RESUMO
Diversified anionic phospholipids, phosphatidylserines (PS), externalized to the surface of apoptotic cells are universal phagocytic signals. However, the role of major PS metabolites, such as peroxidized species of PS (PSox) and lyso-PS, in the clearance of apoptotic cells has not been rigorously evaluated. Here, we demonstrate that H2O2 was equally effective in inducing apoptosis and externalization of PS in naive HL60 cells and in cells enriched with oxidizable polyunsaturated species of PS (supplemented with linoleic acid (LA)). Despite this, the uptake of LA-supplemented cells by RAW264.7 and THP-1 macrophages was more than an order of magnitude more effective than that of naive cells. A similar stimulation of phagocytosis was observed with LA-enriched HL60 cells and Jurkat cells triggered to apoptosis with staurosporine. This was due to the presence of PSox on the surface of apoptotic LA-supplemented cells (but not of naive cells). This enhanced phagocytosis was dependent on activation of the intrinsic apoptotic pathway, as no stimulation of phagocytosis occurred in LA-enriched cells challenged with Fas antibody. Incubation of apoptotic cells with lipoprotein-associated phospholipase A2 (Lp-PLA2), a secreted enzyme with high specificity towards PSox, hydrolyzed peroxidized PS species in LA-supplemented cells resulting in the suppression of phagocytosis to the levels observed for naive cells. This suppression of phagocytosis by Lp-PLA2 was blocked by a selective inhibitor of Lp-PLA2, SB-435495. Screening of possible receptor candidates revealed the ability of several PS receptors and bridging proteins to recognize both PS and PSox, albeit with diverse selectivity. We conclude that PSox is an effective phagocytic 'eat-me' signal that participates in the engulfment of cells undergoing intrinsic apoptosis.
Assuntos
1-Alquil-2-acetilglicerofosfocolina Esterase/farmacologia , Macrófagos/metabolismo , Fosfatidilserinas/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Células HL-60 , Humanos , Peróxido de Hidrogênio/farmacologia , Macrófagos/efeitos dos fármacos , Oxirredução , Fagocitose/efeitos dos fármacos , Fagocitose/fisiologia , Transdução de SinaisRESUMO
Amyloid and amyloid-like fibrils are self-assembling protein nanostructures, of interest for their robust material properties and inherent biological compatibility as well as their putative role in a number of debilitating mammalian disorders. Understanding fibril formation is essential to the development of strategies to control, manipulate or prevent fibril growth. As such, this area of research has attracted significant attention over the last half century. This review describes a number of different models that have been formulated to describe the kinetics of fibril assembly. We describe the macroscopic implications of mechanisms in which secondary processes such as secondary nucleation, fragmentation or branching dominate the assembly pathway, compared to mechanisms dominated by the influence of primary nucleation. We further describe how experimental data can be analysed with respect to the predictions of kinetic models.
Assuntos
Amiloide/química , Biocatálise , Multimerização Proteica , Animais , Humanos , Cinética , Modelos MolecularesRESUMO
Lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) is an enzyme that is produced by inflammatory cells, is bound to circulating LDL, and is involved in hydrolyzing polar phospholipids, including those found in oxidized low-density lipoproteins. To date, the biological role of Lp-PLA(2) in atherogenesis has been controversial, with initial reports purporting an atheroprotective effect attributable to the degradation of platelet activating factor and similar molecules. However, more recent studies suggest a proatherogenic role for this enzyme, which is attributed to Lp-PLA(2)-mediated hydrolysis of oxidatively modified low-density lipoproteins that results in the accumulation of proinflammatory products. The liberation of lysophosphatidylcholine and oxidized nonesterified fatty acids from oxidized phospholipids by the action of Lp-PLA(2) results in diverse inflammatory effects on various cell types involved in atherogenesis. This concept is further supported by a number of recently published epidemiology studies suggesting that plasma levels of the enzyme predict future cardiovascular events independent of conventional risk factors. The development of selective inhibitors of Lp-PLA(2) that inhibit enzyme activity in the circulation as well as within human atherosclerotic lesions opens the possibility of therapeutic manipulation of vascular inflammatory processes to reduce residual cardiovascular events in high risk individuals who continue to suffer fatal and nonfatal events despite the current standard of care.
Assuntos
Aterosclerose/tratamento farmacológico , Inibidores Enzimáticos/uso terapêutico , Fosfolipases A/antagonistas & inibidores , 1-Alquil-2-acetilglicerofosfocolina Esterase , Aterosclerose/enzimologia , Inibidores Enzimáticos/farmacologia , Humanos , Fosfolipases A/metabolismo , Fosfolipases A2 , Fatores de RiscoRESUMO
The tumour suppressor protein, PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a member of the mixed function, serine/threonine/tyrosine phosphatase subfamily of protein phosphatases. Its physiological substrates, however, are primarily 3-phosphorylated inositol phospholipids, which are products of phosphoinositide 3-kinases. PTEN thus antagonizes PI 3-kinase-dependent signalling pathways, which explains to a large extent its tumour suppressor status. We have examined the kinetic behaviour, substrate specificity and regulation of PTEN both in vitro and in a variety of cellular models. Although PTEN can utilize both phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)] and its water-soluble headgroup, inositol 1,3,4,5-tetrakisphosphate, as substrates, it displays classical features of interfacial catalysis, which greatly favour the lipid substrate (by as much as 1000-fold as judged by K(cat)/K(m) values). Expression of PTEN in U87 cells (which lack endogenous PTEN) and measuring the levels of all known 3-phosphorylated lipids suggests that phosphatidylinositol 3,4-bisphosphate and PtdIns(3,4,5)P(3) are both substrates, but that phosphatidylinositol 3-phosphate and phosphatidylinositol 3,5-bisphosphate are not. PTEN binds to several PDZ-domain-containing proteins via a consensus sequence at its extreme C-terminus. Disruption of targeting to PDZ-domain proteins selectively blocks some PTEN functions, but not others, suggesting the existence of spatially localized, functionally dedicated pools of signalling lipids. We have also shown recently that PTEN expression is controlled at the transcriptional level and is profoundly upregulated by peroxisome proliferator activated receptor gamma agonists, thereby providing possible implications for these drugs in diabetes, inflammation and cancer.
Assuntos
Fosfatidilinositol 3-Quinases/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Monoéster Fosfórico Hidrolases/fisiologia , Proteínas Supressoras de Tumor/metabolismo , Proteínas Supressoras de Tumor/fisiologia , Animais , Humanos , Cinética , Modelos Químicos , Modelos Moleculares , PTEN Fosfo-Hidrolase , Fosfatos de Fosfatidilinositol/biossíntese , Inibidores de Fosfoinositídeo-3 Quinase , Monoéster Fosfórico Hidrolases/química , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Tirosina Fosfatases/metabolismo , Transdução de Sinais , Transcrição Gênica , Proteínas Supressoras de Tumor/química , Regulação para CimaRESUMO
The recognition that atherosclerosis represents an inflammatory disease has begun to shift interest towards novel therapies that could specifically target the underlying inflammatory component of atherogenesis. Like low-density lipoprotein, an ideal new drug target would be a modifiable plasma risk factor that not only reflects the ongoing inflammatory process but also actively promotes it. Lipoprotein-associated phospholipase A2, also known as platelet-activating factor acetylhydrolase, is a new risk factor that may have the potential to fulfil these requirements.
Assuntos
Doença da Artéria Coronariana/etiologia , Inflamação/etiologia , Fosfolipases A/fisiologia , 1-Alquil-2-acetilglicerofosfocolina Esterase , Animais , Biomarcadores/sangue , Doença da Artéria Coronariana/enzimologia , Doença da Artéria Coronariana/metabolismo , Inibidores Enzimáticos/farmacologia , Ácidos Graxos não Esterificados/metabolismo , Humanos , Infecções/complicações , Infecções/metabolismo , Inflamação/enzimologia , Inflamação/metabolismo , Lipoproteínas LDL/metabolismo , Lisofosfatidilcolinas/metabolismo , Oxirredução , Fosfolipases A/antagonistas & inibidores , Fosfolipases A/metabolismo , Fosfolipases A2 , Fator de Ativação de Plaquetas/antagonistas & inibidores , Fatores de RiscoRESUMO
The peroxisome proliferator-activated receptors (PPARs) are a family of fatty acid-activated transcription factors which control lipid homeostasis and cellular differentiation. PPARalpha (NR1C1) controls lipid oxidation and clearance in hepatocytes and PPARgamma (NR1C3) promotes preadipocyte differentiation and lipogenesis. Drugs that activate PPARalpha are effective in lowering plasma levels of lipids and have been used in the management of hyperlipidemia. PPARgamma agonists increase insulin sensitivity and are used in the management of type 2 diabetes. In contrast, there are no marketed drugs that selectively target PPARdelta (NR1C2) and the physiological roles of PPARdelta are unclear. In this report we demonstrate that the expression of PPARdelta is increased during the differentiation of human macrophages in vitro. In addition, a highly selective agonist of PPARdelta (compound F) promotes lipid accumulation in primary human macrophages and in macrophages derived from the human monocytic cell line, THP-1. Compound F increases the expression of genes involved in lipid uptake and storage such as the class A and B scavenger receptors (SRA, CD36) and adipophilin. PPARdelta activation also represses key genes involved in lipid metabolism and efflux, i.e. cholesterol 27-hydroxylase and apolipoprotein E. We have generated THP-1 sublines that overexpress PPARdelta and have confirmed that PPARdelta is a powerful promoter of macrophage lipid accumulation. These data suggest that PPARdelta may play a role in the pathology of diseases associated with lipid-filled macrophages, such as atherosclerosis, arthritis, and neurodegeneration.
Assuntos
Metabolismo dos Lipídeos , Macrófagos/metabolismo , Receptores Citoplasmáticos e Nucleares/fisiologia , Fatores de Transcrição/fisiologia , Animais , Linhagem Celular , Colesterol/metabolismo , Regulação da Expressão Gênica/fisiologia , HumanosRESUMO
The death of macrophages contributes to atheroma formation. Oxidation renders low-density lipoprotein (LDL) cytotoxic to human monocyte-macrophages. Lipoprotein-associated phospholipase A2 (Lp-PLA2), also termed platelet-activating factor acetylhydrolase, hydrolyses oxidised phospholipids. Inhibition of Lp-PLA2 by diisopropyl fluorophosphate or Pefabloc (broad-spectrum serine esterase/protease inhibitors), or SB222657 (a specific inhibitor of Lp-PLA2) did not prevent LDL oxidation, but diminished the ensuing toxicity and apoptosis induction when the LDL was oxidised, and inhibited the rise in lysophosphatidylcholine levels that occurred in the inhibitors' absence. Hydrolysis products of oxidised phospholipids thus account for over a third of the cytotoxic and apoptosis-inducing effects of oxidised LDL on macrophages.
Assuntos
Apoptose/fisiologia , Lipoproteínas LDL/fisiologia , Lipoproteínas/metabolismo , Macrófagos/citologia , Monócitos/citologia , Fosfolipases A/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Azetidinas/farmacologia , Inibidores Enzimáticos/farmacologia , Humanos , Isoflurofato/farmacologia , Macrófagos/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Fosfolipases A/metabolismo , Fosfolipases A2 , Sulfonas/farmacologia , Sulfóxidos/farmacologiaRESUMO
The lipophilic 1-substituent in a series of 1-((amidolinked)-alkyl)-pyrimidones, inhibitors of recombinant lipoprotein-associated phospholipase A(2), has been modified to give inhibitors of high potency in human plasma and enhanced physicochemical properties. Phenylpiperazineacetamide derivative 23 shows very promising oral activity.
Assuntos
Inibidores Enzimáticos/farmacologia , Fosfolipases A/antagonistas & inibidores , Fosfolipases A/sangue , 1-Alquil-2-acetilglicerofosfocolina Esterase , Administração Oral , Animais , Inibidores Enzimáticos/síntese química , Humanos , Concentração Inibidora 50 , Taxa de Depuração Metabólica/fisiologia , Microssomos Hepáticos/metabolismo , Piperazinas/síntese química , Piperazinas/farmacologia , Pirimidinonas/síntese química , Pirimidinonas/farmacologia , Coelhos , RatosRESUMO
The PTEN tumor suppressor gene modulates several cellular functions, including cell migration, survival, and proliferation [1] by antagonizing phosphatidylinositol 3-kinase (PI 3-kinase)-mediated signaling cascades. Mechanisms by which the expression of PTEN is regulated are, however, unclear. The ligand-activated nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) [2] has been shown to regulate differentiation and/or cell growth in a number of cell types [3, 4, 5], which has led to the suggestion that PPARgamma, like PTEN [1, 6], could act as a tumor suppressor. PPARgamma has also been implicated in anti-inflammatory responses [7, 8], although downstream mediators of these effects are not well defined. Here, we show that the activation of PPARgamma by its selective ligand, rosiglitazone, upregulates PTEN expression in human macrophages, Caco2 colorectal cancer cells, and MCF7 breast cancer cells. This upregulation correlated with decreased PI 3-kinase activity as measured by reduced phosphorylation of protein kinase B. One consequence of this was that rosiglitazone treatment reduced the proliferation rate of Caco2 and MCF7 cells. Antisense-mediated disruption of PPARgamma expression prevented the upregulation of PTEN that normally accompanies monocyte differentiation and reduced the proportion of macrophages undergoing apoptosis, while electrophoretic mobility shift assays showed that PPARgamma is able to bind two response elements in the genomic sequence upstream of PTEN. Our results demonstrate a role for PPARgamma in regulating PI 3-kinase signaling by modulating PTEN expression in inflammatory and tumor-derived cells.
Assuntos
Anti-Inflamatórios/farmacologia , Antineoplásicos/farmacologia , Genes Supressores de Tumor , Monoéster Fosfórico Hidrolases/genética , Receptores Citoplasmáticos e Nucleares/agonistas , Tiazóis/farmacologia , Tiazolidinedionas , Fatores de Transcrição/agonistas , Proteínas Supressoras de Tumor , Regulação para Cima , Humanos , PTEN Fosfo-Hidrolase , Pioglitazona , Rosiglitazona , Células Tumorais CultivadasRESUMO
Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a serine lipase that is associated with low density lipoprotein (LDL) in human plasma. Substrates include oxidised phosphatidylcholine (PC), which is hydrolysed by Lp-PLA2 to lyso-PC and oxidised fatty acids. Both products are bioactive and proinflammatory, and implicated in monocyte infiltration into the developing plaque, deposition of foam cells, and plaque progression and instability. Lp-PLA2 has recently been shown to be a risk factor for coronary events in previously asymptomatic, hypercholesterolaemic men. A series of azetidinones was designed as potent and selective inhibitors of this enzyme; SB-222657 inhibited release of the chemotactic cleavage products from oxidised LDL, and SB-244323 reduced atherosclerotic plaque development in a 3 month rabbit study. A series of pyrimidones has been designed from a screening hit, and nanomolar inhibitors identified. Oral efficacy in inhibiting plasma Lp-PLA2 in rabbits has been demonstrated with a variety of structural classes.
Assuntos
Arteriosclerose/tratamento farmacológico , Inibidores Enzimáticos/uso terapêutico , Fosfolipases A/antagonistas & inibidores , 1-Alquil-2-acetilglicerofosfocolina Esterase , Arteriosclerose/etiologia , Humanos , Fosfolipases A2 , Relação Estrutura-AtividadeRESUMO
Emerging evidence indicates that chemokine receptor expression patterns are critical in determining the spectrum of action of the chemokines. We have analysed the expression patterns of 17 chemokine receptors and two orphan chemokine receptor-like genes in various freshly prepared human peripheral blood leucocyte populations, including neutrophils, lymphocytes, and naïve and differentiated monocytes using real-time quantitative polymerase chain reaction (TaqMan). This is the first comprehensive study of chemokine receptor expression in such a wide variety of cell types. Human peripheral blood leukocyte populations were found to express a wide range of chemokine receptors that varies depending on cell type and differentiation state. Novel expression patterns of certain chemokine receptors were seen during our analysis. For example, the orphan chemokine receptor HCR was expressed at very high levels by both primary neutrophils and primary monocytes, and was further upregulated on neutrophil activation and during monocyte to macrophage differentiation. When neutrophil calcium transients were measured in response to a panel of 30 different chemokines the results clearly correlated with the chemokine receptor expression profile. For example strong calcium responses were seen in neutrophils following stimulation with the CXCR1 and CXCR2 ligands, interleukin (IL-)8, GCP-2 and Gro-beta. These data have implications for the study of the functional responses of leukocytes to external stimuli and will aid in our understanding of general leukocyte biology.
Assuntos
Leucócitos/metabolismo , Receptores de Quimiocinas/sangue , Receptores de Quimiocinas/metabolismo , Sinalização do Cálcio , Diferenciação Celular , Humanos , Ligantes , Linfócitos/metabolismo , Macrófagos/metabolismo , Monócitos/metabolismo , Neutrófilos/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/genética , Receptores de Quimiocinas/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Modification of the pyrimidone 5-substituent in a series of 1-((amidolinked)-alkyl)-pyrimidones, lipophilic inhibitors of lipoprotein-associated phospholipase A2, has given inhibitors of nanomolar potency and improved physicochemical properties. Compound 23 was identified as a potent, highly water soluble. CNS penetrant inhibitor suitable for intravenous administration.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Fosfolipases A/antagonistas & inibidores , Pirimidinonas/química , Pirimidinonas/farmacologia , Animais , Arteriosclerose/tratamento farmacológico , Vias de Administração de Medicamentos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacocinética , Humanos , Masculino , Estrutura Molecular , Fosfolipases A/metabolismo , Fosfolipases A2 , Pirimidinonas/síntese química , Coelhos , Ratos , Solubilidade , Água/químicaRESUMO
Atomic force microscopy has been employed to investigate the structural organization of amyloid fibrils produced in vitro from three very different polypeptide sequences. The systems investigated are a 10-residue peptide derived from the sequence of transthyretin, the 90-residue SH3 domain of bovine phosphatidylinositol-3'-kinase, and human wild-type lysozyme, a 130-residue protein containing four disulfide bridges. The results demonstrate distinct similarities between the structures formed by the different classes of fibrils despite the contrasting nature of the polypeptide species involved. SH3 and lysozyme fibrils consist typically of four protofilaments, exhibiting a left-handed twist along the fibril axis. The substructure of TTR(10-19) fibrils is not resolved by atomic force microscopy and their uniform appearance is suggestive of a regular self-association of very thin filaments. We propose that the exact number and orientation of protofilaments within amyloid fibrils is dictated by packing of the regions of the polypeptide chains that are not directly involved in formation of the cross-beta core of the fibrils. The results obtained for these proteins, none of which is directly associated with any human disease, are closely similar to those of disease-related amyloid fibrils, supporting the concept that amyloid is a generic structure of polypeptide chains. The detailed architecture of an individual fibril, however, depends on the manner in which the protofilaments assemble into the fibrillar structure, which in turn is dependent on the sequence of the polypeptide and the conditions under which the fibril is formed.
Assuntos
Amiloide/ultraestrutura , Fragmentos de Peptídeos/química , Pré-Albumina/ultraestrutura , Amiloide/química , Animais , Bovinos , Dissulfetos/química , Humanos , Microscopia de Força Atômica/métodos , Microscopia Eletrônica , Muramidase/química , Muramidase/ultraestrutura , Peptídeos/química , Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol 3-Quinases/ultraestrutura , Pré-Albumina/química , Domínios de Homologia de srcRESUMO
From two related series of 2-(alkylthio)-pyrimidones, a novel series of 1-((amidolinked)-alkyl)-pyrimidones has been designed as nanomolar inhibitors of human lipoprotein-associated phospholipase A2. These compounds show greatly enhanced activity in isolated plasma. Selected derivatives such as compounds 51 and 52 are orally active with a good duration of action.
Assuntos
Inibidores Enzimáticos/farmacologia , Lipoproteínas/metabolismo , Fosfolipases A/antagonistas & inibidores , Pirimidinonas/farmacologia , Administração Oral , Animais , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/química , Estrutura Molecular , Fosfolipases A/metabolismo , Fosfolipases A2 , Pirimidinonas/administração & dosagem , Pirimidinonas/química , CoelhosRESUMO
BACKGROUND: Chronic inflammation is believed to increase the risk of coronary events by making atherosclerotic plaques in coronary vessels prone to rupture. We examined blood constituents potentially affected by inflammation as predictors of risk in men with hypercholesterolemia who were enrolled in the West of Scotland Coronary Prevention Study, a trial that evaluated the value of pravastatin in the prevention of coronary events. METHODS: A total of 580 men who had had a coronary event (nonfatal myocardial infarction, death from coronary heart disease, or a revascularization procedure) were each matched for age and smoking status with 2 control subjects (total, 1160) from the same cohort who had not had a coronary event. Lipoprotein-associated phospholipase A2, C-reactive protein, and fibrinogen levels, and the white-cell count were measured at base line, along with other traditional risk factors. The association of these variables with the risk of coronary events was tested in regression models and by dividing the range of values according to quintiles. RESULTS: Levels of C-reactive protein, the white-cell count, and fibrinogen levels were strong predictors of the risk of coronary events; the risk in the highest quintile of the study cohort for each variable was approximately twice that in the lowest quintile. However, the association of these variables with risk was markedly attenuated when age, systolic blood pressure, and lipoprotein levels were included in multivariate models. Levels of lipoprotein-associated phospholipase A2 (platelet-activating factor acetylhydrolase), the expression of which is regulated by mediators of inflammation, had a strong, positive association with risk that was not confounded by other factors. It was associated with almost a doubling of the risk in the highest quintile as compared with the lowest quintile. CONCLUSIONS: Inflammatory markers are predictors of the risk of coronary events, but their predictive ability is attenuated by associations with other coronary risk factors. Elevated levels of lipoprotein-associated phospholipase A2 appear to be a strong risk factor for coronary heart disease, a finding that has implications for atherogenesis and the assessment of risk.
Assuntos
Proteína C-Reativa/análise , Doença das Coronárias/etiologia , Hipercolesterolemia/sangue , Fosfolipases A/sangue , 1-Alquil-2-acetilglicerofosfocolina Esterase , Anticolesterolemiantes/uso terapêutico , Biomarcadores/sangue , Estudos de Casos e Controles , Colesterol/sangue , Doença das Coronárias/mortalidade , Fibrinogênio/análise , Humanos , Hipercolesterolemia/complicações , Hipercolesterolemia/tratamento farmacológico , Inflamação/sangue , Inflamação/complicações , Inflamação/diagnóstico , Contagem de Leucócitos , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/etiologia , Fosfolipases A2 , Pravastatina/uso terapêutico , Fatores de Risco , Triglicerídeos/sangueRESUMO
Human apolipoprotein C-II (apoC-II) self-associates in solution to form aggregates with the characteristics of amyloid including red-green birefringence in the presence of Congo Red under cross-polarized light, increased fluorescence in the presence of thioflavin T, and a fibrous structure when examined by electron microscopy. ApoC-II was expressed and purified from Escherichia coli and rapidly exchanged from 5 M guanidine hydrochloride into 100 mM sodium phosphate, pH 7.4, to a final concentration of 0.3 mg/mL. This apoC-II was initially soluble, eluting as low molecular weight species in gel filtration experiments using Sephadex G-50. Circular dichroism (CD) spectroscopy indicated predominantly unordered structure. Upon incubation for 24 h, apoC-II self-associated into high molecular weight aggregates as indicated by elution in the void volume of a Sephadex G-50 column, by rapid sedimentation in an analytical ultracentrifuge, and by increased light scattering. CD spectroscopy indicated an increase in beta-sheet content, while fluorescence emission spectroscopy of the single tryptophan revealed a blue shift and an increase in maximum intensity, suggesting repositioning of the tryptophan into a less polar environment. Electron microscopy of apoC-II aggregates revealed a novel looped-ribbon morphology (width 12 nm) and several isolated closed loops. Like all of the conserved plasma apolipoproteins, apoC-II contains amphipathic helical regions that account for the increase in alpha-helix content on lipid binding. The increase in beta-structure accompanying apoC-II fibril formation points to an alternative folding pathway and an in vitro system to explore the general tendency of apolipoproteins to form amyloid in vivo.
Assuntos
Amiloide/metabolismo , Apolipoproteínas C/química , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/química , Apolipoproteína C-II , Apolipoproteínas C/ultraestrutura , Benzotiazóis , Cromatografia em Gel , Dicroísmo Circular , Vermelho Congo/metabolismo , Humanos , Microscopia Eletrônica , Conformação Proteica , Espectrometria de Fluorescência , Tiazóis/metabolismo , Triptofano/química , UltracentrifugaçãoRESUMO
A specific and robust immunoassay for the lipoprotein-associated phospholipase A(2) (Lp-PLA(2)), platelet-activating factor acetylhydrolase, is described for the first time. The immunoassay was used to evaluate possible links between plasma Lp-PLA(2) levels and atherosclerosis risk amongst susceptible individuals. Such an investigation was important because Lp-PLA(2) participates in the oxidative modification of low density lipoprotein by cleaving oxidised phosphatidylcholines, generating lysophosphatidylcholine and oxidised free fatty acids. The majority of Lp-PLA(2) was found associated with LDL (approximately 80%) and, as expected, enzyme levels were significantly positively correlated to LDL cholesterol. Plasma Lp-PLA(2) levels were significantly elevated in patients with angiographically proven coronary artery disease (CAD) when compared with age-matched controls, even though LDL cholesterol levels did not differ significantly. Indeed, when included in a general linear model with LDL cholesterol and other risk factors, Lp-PLA(2) appeared to be an independent predictor of disease status. We propose, therefore, that plasma Lp-PLA(2) mass should be viewed as a potential novel risk factor for CAD that provides information related to but additional to traditional lipoprotein measurements.
Assuntos
Arteriosclerose/enzimologia , Fosfolipases A/sangue , Fator de Ativação de Plaquetas/metabolismo , 1-Alquil-2-acetilglicerofosfocolina Esterase , Adulto , Idoso , Anticorpos Monoclonais , Arteriosclerose/diagnóstico por imagem , Arteriosclerose/etiologia , Biomarcadores/sangue , Angiografia Coronária , Ensaio de Imunoadsorção Enzimática , Humanos , Lipídeos/sangue , Masculino , Pessoa de Meia-Idade , Fosfolipases A/imunologia , Fator de Ativação de Plaquetas/imunologia , Ativação Plaquetária , Prognóstico , Fatores de RiscoRESUMO
We have examined the chemical dissection and subsequent reassembly of fibrils formed by a ten-residue peptide to probe the forces that drive the formation of amyloid. The peptide, TTR(10-19), encompasses the A strand of the inner beta-sheet structure that lines the thyroid hormone binding site of the human plasma protein transthyretin. When dissolved in water under low pH conditions the peptide readily forms amyloid fibrils. Electron microscopy of these fibrils indicates the presence of long (>1000 nm) rigid structures of uniform diameter (approximately 14 nm). Addition of urea (3 M) to preformed fibrils disrupts these rigid structures. The partially disrupted fibrils form flexible ribbon-like arrays, which are composed of a number of clearly visible protofilaments (3-4 nm diameter). These protofilaments are highly stable, and resist denaturation in 6 M urea at 75 degrees C over a period of hours. High concentrations (>50%, v/v) of 2,2,2-trifluoroethanol also dissociate TTR(10-19) fibrils to the constituent protofilaments, but these slowly dissociate to monomeric, soluble peptides with extensive alpha-helical structure. Dilution of the denaturant or co-solvent at the stage when dissociation to protofilaments has occurred results in the efficient reassembly of fibrils. These results indicate that assembly of fibrils from protofilaments involves relatively weak and predominantly hydrophobic interactions, whereas assembly of peptides into protofilaments involves both electrostatic and hydrophobic forces, resulting in a highly stable and compact structures.
Assuntos
Biopolímeros/metabolismo , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/ultraestrutura , Placa Amiloide/ultraestrutura , Pré-Albumina/metabolismo , Pré-Albumina/ultraestrutura , Ácido Acético/farmacologia , Sequência de Aminoácidos , Biopolímeros/química , Dicroísmo Circular , Temperatura Alta , Humanos , Concentração de Íons de Hidrogênio , Cinética , Metanol/farmacologia , Microscopia Eletrônica , Fragmentos de Peptídeos/química , Placa Amiloide/química , Placa Amiloide/metabolismo , Pré-Albumina/química , Ligação Proteica/efeitos dos fármacos , Desnaturação Proteica/efeitos dos fármacos , Estrutura Quaternária de Proteína/efeitos dos fármacos , Estrutura Secundária de Proteína/efeitos dos fármacos , Cloreto de Sódio/farmacologia , Solubilidade , Solventes , Eletricidade Estática , Fatores de Tempo , Trifluoretanol/farmacologia , Ureia/farmacologiaRESUMO
Recombinant fractalkine possesses both chemoattractive and adhesive properties in vitro. Previous studies have demonstrated an upregulation of this molecule on the membranes of activated human endothelial cells and hypothesised that fractalkine plays a role in the recruitment and adherence of monocytes to the activated endothelium. Here we present data analysing both the adhesive and chemoattractive properties of this chemokine expressed by activated human umbilical vein endothelial cells. We demonstrate that both recombinant fractalkine and endogenously produced fractalkine function as adhesion molecules, tethering monocytes to the endothelium. However, our data demonstrate that although recombinant fractalkine has the potential to function as a potent monocyte chemoattractant, the endogenous fractalkine cleaved from activated human umbilical vein endothelial cells is not responsible for the observed chemotaxis in this model. Instead, we show that monocyte chemoattractant protein-1 (MCP-1), secreted from the activated human umbilical vein endothelial cells, is responsible for the chemotaxis of these monocytes.
Assuntos
Quimiocinas CX3C , Quimiocinas CXC/fisiologia , Endotélio Vascular/fisiologia , Proteínas de Membrana/fisiologia , Monócitos/citologia , Western Blotting , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Quimiocina CX3CL1 , Quimiocinas CXC/genética , Quimiocinas CXC/farmacologia , Fatores Quimiotáticos/fisiologia , Quimiotaxia/efeitos dos fármacos , Relação Dose-Resposta a Droga , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/farmacologia , Monócitos/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/farmacologia , Fatores de Tempo , Fator de Necrose Tumoral alfa/farmacologia , Veias Umbilicais/citologia , Veias Umbilicais/metabolismoRESUMO
Apolipoprotein C-II (apoC-II) is an exchangeable plasma apolipoprotein and an endogenous activator of lipoprotein lipase (LpL). Genetic deficiencies of apoC-II and overexpression of apoC-II in transgenic mice are both associated with severe hyperlipidemia, indicating a complex role for apoC-II in the regulation of blood lipid levels. ApoC-II exerts no effect on the activity of LpL for soluble substrates, suggesting that activation occurs via the formation of a lipid-bound complex. We have synthesized a peptide corresponding to amino acid residues 39-62 of mature human apoC-II. This peptide does not bind to model lipid surfaces but retains the ability to activate LpL. Conjugation of the fluorophore 7-nitrobenz-2-oxa-1,3-diazole (NBD) to the N-terminal alpha-amino group of apoC-II39-62 facilitated determination of the affinity of the peptide for LpL using fluorescence anisotropy measurements. The dissociation constant describing this interaction was 0.23 microM, and was unchanged when LpL was lipid-bound. Competitive binding studies showed that apoC-II39-62 and full-length apoC-II exhibited the same affinity for LpL in aqueous solution, whereas the affinity for full-length apoC-II was increased at least 1 order of magnitude in the presence of lipid. We suggest that while the binding of apoC-II to the lipid surface promotes the formation of a high-affinity complex of apoC-II and LpL, activation occurs via direct helix-helix interactions between apoC-II39-62 and the loop covering the active site of LpL.