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1.
Biochem Biophys Res Commun ; 359(3): 723-8, 2007 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-17553462

RESUMO

Uptake of modified lipoproteins by macrophages causes foam cell formation and promotes atherosclerosis. Atherogenic lipoproteins are cytotoxic and induce cell death under certain conditions but may also enhance macrophage survival. Macrophages treated with enzymatically modified LDL (E-LDL) were subjected to GeneChip analysis and the antiapoptotic gene TOSO was found induced. TOSO mRNA is upregulated and apoptosis is reduced in E-LDL but not in oxidized LDL (Ox-LDL) loaded macrophages. FLIP(L) abundance was suggested to mediate the antiapoptotic properties of TOSO; however, FLIP(L) was not changed. Ox-LDL is internalized predominantly by scavenger receptors such as CD36 while E-LDL particles are preferentially internalized by Fc- and complement-receptor dependent phagocytosis and internalization of phagobeads by macrophages upregulates TOSO. In COS-7 cells however, phagocytotic activity was not affected by TOSO. These data indicate that E-LDL-generated foam cells are protected from cell death most likely through the expression of TOSO by a FLIP(L) independent mechanism.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Lipoproteínas LDL/metabolismo , Lipoproteínas LDL/farmacologia , Macrófagos/citologia , Macrófagos/metabolismo , Proteínas de Membrana/metabolismo , Regulação para Cima , Animais , Proteínas Reguladoras de Apoptose/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Chlorocebus aethiops , Proteína de Domínio de Morte Associada a Fas/metabolismo , Humanos , Hidroxiesteroide Desidrogenases/metabolismo , Macrófagos/efeitos dos fármacos , Proteínas de Membrana/genética , Especificidade de Órgãos , Oxirredução , Fagocitose/efeitos dos fármacos , RNA Mensageiro/genética , Tripsina/metabolismo
2.
Cytometry A ; 69(3): 189-91, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16479605

RESUMO

Atherosclerosis is characterized by the generation of lipid-loaded macrophage-derived foam cells. To study the effect of different types of atherogenic lipoproteins, human macrophages were loaded with enzymatically degraded low density lipoprotein (E-LDL) or oxidized LDL (Ox-LDL). Cellular cholesterol content was increased by E-LDL, whereas Ox-LDL increased the ceramide content. Cell surface expression analysis by flow cytometry and confocal microscopy revealed that Ox-LDL increased ceramide and lactosylceramide expression compared to E-LDL loading and induced ceramide rafts, whereas loading with E-LDL induced cholesterol-rich microdomains. Formation of different rafts may have consequences for raft associated signaling in cholesterol homeostasis and apoptosis in human macrophages.


Assuntos
Ceramidas/metabolismo , Colesterol/metabolismo , Lipoproteínas LDL/farmacologia , Macrófagos/metabolismo , Microdomínios da Membrana/metabolismo , Antígenos CD/análise , Antígenos CD/metabolismo , Diferenciação Celular/efeitos dos fármacos , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Ceramidas/análise , Colesterol/análise , Citometria de Fluxo , Humanos , Lactosilceramidas/análise , Lactosilceramidas/metabolismo , Lipoproteínas LDL/química , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Lipídeos de Membrana/análise , Lipídeos de Membrana/metabolismo , Microdomínios da Membrana/química , Microdomínios da Membrana/efeitos dos fármacos , Microscopia Confocal , Microscopia de Fluorescência , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo
3.
Mol Biol Cell ; 15(12): 5399-407, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15469992

RESUMO

The ATP-binding cassette transporter A1 (ABCA1) facilitates the cellular release of cholesterol and choline-phospholipids to apolipoprotein A-I (apoA-I) and several studies indicate that vesicular transport is associated with ABCA1 function. Syntaxins play a major role in vesicular fusion and have also been demonstrated to interact with members of the ABC-transporter family. Therefore, we focused on the identification of syntaxins that directly interact with ABCA1. The expression of syntaxins and ABCA1 in cultured human monocytes during M-CSF differentiation and cholesterol loading was investigated and syntaxins 3, 6, and 13 were found induced in foam cells together with ABCA1. Immunoprecipitation experiments revealed a direct association of syntaxin 13 and full-length ABCA1, whereas syntaxin 3 and 6 failed to interact with ABCA1. The colocalization of ABCA1 and syntaxin 13 was also shown by immunofluorescence microscopy. Silencing of syntaxin 13 by small interfering RNA (siRNA) led to reduced ABCA1 protein levels and hence to a significant decrease in apoA-I-dependent choline-phospholipid efflux. ABCA1 is localized in Lubrol WX-insoluble raft microdomains in macrophages and syntaxin 13 and flotillin-1 were also detected in these detergent resistant microdomains along with ABCA1. Syntaxin 13, flotillin-1, and ABCA1 were identified as phagosomal proteins, indicating the involvement of the phagosomal compartment in ABCA1-mediated lipid efflux. In addition, the uptake of latex phagobeads by fibroblasts with mutated ABCA1 was enhanced when compared with control cells and the recombinant expression of functional ABCA1 normalized the phagocytosis rate in Tangier fibroblasts. It is concluded that ABCA1 forms a complex with syntaxin 13 and flotillin-1, residing at the plasma membrane and in phagosomes that are partially located in raft microdomains.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Membrana/metabolismo , Fagocitose , Doença de Tangier/metabolismo , Doença de Tangier/patologia , Transportador 1 de Cassete de Ligação de ATP , Adulto , Células Cultivadas , Humanos , Macrófagos/metabolismo , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Monócitos/metabolismo , Ligação Proteica , Proteínas Qa-SNARE , RNA Interferente Pequeno
4.
Genomics ; 83(6): 1116-24, 2004 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15177564

RESUMO

The highly homologous genes NIPSNAP3 and NIPSNAP4, with 87% amino acid identity, are members of the NIPSNAP family with putative roles in vesicular trafficking. NIPSNAP3 mRNA and NIPSNAP4 mRNA and protein were detected in multiple tissues and cells at varying degrees. Interestingly, NIPSNAP3 is most highly expressed in skeletal muscle, where NIPSNAP4 has a low mRNA abundance. NIPSNAP4 was found associated with membranes and partly localized in rafts. The ubiquitous expression of the highly conserved NIPSNAPs and their association with membranes further support an important cellular function of these proteins probably linked to vesicular trafficking. The NIPSNAP3 and NIPSNAP4 genes are located in close proximity to the 3' end of the ATP-binding cassette transporter A1 (ABCA1), whose mutations cause familial high-density lipoprotein deficiency syndromes. The adjacent genomic location and the finding that ABCA1 is a regulator of vesicular trafficking may indicate a functional relation of these proteins, even though NIPSNAP4 does not interact directly with ABCA1 nor is its expression altered in cells with mutated ABCA1.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Microdomínios da Membrana/metabolismo , Proteínas/genética , Proteínas/metabolismo , Doença de Tangier/genética , Transportador 1 de Cassete de Ligação de ATP , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Sequência de Aminoácidos , Animais , Células CHO , Células CACO-2 , Mapeamento Cromossômico , Cromossomos Humanos Par 9/genética , Cricetinae , Cricetulus , Células HT29 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Dados de Sequência Molecular , Polietilenoglicóis/química , Alinhamento de Sequência , Análise de Sequência de Proteína , Vesículas Transportadoras/fisiologia , Proteínas de Transporte Vesicular
5.
Biochim Biophys Acta ; 1682(1-3): 112-9, 2004 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-15158762

RESUMO

Some of the biological effects of lipoproteins have been attributed to their association with lysophosphatidic acid (LPA), lysophosphatidylcholine (LPC), sphingosine-1-phosphate (S1P) and sphingosylphosphorylcholine (SPC). These lysophospholipids mediate multiple biological responses via several G protein-coupled receptors (GPR). The expression of these receptors, however, has not been systematically investigated in primary human monocytes and macrophages as major cells involved in atherosclerosis. The mRNAs for all 15 receptors described so far were detected in monocytes, macrophages, foam cells and high density lipoprotein (HDL(3))-treated cells using real time RT-PCR. Immunoblots revealed that S1P(1), S1P(2), S1P(4), LPA(1), LPA(2) and GPR65 are expressed in monocytes and macrophages, while S1P(5) and LPA(3) have not been detected. S1P(3) was induced during differentiation but down-regulated by lipid-loading and HDL(3), whereas LPA(1) was down-regulated in differentiated macrophages. The influence of S1P on macrophages was investigated and the induction of CD32 indicates an enhanced phagocytic activity. Altogether, these data give insights into the expression and regulation of lysophospholipid receptors in primary human monocytes, macrophages and foam cells.


Assuntos
Lisofosfolipídeos/metabolismo , Macrófagos/metabolismo , Monócitos/metabolismo , Receptores Acoplados a Proteínas G/genética , Esfingosina/metabolismo , Adulto , Antígenos CD/biossíntese , Antígenos CD/genética , Antígenos de Diferenciação Mielomonocítica/biossíntese , Antígenos de Diferenciação Mielomonocítica/genética , Apolipoproteínas E/biossíntese , Apolipoproteínas E/genética , Diferenciação Celular/fisiologia , Células Espumosas/metabolismo , Humanos , Pessoa de Meia-Idade , Família Multigênica , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/genética , Receptores Acoplados a Proteínas G/biossíntese , Receptores de IgG/biossíntese , Receptores de IgG/genética , Receptores de Lisofosfolipídeos , Esfingosina/análogos & derivados
6.
Biochim Biophys Acta ; 1642(1-2): 25-31, 2003 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-12972290

RESUMO

The association of elevated lipoprotein (a) (Lp(a)) with an increased risk for coronary events is clearly established. This increased risk may in part be due to the activation of monocytes as major cells involved in atherogenesis. High concentrations of plasma Lp(a) were shown to influence the gene expression of human blood monocytes and in the present study we demonstrate a reduced abundance of the lysosomal acid lipase (LAL) mRNA in monocytes of patients with coronary disease and selective Lp(a) hyperlipidemia. This is also supported by in vitro studies where purified Lp(a) but not low-density lipoprotein (LDL) was shown to downregulate mRNA levels of the LAL in control monocytes. A correlation of Lp(a) serum levels and the proinflammatory cytokine IL-6 was recently also described. Therefore, we investigated whether Lp(a) is capable to enhance the release of this acute phase cytokine from human blood monocytes. Purified Lp(a) led to an increased secretion of IL-6, but not TNF-alpha arguing against a general activation of these cells. The association of reduced LAL activity with the premature development of coronary artery disease has been demonstrated in patients with hypercholesterolemia, and in the present study we show for the first time that LAL expression is suppressed in monocytes from patients with Lp(a) hyperlipidemia and by purified Lp(a). In addition, increased levels of IL-6 also predict future cardiovascular events and IL-6 secretion was also induced by purified Lp(a).


Assuntos
Regulação para Baixo/imunologia , Predisposição Genética para Doença/genética , Interleucina-6/metabolismo , Lipase/metabolismo , Lipoproteína(a)/metabolismo , Lisossomos/metabolismo , Monócitos/enzimologia , Células Cultivadas , Doença da Artéria Coronariana/enzimologia , Doença da Artéria Coronariana/genética , Regulação para Baixo/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/genética , Humanos , Hiperlipidemias/enzimologia , Hiperlipidemias/genética , Lipase/genética , Lipoproteína(a)/farmacologia , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Análise de Sequência com Séries de Oligonucleotídeos , Valor Preditivo dos Testes , RNA Mensageiro/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
7.
J Biol Chem ; 277(44): 41307-10, 2002 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-12235128

RESUMO

ATP-binding cassette transporter A1 (ABCA1) is a major regulator of cellular cholesterol and phospholipid homeostasis. Its function has not been fully characterized and may depend on the association with additional proteins. To identify ABCA1-interacting proteins a human liver yeast two-hybrid library was screened with the 144 C-terminal amino acids of ABCA1. Fas-associated death domain protein (FADD) was identified to bind to ABCA1, and this interaction was confirmed by pull-down assays and co-immunoprecipitations. Recombinant expression of a dominant negative form of FADD or the C terminus of ABCA1 in the human hepatoma cell line HepG2 markedly reduced the transfer of phospholipids to apoA-I. This indicates that the binding of additional proteins, one of them being full-length FADD, is required for ABCA1 function. The association of FADD with ABCA1 provides an unexpected link between high density lipoprotein metabolism and an adaptor molecule mainly described in death receptor signal transduction.


Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/fisiologia , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/química , Apolipoproteína A-I/metabolismo , Apoptose , Proteínas de Transporte/química , Células Cultivadas , Proteína de Domínio de Morte Associada a Fas , Humanos , Lipoproteínas HDL/metabolismo , Fosfatidilserinas/metabolismo , Fosfolipídeos/metabolismo , Transdução de Sinais
8.
Biochem Biophys Res Commun ; 293(2): 759-65, 2002 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-12054535

RESUMO

Recent work identified ABCA1 as the major regulator of plasma HDL-cholesterol responsible for the removal of excess choline-phospholipids and cholesterol from peripheral cells and tissues. ABCA1 function may depend on the association with heteromeric proteins and to identify these candidates a human liver yeast two-hybrid library was screened with the carboxyterminal 144 amino acids of ABCA1. Beta2-syntrophin was found to interact with ABCA1 and the C-terminal five amino acids of ABCA1 proned to represent a perfect tail for binding to syntrophin PDZ domains. Immunoprecipitation further confirmed the association of ABCA1 and beta2-syntrophin and in addition utrophin, known to couple beta2-syntrophin and its PDZ ligands to the F-actin cytoskeleton, was identified as a constituent of this complex. ABCA1 in the plasmamembrane of human macrophages was found to be partially associated with Lubrol rafts and effluxed choline-phospholipids involve these microdomains. Beta2-syntrophin does not colocalize in these rafts indicating that beta2-syntrophin may participate in the retaining of ABCA1 in cytoplasmic vesicles and for the targeting of ABCA1 to plasmamembrane microdomains when ABCA1 is released from beta2-syntrophin.


Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas de Membrana/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/análise , Adulto , Sequência de Aminoácidos , Membrana Celular/química , Células Cultivadas , Vesículas Citoplasmáticas/química , Proteínas Associadas à Distrofina , Fibroblastos/metabolismo , Humanos , Substâncias Macromoleculares , Pessoa de Meia-Idade , Dados de Sequência Molecular , Testes de Precipitina , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Técnicas do Sistema de Duplo-Híbrido , Utrofina
9.
Genomics ; 79(5): 693-702, 2002 May.
Artigo em Inglês | MEDLINE | ID: mdl-11991719

RESUMO

Apolipoprotein A-I (apoA-I) is the major apolipoprotein of high-density lipoproteins (HDL) and has an important role in the regulation of the stability, lipid transport, and metabolism of HDL particles. To identify novel proteins that are involved in HDL metabolism, we used mature apoA-I (amino acids 25-267) as a bait for the screening of a human liver two-hybrid cDNA library. Among the identified genes, several encoded known proteins, including serum amyloid A(2a) (SAA(2a)), apoC-I, and phosphodiesterase HCAM1 (PDE1A), found to interact with apoA-I. In addition, we have cloned a novel 29 kDa apoA-I interacting protein, which we named AI-BP (apoA-I binding protein). The AI-BP encoding gene, APOA1BP, which is located on chromosome 1q21, is composed of six exons and five introns and spans 2.5 kb. Northern blot analysis demonstrated ubiquitous expression of the APOA1BP mRNA with the highest expression in kidney, heart, liver, thyroid gland, adrenal gland, and testis. AI-BP protein is not detectable in serum of healthy probands, but serum samples of patients with septic syndromes may contain elevated levels of AI-BP. Significant amounts of AI-BP protein are found in cerebrospinal fluid and urine of healthy probands. The stimulation of cells derived from the kidney proximal tubules with apoA-I or HDL induces a concentration-dependent secretion of AI-BP indicating an important role for AI-BP, in the renal tubular degradation or resorption of apoA-I.


Assuntos
Apolipoproteína A-I/farmacologia , Proteínas de Transporte/genética , Túbulos Renais Proximais/efeitos dos fármacos , Lipoproteínas HDL/farmacologia , Proteínas da Gravidez , Sequência de Aminoácidos , Animais , Apolipoproteína A-I/genética , Apolipoproteína A-I/metabolismo , Sequência de Bases , Células CACO-2 , Proteínas de Transporte/metabolismo , Linhagem Celular , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Relação Dose-Resposta a Droga , Expressão Gênica , Genes/genética , Humanos , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/metabolismo , Camundongos , Dados de Sequência Molecular , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Racemases e Epimerases , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Células Tumorais Cultivadas , Técnicas do Sistema de Duplo-Híbrido
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