Concomitantly, peroxisomal fatty acid (FA) oxidation was reduced, thereby causing accumulation of long- and very long-chain (polyunsaturated) FAs and reduction of odd-chain FAs. Further, Pex16-silencing decreased cellular oxygen consumption and increased FA release. Additionally, silencing of Pex16 impaired adipocyte differentiation, lipogenic and adipogenic marker gene expression, and cellular triglyceride stores. Addition of PPARγ agonist rosiglitazone and peroxisome-related lipid species to Pex16-silenced 3T3-L1 cells rescued adipogenesis. These data provide evidence that PEX16 is required for peroxisome biogenesis and highlights the relevance of peroxisomes for adipogenesis and adipocyte lipid metabolism.
Assuntos
Adipócitos Brancos/metabolismo , Homeostase/fisiologia , Metabolismo dos Lipídeos/fisiologia , Lipídeos/fisiologia , Proteínas de Membrana/metabolismo , Peroxissomos/metabolismo , Células 3T3-L1 , Adipogenia/fisiologia , Animais , Células COS , Diferenciação Celular/fisiologia , Linhagem Celular , Chlorocebus aethiops , Ácidos Graxos/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Consumo de Oxigênio/fisiologia , PPAR gama/metabolismo , Regulação para Cima/fisiologiaRESUMO
Conjugative transfer plays a major role in the transmission of antibiotic resistance in bacteria. pIP501 is a Gram-positive conjugative model plasmid with the broadest transfer host-range known so far and is frequently found in Enterococcus faecalis and Enterococcus faecium clinical isolates. The pIP501 type IV secretion system is encoded by 15 transfer genes. In this work, we focus on the VirB1-like protein TraG, a modular peptidoglycan metabolizing enzyme, and the VirB8-homolog TraM, a potential member of the translocation channel. By providing full-length traG in trans, but not with a truncated variant, we achieved full recovery of wild type transfer efficiency in the traG-knockout mutant E. faecalis pIP501ΔtraG. With peptidoglycan digestion experiments and tandem mass spectrometry we could assign lytic transglycosylase and endopeptidase activity to TraG, with the CHAP domain alone displaying endopeptidase activity. We identified a novel interaction between TraG and TraM in a bacterial-2-hybrid assay. In addition we found that both proteins localize in focal spots at the E. faecalis cell membrane using immunostaining and fluorescence microscopy. Extracellular protease digestion to evaluate protein cell surface exposure revealed that correct membrane localization of TraM requires the transmembrane helix of TraG. Thus, we suggest an essential role for TraG in the assembly of the pIP501 type IV secretion system.
Assuntos
Proteínas de Bactérias/genética , Sequência de Bases , Proteínas de Transporte/genética , Enterococcus faecalis/genética , Regulação Bacteriana da Expressão Gênica , Plasmídeos/química , Deleção de Sequência , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Transporte Biológico , Proteínas de Transporte/metabolismo , Parede Celular/metabolismo , Parede Celular/ultraestrutura , Conjugação Genética , Endopeptidases/genética , Endopeptidases/metabolismo , Enterococcus faecalis/metabolismo , Enterococcus faecalis/ultraestrutura , Peptidoglicano Glicosiltransferase/genética , Peptidoglicano Glicosiltransferase/metabolismo , Plasmídeos/metabolismo , Ligação Proteica , Domínios Proteicos , Sistemas de Secreção Tipo IV/metabolismoRESUMO
Triacylglycerol (TAG) and glycogen are the two major metabolites for carbon storage in most eukaryotic organisms. We investigated the glycogen metabolism of the oleaginous Yarrowia lipolytica and found that this yeast accumulates up to 16% glycogen in its biomass. Assuming that elimination of glycogen synthesis would result in an improvement of lipid accumulation, we characterized and deleted the single gene coding for glycogen synthase, YlGSY1. The mutant was grown under lipogenic conditions with glucose and glycerol as substrates and we obtained up to 60% improvement in TAG accumulation compared to the wild-type strain. Additionally, YlGSY1 was deleted in a background that was already engineered for high lipid accumulation. In this obese background, TAG accumulation was also further increased. The highest lipid content of 52% was found after 3 days of cultivation in nitrogen-limited glycerol medium. Furthermore, we constructed mutants of Y. lipolytica and Saccharomyces cerevisiae that are deleted for both glycogen and TAG synthesis, demonstrating that the ability to store carbon is not essential. Overall, this work showed that glycogen synthesis is a competing pathway for TAG accumulation in oleaginous yeasts and that deletion of the glycogen synthase has beneficial effects on neutral lipid storage.
Assuntos
Proteínas Fúngicas/genética , Glicogênio Sintase/genética , Glicogênio/biossíntese , Engenharia Metabólica/métodos , Triglicerídeos/biossíntese , Yarrowia/metabolismo , Biomassa , Carbono/metabolismo , Fermentação , Proteínas Fúngicas/metabolismo , Deleção de Genes , Expressão Gênica , Glucose/metabolismo , Glicerol/metabolismo , Glicogênio/antagonistas & inibidores , Glicogênio Sintase/deficiência , Cinética , Metabolismo dos Lipídeos , Nitrogênio/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Yarrowia/genéticaRESUMO
The coordinated breakdown of intracellular triglyceride (TG) stores requires the exquisitely regulated interaction of lipolytic enzymes with regulatory, accessory, and scaffolding proteins. Together they form a dynamic multiprotein network designated as the "lipolysome." Adipose triglyceride lipase (Atgl) catalyzes the initiating step of TG hydrolysis and requires comparative gene identification-58 (Cgi-58) as a potent activator of enzyme activity. Here, we identify adipocyte-type fatty acid-binding protein (A-Fabp) and other members of the fatty acid-binding protein (Fabp) family as interaction partners of Cgi-58. Co-immunoprecipitation, microscale thermophoresis, and solid phase assays proved direct protein/protein interaction between A-Fabp and Cgi-58. Using nuclear magnetic resonance titration experiments and site-directed mutagenesis, we located a potential contact region on A-Fabp. In functional terms, A-Fabp stimulates Atgl-catalyzed TG hydrolysis in a Cgi-58-dependent manner. Additionally, transcriptional transactivation assays with a luciferase reporter system revealed that Fabps enhance the ability of Atgl/Cgi-58-mediated lipolysis to induce the activity of peroxisome proliferator-activated receptors. Our studies identify Fabps as crucial structural and functional components of the lipolysome.
Assuntos
1-Acilglicerol-3-Fosfato O-Aciltransferase/metabolismo , Proteínas de Ligação a Ácido Graxo/metabolismo , Lipase/metabolismo , Complexos Multiproteicos/metabolismo , Triglicerídeos/metabolismo , 1-Acilglicerol-3-Fosfato O-Aciltransferase/genética , Tecido Adiposo/metabolismo , Animais , Células COS , Chlorocebus aethiops , Proteínas de Ligação a Ácido Graxo/genética , Humanos , Ligantes , Lipase/genética , Lipólise/genética , Lipossomos/metabolismo , Camundongos , Complexos Multiproteicos/genética , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , ProteóliseRESUMO
Yeast Fld1 and Ldb16 resemble mammalian seipin, implicated in neutral lipid storage. Both proteins form a complex at the endoplasmic reticulum-lipid droplet (LD) interface. Malfunction of this complex either leads to LD clustering or to the generation of supersized LD (SLD) in close vicinity to the nuclear envelope, in response to altered phospholipid (PL) composition. We show that similar to mutants lacking Fld1, deletion of LDB16 leads to abnormal proliferation of a subdomain of the nuclear envelope, which is tightly associated with clustered LD. The human lipin-1 ortholog, the PAH1 encoded phosphatidic acid (PA) phosphatase, and its activator Nem1 are highly enriched at this site. The specific accumulation of PA-binding marker proteins indicates a local enrichment of PA in the fld1 and ldb16 mutants. Furthermore, we demonstrate that clustered LD in fld1 or ldb16 mutants are transformed to SLD if phosphatidylcholine synthesis is compromised by additional deletion of the phosphatidylethanolamine methyltransferase, Cho2. Notably, treatment of wild-type cells with oleate induced a similar LD clustering and nuclear membrane proliferation phenotype as observed in fld1 and ldb16 mutants. These data suggest that the Fld1-Ldb16 complex affects PA homeostasis at an LD-forming subdomain of the nuclear envelope. Lack of Fld1-Ldb16 leads to locally elevated PA levels that induce an abnormal proliferation of nER membrane structures and the clustering of associated LD. We suggest that the formation of SLD is a consequence of locally altered PL metabolism at this site.
Assuntos
Subunidades gama da Proteína de Ligação ao GTP/genética , Regulação Fúngica da Expressão Gênica , Proteínas Mitocondriais/genética , Membrana Nuclear/metabolismo , Ácidos Fosfatídicos/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Subunidades gama da Proteína de Ligação ao GTP/deficiência , Gotículas Lipídicas/efeitos dos fármacos , Gotículas Lipídicas/metabolismo , Gotículas Lipídicas/ultraestrutura , Metabolismo dos Lipídeos/efeitos dos fármacos , Proteínas Mitocondriais/deficiência , Mutação , Membrana Nuclear/efeitos dos fármacos , Membrana Nuclear/genética , Membrana Nuclear/ultraestrutura , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ácido Oleico/farmacologia , Fosfatidato Fosfatase/genética , Fosfatidato Fosfatase/metabolismo , Fosfatidilcolinas/metabolismo , Fosfatidiletanolamina N-Metiltransferase/genética , Fosfatidiletanolamina N-Metiltransferase/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de SinaisRESUMO
Scavenging of nitric oxide (NO) often interferes with studies on NO signaling in cell-free preparations. We observed that formation of cGMP by NO-stimulated purified soluble guanylate cyclase (sGC) was virtually abolished in the presence of cytosolic preparations of porcine coronary arteries, with the scavenging activity localized in the tunica media (smooth muscle layer). Electrochemical measurement of NO release from a donor compound and light absorbance spectroscopy showed that cytosolic preparations contained a reduced heme protein that scavenged NO. This protein, which reacted with anti-human hemoglobin antibodies, was efficiently removed from the preparations by haptoglobin affinity chromatography. The cleared cytosols showed only minor scavenging of NO according to electrochemical measurements and did not decrease cGMP formation by NO-stimulated sGC. In contrast, the column flow-through caused a nearly 2-fold increase of maximal sGC activity (from 33.1 ± 1.6 to 54.9 ± 2.2 µmol × min(-1) × mg(-1)). The proteins retained on the affinity column were identified as hemoglobin α and ß subunits. The results indicate that hemoglobin, presumably derived from vasa vasorum erythrocytes, is present and scavenges NO in preparations of porcine coronary artery smooth muscle. Selective removal of hemoglobin-mediated scavenging unmasked stimulation of maximal NO-stimulated sGC activity by a soluble factor expressed in vascular tissue.
Assuntos
Vasos Coronários/metabolismo , Hemoglobinas/metabolismo , Óxido Nítrico/metabolismo , Túnica Média/metabolismo , Animais , Bovinos , GMP Cíclico/metabolismo , Citoglobina , Globinas/metabolismo , Haptoglobinas/metabolismo , Humanos , Técnicas In Vitro , Guanilil Ciclase Solúvel/metabolismo , SuínosRESUMO
Lipid droplets (LDs) of hepatic stellate cells (HSCs) contain large amounts of vitamin A [in the form of retinyl esters (REs)] as well as other neutral lipids such as TGs. During times of insufficient vitamin A availability, RE stores are mobilized to ensure a constant supply to the body. To date, little is known about the enzymes responsible for the hydrolysis of neutral lipid esters, in particular of REs, in HSCs. In this study, we aimed to identify LD-associated neutral lipid hydrolases by a proteomic approach using the rat stellate cell line HSC-T6. First, we loaded cells with retinol and FAs to promote lipid synthesis and deposition within LDs. Then, LDs were isolated and lipid composition and the LD proteome were analyzed. Among other proteins, we found perilipin 2, adipose TG lipase (ATGL), and comparative gene identification-58 (CGI-58), known and established LD proteins. Bioinformatic search of the LD proteome for α/ß-hydrolase fold-containing proteins revealed no yet uncharacterized neutral lipid hydrolases. In in vitro activity assays, we show that rat (r)ATGL, coactivated by rat (r)CGI-58, efficiently hydrolyzes TGs and REs. These findings suggest that rATGL and rCGI-58 are LD-resident proteins in HSCs and participate in the mobilization of both REs and TGs.
Assuntos
Aciltransferases/metabolismo , Células Estreladas do Fígado/metabolismo , Lipase/metabolismo , Gotículas Lipídicas/metabolismo , Adipócitos/metabolismo , Animais , Linhagem Celular , Lipólise , Lipoproteínas/metabolismo , Proteínas de Membrana/metabolismo , Perilipina-2 , Proteoma/metabolismo , Ratos , Proteínas de Ligação ao Retinol/metabolismo , Ésteres de Retinil , Triglicerídeos/metabolismo , Vitamina A/farmacologiaRESUMO
Mitochondrial outer membrane permeabilization is a watershed event in the process of apoptosis, which is tightly regulated by a series of pro- and anti-apoptotic proteins belonging to the BCL-2 family, each characteristically possessing a BCL-2 homology domain 3 (BH3). Here, we identify a yeast protein (Ybh3p) that interacts with BCL-X(L) and harbours a functional BH3 domain. Upon lethal insult, Ybh3p translocates to mitochondria and triggers BH3 domain-dependent apoptosis. Ybh3p induces cell death and disruption of the mitochondrial transmembrane potential via the mitochondrial phosphate carrier Mir1p. Deletion of Mir1p and depletion of its human orthologue (SLC25A3/PHC) abolish stress-induced mitochondrial targeting of Ybh3p in yeast and that of BAX in human cells, respectively. Yeast cells lacking YBH3 display prolonged chronological and replicative lifespans and resistance to apoptosis induction. Thus, the yeast genome encodes a functional BH3 domain that induces cell death through phylogenetically conserved mechanisms.