RESUMO
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is involved in the degradation of the low-density lipoprotein receptor. PCSK9 also targets proteins involved in lipid metabolism (very low-density lipoprotein receptor), immunity (major histocompatibility complex I), and viral infection (cluster of differentiation 81). Recent studies have also indicated that PCSK9 loss-of-function mutations are associated with an increased incidence of diabetes; however, the expression and function of PCSK9 in insulin-producing pancreatic beta cells remain unclear. Here, we studied PCSK9 regulation and function by performing loss- and gain-of-function experiments in the human beta cell line EndoC-ßH1. We demonstrate that PCSK9 is expressed and secreted by EndoC-ßH1 cells. We also found that PCSK9 expression is regulated by cholesterol and sterol regulatory element-binding protein transcription factors, as previously demonstrated in other cell types such as hepatocytes. Importantly, we show that PCSK9 knockdown using siRNA results in deregulation of various elements of the transcriptome, proteome, and secretome, and increases insulin secretion. We also observed that PCSK9 decreases low-density lipoprotein receptor and very low-density lipoprotein receptor levels via an extracellular signaling mechanism involving exogenous PCSK9, as well as levels of cluster of differentiation 36, a fatty acid transporter, through an intracellular signaling mechanism. Finally, we found that PCSK9 regulates the cell surface expression of PDL1 and HLA-ABC, proteins involved in cell-lymphocyte interaction, also via an intracellular mechanism. Collectively, these results highlight PCSK9 as a regulator of multiple cell surface receptors in pancreatic beta cells.
Assuntos
Células Secretoras de Insulina , Proteínas de Membrana , Pró-Proteína Convertase 9 , Antígenos CD36/metabolismo , Linhagem Celular , Mutação com Ganho de Função , Humanos , Células Secretoras de Insulina/metabolismo , Lipoproteínas VLDL/metabolismo , Mutação com Perda de Função , Proteínas de Membrana/metabolismo , Pró-Proteína Convertase 9/metabolismo , Receptores de LDL/metabolismoRESUMO
While low concentrations of high-density lipoprotein-cholesterol (HDL-C) are widely accepted as an independent cardiovascular risk factor, HDL-C-rising therapies largely failed, suggesting the importance of both HDL functions and individual subspecies. Indeed HDL particles are highly heterogeneous, with small, dense pre-beta-HDLs being considered highly biologically active but remaining poorly studied, largely reflecting difficulties for their purification. We developed an original experimental approach allowing the isolation of sufficient amounts of human pre-beta-HDLs and revealing the specificity of their proteomic and lipidomic profiles and biological activities. Pre-beta-HDLs were enriched in highly poly-unsaturated species of phosphatidic acid and phosphatidylserine, and in an unexpectedly high number of proteins implicated in the inflammatory response, including serum paraoxonase/arylesterase-1, vitronectin and clusterin, as well as in complement regulation and immunity, including haptoglobin-related protein, complement proteins and those of the immunoglobulin class. Interestingly, amongst proteins associated with lipid metabolism, phospholipid transfer protein, cholesteryl ester transfer protein and lecithin:cholesterol acyltransferase were strongly enriched in, or restricted to, pre-beta-HDL. Furthermore, pre-beta-HDL potently mediated cellular cholesterol efflux and displayed strong anti-inflammatory activities. A correlational network analysis between lipidome, proteome and biological activities highlighted 15 individual lipid and protein components of pre-beta-HDL relevant to cardiovascular disease, which may constitute novel diagnostic targets in a pathological context of altered lipoprotein metabolism.
Assuntos
Doenças Cardiovasculares , Humanos , Proteômica , HDL-Colesterol , Fatores de Risco de Doenças Cardíacas , Metabolismo dos LipídeosRESUMO
Phosphatidylserine (PS) is a minor phospholipid constituent of high-density lipoprotein (HDL) that exhibits potent anti-inflammatory activity. It remains indeterminate whether PS incorporation can enhance anti-inflammatory effects of reconstituted HDL (rHDL). Human macrophages were treated with rHDL containing phosphatidylcholine alone (PC-rHDL) or PC and PS (PC/PS-rHDL). Interleukin (IL)-6 secretion and expression was more strongly inhibited by PC/PS-rHDL than PC-rHDL in both tumor necrosis factor (TNF)-α- and lipopolysaccharide (LPS)-stimulated macrophages. siRNA experiments revealed that the enhanced anti-inflammatory effects of PC/PS-rHDL required scavenger receptor class B type I (SR-BI). Furthermore, PC/PS-rHDL induced a greater increase in Akt1/2/3 phosphorylation than PC-rHDL. In addition, PC/PS but not PC-rHDL decreased the abundance of plasma membrane lipid rafts and p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation. Finally, when these rHDL formulations were administered to dyslipidemic low-density lipoprotein (LDL)-receptor knockout mice fed a high-cholesterol diet, circulating IL-6 levels were significantly reduced only in PC/PS-rHDL-treated mice. In parallel, enhanced Akt1/2/3 phosphorylation by PC/PS-rHDL was observed in the mouse aortic tissue using immunohistochemistry. We concluded that the incorporation of PS into rHDLs enhanced their anti-inflammatory activity by modulating Akt1/2/3- and p38 MAPK-mediated signaling through SR-BI in stimulated macrophages. These data identify PS as a potent anti-inflammatory component capable of enhancing therapeutic potential of rHDL-based therapy.
Assuntos
Lipoproteínas HDL , Fosfatidilserinas , Animais , Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/farmacologia , Espaço Intracelular/metabolismo , Lipoproteínas HDL/metabolismo , Macrófagos/metabolismo , Camundongos , Fosfatidilserinas/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
AIMS: High-density lipoprotein (HDL) contains multiple components that endow it with biological activities. Apolipoprotein A-I (apoA-I) and surface phospholipids contribute to these activities; however, structure-function relationships in HDL particles remain incompletely characterised. METHODS: Reconstituted HDLs (rHDLs) were prepared from apoA-I and soy phosphatidylcholine (PC) at molar ratios of 1:50, 1:100 and 1:150. Oxidative status of apoA-I was varied using controlled oxidation of Met112 residue. HDL-mediated inactivation of PC hydroperoxides (PCOOH) derived from mildly pre-oxidized low-density lipoprotein (LDL) was evaluated by HPLC with chemiluminescent detection in HDL+LDL mixtures and re-isolated LDL. Cellular cholesterol efflux was characterised in RAW264.7 macrophages. RESULTS: rHDL inactivated LDL-derived PCOOH in a dose- and time-dependent manner. The capacity of rHDL to both inactivate PCOOH and efflux cholesterol via ATP-binding cassette transporter A1 (ABCA1) increased with increasing apoA-I/PC ratio proportionally to the apoA-I content in rHDL. Controlled oxidation of apoA-I Met112 gradually decreased PCOOH-inactivating capacity of rHDL but increased ABCA1-mediated cellular cholesterol efflux. CONCLUSIONS: Increasing apoA-I content in rHDL enhanced its antioxidative activity towards oxidized LDL and cholesterol efflux capacity via ABCA1, whereas oxidation of apoA-I Met112 decreased the antioxidative activity but increased the cholesterol efflux. These findings provide important considerations in the design of future HDL therapeutics. Non-standard abbreviations and acronyms: AAPH, 2,2'-azobis(-amidinopropane) dihydrochloride; ABCA1, ATP-binding cassette transporter A1; apoA-I, apolipoprotein A-I; BHT, butylated hydroxytoluene; CV, cardiovascular; EDTA, ethylenediaminetetraacetic acid; HDL-C, high-density lipoprotein cholesterol; LOOH, lipid hydroperoxides; Met(O), methionine sulfoxide; Met112, methionine 112 residue; Met86, methionine 86 residue; oxLDL, oxidized low-density lipoprotein; PBS, phosphate-buffered saline; PC, phosphatidylcholine; PL, phospholipid; PCOOH, phosphatidylcholine hydroperoxide; PLOOH, phospholipid hydroperoxide.
Assuntos
Antioxidantes/metabolismo , Colesterol/metabolismo , Lipoproteínas HDL/metabolismo , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Animais , Apolipoproteína A-I/metabolismo , Transporte Biológico/fisiologia , Linhagem Celular , Humanos , Lipoproteínas LDL/metabolismo , Macrófagos/metabolismo , Macrófagos/fisiologia , Camundongos , Oxirredução , Fosfatidilcolinas/metabolismo , Células RAW 264.7RESUMO
Erwinia amylovora, the bacterium responsible for fire blight, relies on a type III secretion system and a single injected effector, DspA/E, to induce disease in host plants. DspA/E belongs to the widespread AvrE family of type III effectors that suppress plant defense responses and promote bacterial growth following infection. Ectopic expression of DspA/E in plant or in Saccharomyces cerevisiae is toxic, indicating that DspA/E likely targets a cellular process conserved between yeast and plant. To unravel the mode of action of DspA/E, we screened the Euroscarf S. cerevisiae library for mutants resistant to DspA/E-induced growth arrest. The most resistant mutants (Δsur4, Δfen1, Δipt1, Δskn1, Δcsg1, Δcsg2, Δorm1, and Δorm2) were impaired in the sphingolipid biosynthetic pathway. Exogenously supplied sphingolipid precursors such as the long chain bases (LCBs) phytosphingosine and dihydrosphingosine also suppressed the DspA/E-induced yeast growth defect. Expression of DspA/E in yeast down-regulated LCB biosynthesis and induced a rapid decrease in LCB levels, indicating that serine palmitoyltransferase (SPT), the first and rate-limiting enzyme of the sphingolipid biosynthetic pathway, was repressed. SPT down-regulation was mediated by dephosphorylation and activation of Orm proteins that negatively regulate SPT. A Δcdc55 mutation affecting Cdc55-PP2A protein phosphatase activity prevented Orm dephosphorylation and suppressed DspA/E-induced growth arrest.
Assuntos
Proteínas de Bactérias/genética , Regulação para Baixo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Serina C-Palmitoiltransferase/genética , Esfingolipídeos/biossíntese , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/toxicidade , Vias Biossintéticas , Expressão Gênica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Serina C-Palmitoiltransferase/metabolismo , Esfingolipídeos/químicaRESUMO
Phosphatidylinositol phosphates are important regulators of processes such as the cytoskeleton organization, membrane trafficking and gene transcription, which are all crucial for polarized cell growth. In particular, phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] has essential roles in polarized growth as well as in cellular responses to stress. In the yeast Saccharomyces cerevisiae, the sole phosphatidylinositol-4-phosphate 5-kinase (PI4P5K) Mss4p is essential for generating plasma membrane PtdIns(4,5)P2. Here, we show that Mss4p is required for yeast invasive growth in low-nutrient conditions. We isolated specific mss4 mutants that were defective in cell elongation, induction of the Flo11p flocculin, adhesion and cell wall integrity. We show that mss4-f12 cells have reduced plasma membrane PtdIns(4,5)P2 levels as well as a defect in its polarized distribution, yet Mss4-f12p is catalytically active in vitro. In addition, the Mss4-f12 protein was defective in localizing to the plasma membrane. Furthermore, addition of cAMP, but not an activated MAPKKK allele, partially restored the invasive growth defect of mss4-f12 cells. Taken together, our results indicate that plasma membrane PtdIns(4,5)P2 is crucial for yeast invasive growth and suggest that this phospholipid functions upstream of the cAMP-dependent protein kinase A signaling pathway.
Assuntos
Fosfatos de Fosfatidilinositol/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Adesão Celular/fisiologia , Polaridade Celular/fisiologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Fosfolipídeos/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de SinaisRESUMO
Although sphingolipids emerged as important signals for plant response to low temperature, investigations have been limited so far to the function of long-chain base intermediates. The formation and function of ceramide phosphates (Cer-Ps) in chilled Arabidopsis were explored. Cer-Ps were analysed by thin layer chromatography (TLC) following in vivo metabolic radiolabelling. Ceramide kinase activity, gene expression and growth phenotype were determined in unstressed and cold-stressed wild type (WT) and Arabidopsis ceramide kinase mutant acd5. A rapid and transient formation of Cer-P occurs in cold-stressed WT Arabidopsis plantlets and cultured cells, which is strongly impaired in acd5 mutant. Although concomitant, Cer-P formation is independent of long-chain base phosphate (LCB-P) formation. No variation of ceramide kinase activity was measured in vitro in WT plantlets upon cold stress but the activity in acd5 mutant was further reduced by cold stress. At the seedling stage, acd5 response to cold was similar to that of WT. Nevertheless, acd5 seed germination was hypersensitive to cold and abscisic acid (ABA), and ABA-dependent gene expression was modified in acd5 seeds when germinated at low temperature. Our data involve for the first time Cer-P and ACD5 in low temperature response and further underline the complexity of sphingolipid signalling operating during cold stress.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Regulação da Expressão Gênica de Plantas , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Estresse Fisiológico , Ácido Abscísico/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Ceramidas/metabolismo , Temperatura Baixa , Germinação , Mutação , Fenótipo , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Reguladores de Crescimento de Plantas/metabolismo , Plântula/enzimologia , Plântula/genética , Plântula/crescimento & desenvolvimento , Plântula/fisiologia , Sementes/enzimologia , Sementes/genética , Sementes/crescimento & desenvolvimento , Sementes/fisiologia , Transdução de SinaisRESUMO
BACKGROUND: The capacity of high-density lipoprotein cholesterol (HDL) to acquire free cholesterol (FC) from triglyceride-rich lipoproteins during lipoprotein lipase-dependent lipolysis in a process of reverse remnant cholesterol transport, has been proposed as a key biological function of HDL particles that underlies the U-shaped relationship between HDLcholesterol and cardiovascular diseases. Although reverse remnant cholesterol transport has been evaluated in a fasting state, it has never been explored under nonfasting conditions. METHODS AND RESULTS: FC transfer was evaluated in healthy men (n=78) before and throughout the postprandial phase up to 8 hours after consumption of a test meal. Postprandially, the capacity of HDL to acquire FC increased progressively, reaching a maximal mean value of 98.5%±22.5% 6 hours after meal intake (P<0.05). Analysis of the study population according to tertiles of postprandial variation of FC transfer identified subjects exhibiting reduced capacity of HDL to acquire FC (tertile 1), those for whom the capacity of HDL to acquire FC remained unchanged (tertile 2), and subjects characterized by an enhanced FC transfer during the postprandial phase (tertile 3). Across the tertiles, we found an inverse relationship between the maximal postprandial change in FC transfer to HDL and the degree of postprandial triglyceride response. CONCLUSIONS: Healthy individuals exhibiting exacerbated postprandial triglyceride response and reduced HDL cholesterol levels feature reduced FC transfer to HDL during the postprandial state. These data suggest that to normalize postprandial triglyceride response, 2 conditions need to be fulfilled: notably elevated FC transfer to HDL in the postprandial phase and increased levels of acceptor HDL particles.
Assuntos
Hipertrigliceridemia , Período Pós-Prandial , Triglicerídeos , Humanos , Masculino , Período Pós-Prandial/fisiologia , Triglicerídeos/sangue , Hipertrigliceridemia/sangue , Adulto , Lipoproteínas/sangue , Voluntários Saudáveis , Pessoa de Meia-Idade , HDL-Colesterol/sangue , Colesterol/sangue , Adulto Jovem , Lipoproteínas HDL/sangue , Biomarcadores/sangue , Fatores de TempoRESUMO
BACKGROUND: The role of proprotein convertase subtilisin/kexin type 9 (PCSK9) in dyslipidemia may go beyond its immediate effects on low-density lipoprotein receptor (LDL-R) activity. OBJECTIVE: This study aimed to assess PCSK9-derived alterations of high-density lipoprotein (HDL) physiology, which bear a potential to contribute to cardiovascular risk profile. METHODS: HDL was isolated from 33 patients with familial autosomal dominant hypercholesterolemia (FH), including those carrying PCSK9 gain-of-function (GOF) genetic variants (FH-PCSK9, n = 11), together with two groups of dyslipidemic patients employed as controls and carrying genetic variants in the LDL-R not treated (ntFH-LDLR, n = 11) and treated (tFH-LDLR, n = 11) with statins, and 11 normolipidemic controls. Biological evaluations paralleled by proteomic, lipidomic and glycomic analyses were applied to characterize functional and compositional properties of HDL. RESULTS: Multiple deficiencies in the HDL function were identified in the FH-PCSK9 group relative to dyslipidemic FH-LDLR patients and normolipidemic controls, which involved reduced antioxidative, antiapoptotic, anti-thrombotic and anti-inflammatory activities. By contrast, cellular cholesterol efflux capacity of HDL was unchanged. In addition, multiple alterations of the proteomic, lipidomic and glycomic composition of HDL were found in the FH-PCSK9 group. Remarkably, HDLs from FH-PCSK9 patients were systematically enriched in several lysophospholipids as well as in A2G2S2 (GP13) glycan and apolipoprotein A-IV. Based on network analysis of functional and compositional data, a novel mosaic structure-function model of HDL biology involving FH was developed. CONCLUSION: Several metrics of anti-atherogenic HDL functionality are altered in FH-PCSK9 patients paralleled by distinct compositional alterations. These data provide a first-ever overview of the impact of GOF PCSK9 genetic variants on structure-function relationships in HDL.
Assuntos
Hiperlipoproteinemia Tipo II , Pró-Proteína Convertase 9 , Humanos , Pró-Proteína Convertase 9/genética , Lipoproteínas HDL/genética , Proteômica , Hiperlipoproteinemia Tipo II/genética , Relação Estrutura-Atividade , Receptores de LDL/genética , MutaçãoRESUMO
Dendritic cells (DCs) mature in an immunogenic or tolerogenic manner depending on the context in which an antigen is perceived, preserving the balance between immunity and tolerance. Whereas the pathways driving immunogenic maturation in response to infectious insults are well-characterized, the signals that drive tolerogenic maturation during homeostasis are still poorly understood. We found that the engulfment of apoptotic cells triggered homeostatic maturation of type 1 conventional DCs (cDC1s) within the spleen. This maturation process could be mimicked by engulfment of empty, nonadjuvanted lipid nanoparticles (LNPs), was marked by intracellular accumulation of cholesterol, and was highly specific to cDC1s. Engulfment of either apoptotic cells or cholesterol-rich LNPs led to the activation of the liver X receptor (LXR) pathway, which promotes the efflux of cellular cholesterol, and repressed genes associated with immunogenic maturation. In contrast, simultaneous engagement of TLR3 to mimic viral infection via administration of poly(I:C)-adjuvanted LNPs repressed the LXR pathway, thus delaying cellular cholesterol efflux and inducing genes that promote T cell-mediated immunity. These data demonstrate that conserved cellular cholesterol efflux pathways are differentially regulated in tolerogenic versus immunogenic cDC1s and suggest that administration of nonadjuvanted cholesterol-rich LNPs may be an approach for inducing tolerogenic DC maturation.
Assuntos
Células Dendríticas , Transdução de Sinais , Receptores X do Fígado/metabolismo , Transdução de Sinais/genética , Homeostase , ColesterolRESUMO
Phosphatidylinositol-4-phosphate (PtdIns4P) is the most abundant phosphoinositide in plants and the precursor of phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P(2)]. This lipid is the substrate of phosphoinositide-dependent phospholipase C (PI-PLC) that produces diacylglycerol (DAG) which can be phosphorylated to phosphatidic acid (PtdOH). In plants, it has been suggested that PtdIns4P may also be a direct substrate of PI-PLC. Whether PtdIns4P is the precursor of PtdIns(4,5)P(2) or a substrate of PI-PLC, its production by phosphatidylinositol-4-kinases (PI4Ks) is the first step in generating the phosphoinositides hydrolyzed by PI-PLC. PI4Ks can be divided into type-II and type-III. In plants, the identity of the PI4K upstream of PI-PLC is unknown. In Arabidopsis, cold triggers PI-PLC activation, resulting in PtdOH production which is paralleled by decreases in PtdIns4P and PtdIns(4,5)P(2). In suspension cells, both the PtdIns4P decrease and the PtdOH increase in response to cold were impaired by 30 µM wortmannin, a type-III PI4K inhibitor. Type-III PI4Ks include AtPI4KIIIα1, ß1 and ß2 isoforms. In this work we show that PtdOH resulting from the PI-PLC pathway is significantly lowered in a pi4kIIIß1ß2 double mutant exposed to cold stress. Such a decrease was not detected in single pi4kIIIß1 and pi4kIIIß2 mutants, indicating that AtPI4KIIIß1 and AtPI4KIIIß2 can both act upstream of the PI-PLC. Although several short-term to long-term responses to cold were unchanged in pi4kIIIß1ß2, cold induction of several genes was impaired in the double mutant and its germination was hypersensitive to chilling. We also provide evidence that de novo synthesis of PtdIns4P by PI4Ks occurs in parallel to PI-PLC activation.
Assuntos
1-Fosfatidilinositol 4-Quinase/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Temperatura Baixa , Fosfoinositídeo Fosfolipase C/metabolismo , Transdução de Sinais , 1-Fosfatidilinositol 4-Quinase/genética , Arabidopsis/citologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Diacilglicerol Quinase/metabolismo , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Microssomos/efeitos dos fármacos , Microssomos/enzimologia , Mutação/genética , Fosfatidilinositóis/metabolismo , Fosfoinositídeo Fosfolipase C/genética , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Plântula/efeitos dos fármacos , Plântula/metabolismo , Transdução de Sinais/efeitos dos fármacos , Suspensões , Fatores de TempoRESUMO
⢠Long-chain bases (LCBs) are pleiotropic sphingolipidic signals in eukaryotes. We investigated the source and function of phytosphingosine-1-phosphate (PHS-P), a phospho-LCB rapidly and transiently formed in Arabidopsis thaliana on chilling. ⢠PHS-P was analysed by thin-layer chromatography following in vivo metabolic radiolabelling. Pharmacological and genetic approaches were used to identify the sphingosine kinase isoforms involved in cold-responsive PHS-P synthesis. Gene expression, mitogen-activated protein kinase activation and growth phenotypes of three LCB kinase mutants (lcbk1, sphk1 and lcbk2) were studied following cold exposure. ⢠Chilling provoked the rapid and transient formation of PHS-P in Arabidopsis cultured cells and plantlets. Cold-evoked PHS-P synthesis was reduced by LCB kinase inhibitors and abolished in the LCB kinase lcbk2 mutant, but not in lcbk1 and sphk1 mutants. lcbk2 presented a constitutive AtMPK6 activation at 22°C. AtMPK6 activation was also triggered by PHS-P treatment independently of PHS/PHS-P balance. lcbk2 mutants grew comparably with wild-type plants at 22 and 4°C, but exhibited a higher root growth at 12°C, correlated with an altered expression of the cold-responsive DELLA gene RGL3. ⢠Together, our data indicate a function for LCBK2 in planta. Furthermore, they connect PHS-P formation with plant response to cold, expanding the field of LCB signalling in plants.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/fisiologia , Congelamento , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Transdução de Sinais , Esfingosina/análogos & derivados , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Células Cultivadas , DNA Bacteriano/genética , Ativação Enzimática/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/genética , Mutagênese Insercional/efeitos dos fármacos , Mutagênese Insercional/genética , Mutação/genética , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , Inibidores de Proteínas Quinases/farmacologia , Regulon/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Esfingosina/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genéticaRESUMO
Chilling triggers rapid molecular responses that permit the maintenance of plant cell homeostasis and plant adaptation. Recent data showed that nitric oxide (NO) is involved in plant acclimation and tolerance to cold. The participation of NO in the early transduction of the cold signal in Arabidopsis thaliana was investigated. The production of NO after a short exposure to cold was assessed using the NO-sensitive fluorescent probe 4, 5-diamino fluoresceine diacetate and chemiluminescence. Pharmacological and genetic approaches were used to analyze NO sources and NO-mediated changes in cold-regulated gene expression, phosphatidic acid (PtdOH) synthesis and sphingolipid phosphorylation. NO production was detected after 1-4h of chilling. It was impaired in the nia1nia2 nitrate reductase mutant. Moreover, NO accumulation was not observed in H7 plants overexpressing the A. thaliana nonsymbiotic hemoglobin Arabidopsis haemoglobin 1 (AHb1). Cold-regulated gene expression was affected in nia1nia2 and H7 plants. The synthesis of PtdOH upon chilling was not modified by NO depletion. By contrast, the formation of phytosphingosine phosphate and ceramide phosphate, two phosphorylated sphingolipids that are transiently synthesized upon chilling, was negatively regulated by NO. Taken together, these data suggest a new function for NO as an intermediate in gene regulation and lipid-based signaling during cold transduction.
Assuntos
Arabidopsis/genética , Arabidopsis/metabolismo , Temperatura Baixa , Regulação da Expressão Gênica de Plantas , Óxido Nítrico/metabolismo , Esfingolipídeos/biossíntese , Arabidopsis/efeitos dos fármacos , Arabidopsis/enzimologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Benzoatos/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Hemoglobinas/genética , Hemoglobinas/metabolismo , Imidazóis/farmacologia , Nitrato Redutase/metabolismo , Ácidos Fosfatídicos/biossíntese , Fosforilação/efeitos dos fármacos , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Folhas de Planta/metabolismo , S-Nitrosoglutationa/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genética , Simbiose/efeitos dos fármacosRESUMO
BACKGROUND AND AIMS: While low concentrations of high-density lipoprotein-cholesterol (HDL-C) represent a well-established cardiovascular risk factor, extremely high HDL-C is paradoxically associated with elevated cardiovascular risk, resulting in the U-shape relationship with cardiovascular disease. Free cholesterol transfer to HDL upon lipolysis of triglyceride-rich lipoproteins (TGRL) was recently reported to underlie this relationship, linking HDL-C to triglyceride metabolism and atherosclerosis. In addition to free cholesterol, other surface components of TGRL, primarily phospholipids, are transferred to HDL during lipolysis. It remains indeterminate as to whether such transfer is linked to HDL-C and cardiovascular disease. METHODS AND RESULTS: When TGRL was labelled with fluorescent phospholipid 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), time- and dose-dependent transfer of DiI to HDL was observed upon incubations with lipoprotein lipase (LPL). The capacity of HDL to acquire DiI was decreased by -36% (p<0.001) in low HDL-C patients with acute myocardial infarction (n = 22) and by -95% (p<0.001) in low HDL-C subjects with Tangier disease (n = 7), unchanged in low HDL-C patients with Type 2 diabetes (n = 17) and in subjects with high HDL-C (n = 20), and elevated in subjects with extremely high HDL-C (+11%, p<0.05) relative to healthy normolipidemic controls. Across all the populations combined, HDL capacity to acquire DiI was directly correlated with HDL-C (r = 0.58, p<0.001). No relationship of HDL capacity to acquire DiI with both overall and cardiovascular mortality obtained from epidemiological studies for the mean HDL-C levels observed in the studied populations was obtained. CONCLUSIONS: These data indicate that the capacity of HDL to acquire phospholipid from TGRL upon LPL-mediated lipolysis is proportional to HDL-C and does not reflect cardiovascular risk in subjects widely differing in HDL-C levels.
Assuntos
Doenças Cardiovasculares , Diabetes Mellitus Tipo 2 , Doenças Cardiovasculares/diagnóstico , Colesterol , Fatores de Risco de Doenças Cardíacas , Humanos , Lipólise , Lipase Lipoproteica/metabolismo , Lipoproteínas HDL/metabolismo , Fosfolipídeos , Fatores de Risco , TriglicerídeosRESUMO
All mature Saccharomyces cerevisiae sphingolipids comprise inositolphosphorylceramides containing C26:0 or C24:0 fatty acids and either phytosphingosine or dihydrosphingosine. Here we analysed the lipid profile of lag1Delta lac1Delta mutants lacking acyl-CoA-dependent ceramide synthesis, which require the reverse ceramidase activity of overexpressed Ydc1p for sphingolipid biosynthesis and viability. These cells, termed 2Delta.YDC1, make sphingolipids containing exclusively dihydrosphingosine and an abnormally wide spectrum of fatty acids with between 18 and 26 carbon atoms. Like wild-type cells, 2Delta.YDC1 cells stop growing when exposed to Aureobasidin A (AbA), an inhibitor of the inositolphosphorylceramide synthase AUR1, yet their ceramide levels remain very low. This finding argues against a current hypothesis saying that yeast cells do not require inositolphosphorylceramides and die in the presence of AbA only because ceramides build up to toxic concentrations. Moreover, W303lag1Delta lac1Delta ypc1Delta ydc1Delta cells, reported to be AbA resistant, stop growing on AbA after a certain number of cell divisions, most likely because AbA blocks the biosynthesis of anomalous inositolphosphorylsphingosides. Thus, data argue that inositolphosphorylceramides of yeast, the equivalent of mammalian sphingomyelins, are essential for growth. Data also clearly confirm that wild-type strains, when exposed to AbA, immediately stop growing because of ceramide intoxication, long before inositolphosphorylceramide levels become subcritical.
Assuntos
Ceramidas/biossíntese , Depsipeptídeos/farmacologia , Glicoesfingolipídeos/biossíntese , Saccharomyces cerevisiae/crescimento & desenvolvimento , Antifúngicos/farmacologia , Inibidores Enzimáticos/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
After glycosylphosphatidylinositols (GPIs) are added to GPI proteins of Saccharomyces cerevisiae, a fatty acid of the diacylglycerol moiety is exchanged for a C(26:0) fatty acid through the subsequent actions of Per1 and Gup1. In most GPI anchors this modified diacylglycerol-based anchor is subsequently transformed into a ceramide-containing anchor, a reaction which requires Cwh43. Here we show that the last step of this GPI anchor lipid remodeling can be monitored in microsomes. The assay uses microsomes from cells that have been grown in the presence of myriocin, a compound that blocks the biosynthesis of dihydrosphingosine (DHS) and thus inhibits the biosynthesis of ceramide-based anchors. Such microsomes, when incubated with [(3)H]DHS, generate radiolabeled, ceramide-containing anchor lipids of the same structure as made by intact cells. Microsomes from cwh43Delta or mcd4Delta mutants, which are unable to make ceramide-based anchors in vivo, do not incorporate [(3)H]DHS into anchors in vitro. Moreover, gup1Delta microsomes incorporate [(3)H]DHS into the same abnormal anchor lipids as gup1Delta cells synthesize in vivo. Thus, the in vitro assay of ceramide incorporation into GPI anchors faithfully reproduces the events that occur in mutant cells. Incorporation of [(3)H]DHS into GPI proteins is observed with microsomes alone, but the reaction is stimulated by cytosol or bovine serum albumin, ATP plus coenzyme A (CoA), or C(26:0)-CoA, particularly if microsomes are depleted of acyl-CoA. Thus, [(3)H]DHS cannot be incorporated into proteins in the absence of acyl-CoA.
Assuntos
Ceramidas/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Glicosilfosfatidilinositóis/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Background Heart attacks and stroke often result from occlusive thrombi following the rupture of vulnerable atherosclerotic plaques. Vascular smooth muscle cells (VSMCs) play a pivotal role in plaque vulnerability because of their switch towards a proinflammatory/macrophage-like phenotype when in the context of atherosclerosis. The prometastatic transcription factor Slug/Snail2 is a critical regulator of cell phenotypic transition. Here, we aimed to investigate the role of Slug in the transdifferentiation process of VSMCs occurring during atherogenesis. Methods and Results In rat and human primary aortic smooth muscle cells, Slug protein expression is strongly and rapidly increased by platelet-derived growth factor-BB (PDGF-BB). PDGF-BB increases Slug protein without affecting mRNA levels indicating that this growth factor stabilizes Slug protein. Immunocytochemistry and subcellular fractionation experiments reveal that PDGF-BB triggers a rapid accumulation of Slug in VSMC nuclei. Using pharmacological tools, we show that the PDGF-BB-dependent mechanism of Slug stabilization in VSMCs involves the extracellular signal-regulated kinase 1/2 pathway. Immunohistochemistry experiments on type V and type VI atherosclerotic lesions of human carotids show smooth muscle-specific myosin heavy chain-/Slug-positive cells surrounding the prothrombotic lipid core. In VSMCs, Slug siRNAs inhibit prostaglandin E2 secretion and prevent the inhibition of cholesterol efflux gene expression mediated by PDGF-BB, known to be involved in plaque vulnerability and/or thrombogenicity. Conclusions Our results highlight, for the first time, a role of Slug in aortic smooth muscle cell transdifferentiation and enable us to consider Slug as an actor playing a role in the atherosclerotic plaque progression towards a life-threatening phenotype. This also argues for common features between acute cardiovascular events and cancer.
Assuntos
Aterosclerose/metabolismo , Becaplermina/farmacologia , Transdiferenciação Celular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Fatores de Transcrição da Família Snail/metabolismo , Animais , Aterosclerose/genética , Aterosclerose/patologia , Células Cultivadas , Dinoprostona/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Cadeias Pesadas de Miosina/metabolismo , Ratos , Transdução de Sinais , Fatores de Transcrição da Família Snail/genéticaRESUMO
BACKGROUND: Low concentrations of high-density lipoprotein cholesterol (HDL-C) represent a well-established cardiovascular risk factor. Paradoxically, extremely high HDL-C levels are equally associated with elevated cardiovascular risk, resulting in the U-shape relationship of HDL-C with cardiovascular disease. Mechanisms underlying this association are presently unknown. We hypothesised that the capacity of high-density lipoprotein (HDL) to acquire free cholesterol upon triglyceride-rich lipoprotein (TGRL) lipolysis by lipoprotein lipase underlies the non-linear relationship between HDL-C and cardiovascular risk. METHODS: To assess our hypothesis, we developed a novel assay to evaluate the capacity of HDL to acquire free cholesterol (as fluorescent TopFluor® cholesterol) from TGRL upon in vitro lipolysis by lipoprotein lipase. RESULTS: When the assay was applied to several populations markedly differing in plasma HDL-C levels, transfer of free cholesterol was significantly decreased in low HDL-C patients with acute myocardial infarction (-45%) and type 2 diabetes (-25%), and in subjects with extremely high HDL-C of >2.59 mmol/L (>100 mg/dL) (-20%) versus healthy normolipidaemic controls. When these data were combined and plotted against HDL-C concentrations, an inverse U-shape relationship was observed. Consistent with these findings, animal studies revealed that the capacity of HDL to acquire cholesterol upon lipolysis was reduced in low HDL-C apolipoprotein A-I knock-out mice and was negatively correlated with aortic accumulation of [3H]-cholesterol after oral gavage, attesting this functional characteristic as a negative metric of postprandial atherosclerosis. CONCLUSIONS: Free cholesterol transfer to HDL upon TGRL lipolysis may underlie the U-shape relationship between HDL-C and cardiovascular disease, linking HDL-C to triglyceride metabolism and atherosclerosis.
Assuntos
Aorta Torácica/metabolismo , Doenças Cardiovasculares/metabolismo , Proteínas de Transferência de Ésteres de Colesterol/metabolismo , Lipólise/fisiologia , Lipoproteínas HDL/metabolismo , Triglicerídeos/metabolismo , Animais , Biomarcadores/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Lipase Lipoproteica/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Período Pós-PrandialRESUMO
Uptake of low-density lipoprotein (LDL) particles by macrophages represents a key step in the development of atherosclerotic plaques, leading to the foam cell formation. Chemical modification of LDL is however necessary to induce this process. Proatherogenic LDL modifications include aggregation, enzymatic digestion and oxidation. LDL oxidation by one-electron (free radicals) and two-electron oxidants dramatically increases LDL affinity to macrophage scavenger receptors, leading to rapid LDL uptake and fatty streak formation. Circulating high-density lipoprotein (HDL) particles, primarily small, dense, protein-rich HDL3, provide potent protection of LDL from oxidative damage by free radicals, resulting in the inhibition of the generation of pro-inflammatory oxidized lipids. HDL-mediated inactivation of lipid hydroperoxides involves their initial transfer from LDL to HDL and subsequent reduction to inactive hydroxides by redox-active Met residues of apolipoprotein A-I. Several HDL-associated enzymes are present at elevated concentrations in HDL3 relative to large, light HDL2 and can be involved in the inactivation of short-chain oxidized phospholipids. Therefore, HDL represents a multimolecular complex capable of acquiring and inactivating proatherogenic lipids. Antioxidative function of HDL can be impaired in several metabolic and inflammatory diseases. Structural and compositional anomalies in the HDL proteome and lipidome underlie such functional deficiency. Concomitant normalization of the metabolism, circulating levels, composition and biological activities of HDL particles, primarily those of small, dense HDL3, can constitute future therapeutic target.