RESUMO
Apolipoprotein (apo) E4 isoform, a major risk factor for Alzheimer disease (AD), is more susceptible to proteolysis than apoE2 and apoE3 isoforms. ApoE4 fragments have been found in AD patients' brain. In the present study, we examined the effect of full-length apoE4 and apoE4 fragments apoE4[Δ(186-299)] and apoE4[Δ(166-299)] on inflammation in human neuroblastoma SK-N-SH and human astrocytoma SW-1783 cells. Western blot and zymography analysis showed that treatment of SK-N-SH cells with apoE4[Δ(186-299)], but not full-length apoE4 or the shorter apoE4[Δ(166-299)] fragment, leads to increased extracellular levels of matrix metalloproteinase 9 (MMP9) and tissue inhibitor of metalloproteinase 1 (TIMP1). Real-time PCR showed that interleukin (IL)-1ß gene expression is also increased in SK-N-SH cells treated with apoE4[Δ(186-299)]. Treatment of SK-N-SH cells with IL-1ß leads to increased MMP9 and TIMP1 extracellular levels, suggesting that the induction of IL-1ß may be the mechanism by which apoE4[Δ(186-299)] regulates MMP9 and TIMP1 levels in these cells. In contrast to SK-N-SH cells, treatment of SW-1783 cells with apoE4[Δ(186-299)], and to a lesser extent with apoE4, leads to increased TIMP1 extracellular levels without affecting MMP9 levels. Additionally, apoE4[Δ(186-299)] leads to decreased IL-10 gene expression in SK-N-SH cells, whereas both apoE4 and apoE4[Δ(186-299)] lead to decreased TNFα gene expression without affecting IL-1ß and IL-10 gene expression in SW-1783 cells. Overall, our findings indicate that a specific apoE4 fragment (apoE4[Δ(186-299)]), with molecular mass similar that of apoE4 fragments detected in AD patients' brain, can influence the level of inflammatory molecules in brain cell lines. It is possible that these phenomena contribute to AD pathogenesis.
Assuntos
Apolipoproteína E4/farmacologia , Encéfalo/efeitos dos fármacos , Citocinas/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Doença de Alzheimer/metabolismo , Apolipoproteína E4/metabolismo , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Western Blotting , Encéfalo/metabolismo , Linhagem Celular Tumoral , Citocinas/efeitos dos fármacos , Humanos , Inflamação/metabolismo , Metaloproteinase 9 da Matriz/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Inibidor Tecidual de Metaloproteinase-1/efeitos dos fármacos , TransfecçãoRESUMO
OBJECTIVES: To determine whether mutations in APOA1 affect levels of high-density lipoprotein (HDL) cholesterol and to predict risk of ischaemic heart disease (IHD) and total mortality in the general population. BACKGROUND: Epidemiologically, risk of IHD is inversely related to HDL cholesterol levels. Mutations in apolipoprotein (apo) A-I, the major protein constituent of HDL, might be associated with low HDL cholesterol and predispose to IHD and early death. DESIGN: We resequenced APOA1 in 190 individuals and examined the effect of mutations on HDL cholesterol, risk of IHD, myocardial infarction (MI) and mortality in 10 440 individuals in the prospective Copenhagen City Heart Study followed for 31 years. Results were validated in an independent case-control study (n = 16 035). Additionally, we determined plasma ratios of mutant to wildtype (WT) apoA-I in human heterozygotes and functional effects of mutations in adenovirus-transfected mice. RESULTS: We identified a new mutation, A164S (1 : 500 in the general population), which predicted hazard ratios for IHD, MI and total mortality of 3.2 [95% confidence interval (CI): 1.6-6.5], 5.5 (95% CI: 2.6-11.7) and 2.5 (95% CI: 1.3-4.8), respectively, in heterozygotes compared with noncarriers. Mean reduction in survival time in heterozygotes was 10 years (P < 0.0001). Results for IHD and MI were confirmed in the case-control study. Furthermore, the ratio of mutant S164 to WT A164 apoA-I in plasma of heterozygotes was reduced. In addition, A164S heterozygotes had normal plasma lipid and lipoprotein levels, including HDL cholesterol and apoA-I, and this finding was confirmed in adenovirus-transfected mice. CONCLUSIONS: A164S is the first mutation in APOA1 to be described that predicts an increased risk of IHD, MI and total mortality without low HDL cholesterol levels.
Assuntos
Apolipoproteína A-I/genética , Lipoproteínas HDL/sangue , Mutação/genética , Isquemia Miocárdica/sangue , Isquemia Miocárdica/genética , Adulto , Idoso , Animais , Estudos de Casos e Controles , Dinamarca/epidemiologia , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Isquemia Miocárdica/mortalidade , Fatores de Risco , Análise de Sequência de DNA , Análise de SobrevidaRESUMO
We have used a new ApoA-I transgenic mouse model to identify by global gene expression profiling, candidate genes that affect lipid and lipoprotein metabolism in response to fenofibrate treatment. Multilevel bioinformatical analysis and stringent selection criteria (2-fold change, 0% false discovery rate) identified 267 significantly changed genes involved in several molecular pathways. The fenofibrate-treated group did not have significantly altered levels of hepatic human APOA-I mRNA and plasma ApoA-I compared with the control group. However, the treatment increased cholesterol levels to 1.95-fold mainly due to the increase in high-density lipoprotein (HDL) cholesterol. The observed changes in HDL are associated with the upregulation of genes involved in phospholipid biosynthesis and lipid hydrolysis, as well as phospholipid transfer protein. Significant upregulation was observed in genes involved in fatty acid transport and beta-oxidation, but not in those of fatty acid and cholesterol biosynthesis, Krebs cycle and gluconeogenesis. Fenofibrate changed significantly the expression of seven transcription factors. The estrogen receptor-related gamma gene was upregulated 2.36-fold and had a significant positive correlation with genes of lipid and lipoprotein metabolism and mitochondrial functions, indicating an important role of this orphan receptor in mediating the fenofibrate-induced activation of a specific subset of its target genes.
Assuntos
Apolipoproteína A-I/genética , Fenofibrato/uso terapêutico , Metabolismo dos Lipídeos/genética , Receptores de Estrogênio/genética , Animais , HDL-Colesterol/sangue , Feminino , Fenofibrato/farmacologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Redes e Vias Metabólicas/efeitos dos fármacos , Redes e Vias Metabólicas/genética , Camundongos , Camundongos Transgênicos , Análise Serial de Proteínas , RNA Mensageiro/metabolismo , Fatores de Transcrição/genética , Regulação para CimaRESUMO
Apolipoproteins, such as apolipoprotein A-I (apoA-I), can stimulate cholesterol efflux from cells expressing the ATP binding cassette transporter A1 (ABCA1). The nature of the molecular interaction between these cholesterol acceptors and ABCA1 is controversial, and models suggesting a direct protein-protein interaction or indirect association have been proposed. To explore this issue, we performed competition binding and chemical cross-linking assays using six amphipathic plasma proteins and an 18 amino acid amphipathic helical peptide. All seven proteins stimulated lipid efflux and inhibited the cross-linking of apoA-I to ABCA1. Cross-linking of apoA-I to ABCA1 was saturable and occurred at high affinity (Kd of 7.0 +/- 1.9 nM), as was cross-linking of apoA-II. After binding to ABCA1, apoA-I rapidly dissociated (half-life of 25 min) from the complex and was released back into the medium. A mutant form of ABCA1 (W590S) that avidly binds apoA-I but fails to promote cholesterol efflux released apoA-I with similar kinetics but without transfer of cholesterol to apoA-I. Thus, a high-affinity, saturable, protein-protein interaction occurs between ABCA1 and all of its amphipathic protein ligands. Dissociation of the complex leads to the cellular release of cholesterol and the apolipoprotein. However, dissociation is not dependent on cholesterol transfer, which is a clearly separable event, distinguishable by ABCA1 mutants.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Apolipoproteínas/metabolismo , Colesterol/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Adenoviridae , Transporte Biológico/fisiologia , Linhagem Celular Transformada , Membrana Celular/metabolismo , Humanos , Metabolismo dos Lipídeos , Substâncias Macromoleculares , Mutação , Peptídeos/metabolismo , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução GenéticaRESUMO
We have used adenovirus-mediated gene transfer in apoA-I-deficient (A-I-/-) mice to probe the in vivo assembly and metabolism of HDL using apoA-I variants, focusing primarily on the role of the C-terminal 32 amino acids (helices 9-10). Lipid, lipoprotein, and apoA-I analyses showed that plasma levels of apoA-I and HDL of the mutants were 40-88% lower than that of wild type (WT) human apoA-I despite comparable levels of expression in the liver. WT apoA-I and mutant 1 (P165A, E172A) formed spherical particles with the size and density of HDL2 and HDL3. Mutant 2 (E234A, E235A, K238A, K239A) generated spherical particles with density between HDL2 and HDL3. Mutant 3 (L211V, L214V, L218V, L219V) and mutant 4 (L222K, F225K, F229K), which have substitutions of hydrophobic residues in the C-terminus, generated discoidal HDL particles indicating a defect in their conversion to mature spherical HDL. Significant amounts of mutant 4 and mutant 5 (truncated at residue 219) were found in the lipid poor fractions after ultracentrifugation of the plasma (18 and 35%, respectively, of total apoA-I). These findings suggest that hydrophobic residues in and/or between helices 9 and 10 are important for the maturation of HDL in vivo.
Assuntos
Apolipoproteína A-I/metabolismo , Lipoproteínas HDL/metabolismo , Fígado/metabolismo , Adenoviridae/genética , Animais , Apolipoproteína A-I/sangue , Apolipoproteína A-I/deficiência , Apolipoproteína A-I/genética , HDL-Colesterol/sangue , Dimiristoilfosfatidilcolina/metabolismo , Deleção de Genes , Técnicas de Transferência de Genes , Vetores Genéticos , Humanos , Lipídeos/sangue , Lipoproteínas HDL/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Fosfatidilcolina-Esterol O-Aciltransferase/metabolismo , Estrutura Secundária de Proteína , RNA Mensageiro/metabolismoAssuntos
Apolipoproteínas E/metabolismo , Hipertrigliceridemia/metabolismo , Adenoviridae/genética , Animais , Apolipoproteína E4 , Apolipoproteínas E/química , Apolipoproteínas E/genética , Deleção de Genes , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Humanos , Hipertrigliceridemia/induzido quimicamente , Lipoproteínas VLDL/metabolismo , Camundongos , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Relação Estrutura-AtividadeRESUMO
Apolipoprotein E (apoE) promotes receptor-mediated catabolism of apoE-containing lipoprotein remnants. Impairments in remnant clearance are associated with type III hyperlipoproteinemia and premature atherosclerosis. In humans, apoE plasma levels correlate with plasma triglyceride levels, suggesting that excess apoE may also affect plasma triglyceride levels. We have used adenovirus-mediated gene transfer in mice to map the domains of apoE required for cholesterol and triglyceride clearance, in vivo. Adenovirus expressing apoE3 and apoE4 at doses of (1-2) x 10(9) pfu increased plasma cholesterol and triglyceride levels in normal C57BL6 mice and failed to normalize the high cholesterol levels of apoE-deficient mice due to induction of hypertriglyceridemia. In contrast, an adenovirus expressing the truncated apoE 1-185 form normalized the cholesterol levels of E(-)(/)(-) mice and did not cause hypertriglyceridemia. Northern blot analysis of hepatic RNA from mice expressing the full-length and the truncated apoE forms showed comparable steady-state apoE mRNA levels of the full-length apoE forms that cause hyperlipidemia and the truncated apoE forms that do not cause hyperlipidemia. The findings suggest that the amino-terminal residues 1-185 of apoE are sufficient for the clearance of apoE-containing lipoprotein remnants by the liver, whereas domains of the carboxy-terminal one-third of apoE are required for apoE-induced hyperlipidemia.
Assuntos
Apolipoproteínas E/fisiologia , Hiperlipidemias/genética , Lipoproteínas/metabolismo , Fragmentos de Peptídeos/fisiologia , Adenoviridae/genética , Animais , Apolipoproteína E3 , Apolipoproteína E4 , Apolipoproteínas E/biossíntese , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Transporte Biológico Ativo/genética , Cromatografia Líquida de Alta Pressão , Feminino , Deleção de Genes , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Hipercolesterolemia/sangue , Hipercolesterolemia/etiologia , Hipercolesterolemia/genética , Hiperlipidemias/sangue , Hiperlipidemias/etiologia , Hipertrigliceridemia/sangue , Hipertrigliceridemia/etiologia , Hipertrigliceridemia/genética , Lipoproteínas/sangue , Lipoproteínas VLDL/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fragmentos de Peptídeos/genética , Estrutura Terciária de Proteína/genética , RNA Mensageiro/metabolismo , Triglicerídeos/metabolismo , Células Tumorais CultivadasRESUMO
Apolipoprotein (apo) E has been implicated in cholesterol and triglyceride homeostasis in humans. At physiological concentration apoE promotes efficient clearance of apoE-containing lipoprotein remnants. However, high apoE plasma levels correlate with high plasma triglyceride levels. We have used adenovirus-mediated gene transfer in apoE-deficient mice (E(-)/-) to define the domains of apoE required for cholesterol and triglyceride homeostasis in vivo. A dose of 2 x 10(9) plaque-forming units of apoE4-expressing adenovirus reduced slightly the cholesterol levels of E(-)/- mice and resulted in severe hypertriglyceridemia, due to accumulation of cholesterol and triglyceride-rich very low density lipoprotein particles in plasma. In contrast, the truncated form apoE4-202 resulted in a 90% reduction in the plasma cholesterol levels but did not alter plasma triglyceride levels in the E(-)/- mice. ApoE secretion by cell cultures, as well as the steady-state hepatic mRNA levels in individual mice expressing apoE4 or apoE4-202, were similar. In contrast, very low density lipoprotein-triglyceride secretion in mice expressing apoE4, but not apoE4-202, was increased 10-fold, as compared with mice infected with a control adenovirus. The findings suggest that the amino-terminal 1-202 region of apoE4 contains the domains required for the in vivo clearance of lipoprotein remnants. Furthermore, the carboxyl-terminal 203-299 residues of apoE promote hepatic very low density lipoprotein-triglyceride secretion and contribute to apoE-induced hypertriglyceridemia.
Assuntos
Apolipoproteínas E/metabolismo , Colesterol/metabolismo , Homeostase , Triglicerídeos/metabolismo , Adenoviridae/genética , Animais , Apolipoproteína E4 , Apolipoproteínas E/sangue , Apolipoproteínas E/química , Apolipoproteínas E/genética , Sequência de Bases , Colesterol/sangue , Cromatografia Líquida , Primers do DNA , Feminino , Humanos , Fígado/metabolismo , Camundongos , Camundongos Knockout , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Triglicerídeos/sangue , Células Tumorais CultivadasRESUMO
To probe the secondary structure of the C-terminus (residues 165-243) of lipid-free human apolipoprotein A-I (apoA-I) and its role in protein stability, recombinant wild-type and seven site-specific mutants have been produced in C127 cells, purified, and studied by circular dichroism and fluorescence spectroscopy. A double substitution (G185P, G186P) increases the protein stability without altering the secondary structure, suggesting that G185 and G186 are located in a loop/disordered region. A triple substitution (L222K, F225K, F229K) leads to a small increase in the alpha-helical content and stability, indicating that L222, F225, and F229 are not involved in stabilizing hydrophobic core contacts. The C-terminal truncation Delta(209-243) does not change the alpha-helical content but reduces the protein stability. Truncation of a larger segment, Delta(185-243), does not affect the secondary structure or stability. In contrast, an intermediate truncation, Delta(198-243), leads to a significant reduction in the alpha-helical content, stability, and unfolding cooperativity. The internal 11-mer deletion Delta(187-197) has no significant effect on the conformation or stability, whereas another internal 11-mer deletion, Delta(165-175), dramatically disrupts and destabilizes the protein conformation, suggesting that the presence of residues 165-175 is crucial for proper apoA-I folding. Overall, the findings suggest the presence of stable helical structure in the C-terminal region 165-243 of lipid-free apoA-I and the involvement of segment 209-243 in stabilizing interactions in the molecule. The effect of the substitution (G185P, G186P) on the exposure of tryptophans located in the N-terminal half suggests an apoA-I tertiary conformation with the C-terminus located close to the N-terminus.
Assuntos
Apolipoproteína A-I/química , Apolipoproteína A-I/genética , Mutagênese Sítio-Dirigida , Substituição de Aminoácidos/genética , Animais , Apolipoproteína A-I/metabolismo , Dicroísmo Circular , Guanidina , Humanos , Metabolismo dos Lipídeos , Camundongos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Espectrometria de Fluorescência , Temperatura , Células Tumorais CultivadasRESUMO
Cotransfection of HepG2 cells with SMADs established that SMAD3 and SMAD3-SMAD4 transactivated (15-70-fold) the -890/+24 apoCIII promoter and shorter promoter segments, whereas cotransfection with a dominant negative SMAD4 mutant repressed the apoCIII promoter activity by 50%, suggesting that SMAD proteins participate in apoCIII gene regulation. Transactivation required the presence of a hormone response element, despite the fact that SMADs could not bind directly to it. Cotransfection of SMAD3-SMAD4 along with hepatocyte nuclear factor-4 resulted in a strong synergistic transactivation of the -890/+24 apoCIII promoter, proximal promoter segments, or synthetic promoters containing either the apoCIII enhancer or the proximal apoCIII hormone response element. Inhibition of endogenous hepatocyte nuclear factor-4 synthesis by an antisense ribozyme construct reduced the constitutive activity of the apoCIII promoter in HepG2 cells to 10% and abolished the SMAD-mediated transactivation. Co-immunoprecipitation and GST pull-down assays provided evidence for physical interactions between SMAD3, SMAD4, and hepatic nuclear factor-4. Our findings indicate that transforming growth factor beta and its signal transducer SMAD proteins can modulate gene transcription by novel mechanisms that involve their physical and functional interaction with hepatocyte nuclear factor-4, suggesting that SMAD proteins may play an important role in apolipoprotein gene expression and lipoprotein metabolism.
Assuntos
Apolipoproteínas C/genética , Proteínas de Ligação a DNA/fisiologia , Fosfoproteínas/fisiologia , Regiões Promotoras Genéticas , Transativadores/fisiologia , Fatores de Transcrição/fisiologia , Ativação Transcricional , Animais , Apolipoproteína C-III , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Sítios de Ligação , Células COS , Fator 4 Nuclear de Hepatócito , Humanos , Elementos de Resposta , Proteína Smad3 , Proteína Smad4 , Fator de Crescimento Transformador beta/fisiologia , Células Tumorais CultivadasRESUMO
The binding of apoA-I-containing ligands to the HDL receptor scavenger receptor class B type I (SR-BI) was characterized using two different assays. The first employed conventional binding or competition assays with (125)I-labeled ligands. The second is a new nonradioactive ligand binding assay, in which the receptor-associated ligand is detected by quantitative immunoblotting ("immunoreceptor assay"). Using both methods, we observed that the K(d) value for spherical HDL (density = 1.1-1.13 g/ml) was approximately 16 microgram of protein/ml, while the values for discoidal reconstituted HDL (rHDL) containing proapoA-I or plasma apoA-I were substantially lower (approximately 0.4-5 microgram of protein/ml). We also observed reduced affinity and/or competition for spherical (125)I-HDL cell association by higher relative to lower density HDL and very poor competition by lipid-free apoA-I and pre-beta-1 HDL. Deletion of either 58 carboxyl-terminal or 59 amino-terminal residues from apoA-I, relative to full-length control apoA-I, resulted in little or no change in the affinity of corresponding rHDL particles. However, rHDL particles containing a double mutant lacking both terminal domains competed poorly with spherical (125)I-HDL for binding to SR-BI. These findings suggest an important role for apoA-I and its conformation/organization within particles in mediating HDL binding to SR-BI and indicate that the NH(2) and COOH termini of apoA-I directly or indirectly contribute independently to binding to SR-BI.
Assuntos
Apolipoproteína A-I/metabolismo , Antígenos CD36/metabolismo , Lipoproteínas HDL/metabolismo , Proteínas de Membrana , Receptores Imunológicos , Animais , Apolipoproteína A-I/química , Apolipoproteína A-I/genética , Sítios de Ligação , Antígenos CD36/química , Éxons , Humanos , Radioisótopos do Iodo , Cinética , Ligantes , Camundongos , Mutagênese Sítio-Dirigida , Ensaio Radioligante , Receptores de Lipoproteínas/metabolismo , Receptores Depuradores , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Receptores Depuradores Classe B , Deleção de Sequência , Transfecção , Células Tumorais CultivadasRESUMO
Recent studies have shown that at physiological conditions (pH 7.6, 37 degrees C), the reactivity of recombinant apoE isoforms secreted by mammalian cells toward amyloid peptide beta (Abeta40) follows the order apoE2 > apoE3 > apoE4 for the apoE monomer and apoE2 > apoE3 for apoE dimer that is formed via that intramolecular disulfide bridges. Different Abeta binding properties have been reported for the plasma-derived apoE and commercially available apoE preparations that differ from the native apoE forms in the degree of their O-glycosylation. To define structural elements of apoE involved in the interaction with Abeta, we have introduced point mutations as well as amino- and carboxy-terminal deletions in the apoE structure. The mutant apoE forms were expressed transiently using the Semliki Forest Virus system, and the culture medium was utilized to study the reactivity of the mutated proteins with Abeta 40. This analysis showed that a mutation in the O-glycosylation site of apoE2 (Thr194-Ala) did not affect the SDS-stable binding of apoE to Abeta. In contrast, introduction of cysteine at position 158 of apoE4 (Arg112, Cys158) increased the SDS-stable binding of apoE to Abeta to the levels similar to those observed in apoE2. Similar analysis showed that apoE truncated at residues 259, 249, 239, and 229 retains the SDS-stable binding to Abeta40, whereas apoE truncated at residues 185 and 165 does not bind to Abeta. The deletion of aminoterminal residues 2-19 reduced the SDS-stable binding of apoE2 to Abeta and deletion of residues 2-81 abolished binding to Abeta. It is also noteworthy that the (Delta2-81) apoE mutant exists predominantly as a dimer, suggesting that removal of residues 2-81 promoted dimerization of apoE. These findings suggest that the amino- and carboxy-terminal residues of apoE are required for SDS-stable binding of apoE to Abeta and that the presence of at least one cysteine contributes to the efficient Abeta binding.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Apolipoproteínas E/metabolismo , Cisteína/metabolismo , Fragmentos de Peptídeos/metabolismo , Treonina/metabolismo , Substituição de Aminoácidos/genética , Peptídeos beta-Amiloides/química , Animais , Apolipoproteínas E/química , Apolipoproteínas E/genética , Arginina/genética , Sítios de Ligação/genética , Configuração de Carboidratos , Linhagem Celular , Cricetinae , Cisteína/química , Cisteína/genética , Eletroforese em Gel de Poliacrilamida , Glicosilação , Humanos , Rim/citologia , Substâncias Macromoleculares , Fragmentos de Peptídeos/química , Mutação Puntual , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Treonina/química , Treonina/genéticaRESUMO
DNase I footprinting of the apoA-II promoter using sterol regulatory element binding protein-2 [(SREBP-2 (1-458)] expressed in bacteria identified four protected regions, designated AIIAB (-64 to -48), AIICD (-178 to -154), AIIDE (-352 to -332) and AIIK (-760 to -743), which bind SREBP-2 and contain either palindromic or direct repeat motifs. Potassium permanganate and dimethyl sulfate interference experiments using the AIIAB region as probe showed that the nucleotides of a decameric palindromic repeat RTCAMVTGMY and two 5' T residues participate in DNA-protein interactions. SREBP-2 transactivated the intact (-911/+29) apoA-II promoter 1.7-fold and truncated apoA-II promoter segments which contain one, two or three SREBP-2 sites 11- to 17-fold in HepG2 cells. Transactivation of a promoter construct containing the binding site AIIAB and the apoA-II enhancer, which includes the binding site AIIK, was abolished by mutations in element AIIAB. An SREBP-2 mutant defective in DNA binding caused a dose-dependent repression of the apoA-II promoter activity. Repression was also caused by an SREBP-2 mutant which lacks the N-terminal activation domain (residues 1-93) but binds normally to its cognate sites. In contrast, a double SREBP-2 mutant which lacks both the DNA binding and the activation domains has no effect on the apoA-II promoter activity. Overall, the findings suggest that SREBP-2 can transactivate the apoA-II promoter by binding to multiple sites. Furthermore, the repression caused by the DNA binding deficient mutants results from squelching of positive activator(s) which appear to recognize the activation domain of SREBP-2.
Assuntos
Apolipoproteína A-II/genética , Proteínas de Ligação a DNA/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Ativação Transcricional , Animais , Sequência de Bases , Sítios de Ligação , DNA Complementar , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Humanos , Dados de Sequência Molecular , Proteína de Ligação a Elemento Regulador de Esterol 2 , Fatores de Transcrição/genética , Células Tumorais CultivadasRESUMO
Human apolipoprotein CIII (apoCIII) is a major determinant of plasma triglyceride metabolism. The regulatory elements that control both hepatic and intestinal transcription of the human apoCIII gene are localized between nucleotides -792 and -25 of the apoCIII promoter. Elements important for apoCIII promoter activity are three hormone response elements (HREs) and three SP1-binding sites. Orphan members of the nuclear hormone receptor superfamily can bind the HREs and strongly enhance or repress apoCIII promoter activity. In the present study we have investigated the ability of ligand-dependent nuclear hormone receptors to bind and modulate the human apoCIII promoter activity. Experiments using DNA binding and competition assays showed that the proximal element B (-87/-72) binds strongly, in addition to HNF-4, ARP-1, EAR-2, and EAR-3, heterodimers of RXRalpha with RARalpha, and less efficiently, homodimers of RARalpha and heterodimers of RXRalpha with T3Rbeta or PPARalpha. Element G (-669/-648), which was shown previously to bind ARP-1 and EAR-3 but not HNF-4, binds strongly heterodimers of RXRalpha with either RARalpha or T3Rbeta. Finally element I4 (-732/-712), which was shown to bind HNF-4, also binds strongly ARP-1 and EAR-3, as well as RXRalpha/RARalpha heterodimers and less efficiently, RXRalpha/T3Rbeta heterodimers. Methylation interference experiments have identified the protein-DNA interactions between different nuclear receptors and the respective HREs on the apoCIII promoter. RXRalpha/RARalpha heterodimers and HNF-4 homodimers bind to DR-1 motifs on elements B and I4, respectively. RXRalpha/T3Rbeta heterodimers and ARP-1 bind to DR-5 and DR-0 motifs respectively on element G. Cotransfection experiments in HepG2 cells showed that RXRalpha or a combination of RXRalpha and RARalpha increased the apoCIII promoter activity approximately 2-fold in the presence of the ligands 9-cis or all-trans RA. In contrast, a combination of RXRalpha and T3Rbeta transactivated the apoCIII promoter 1.5-fold in the presence of 9-cis RA but it repressed the apoCIII promoter activity in the presence of T3. Mutations in the HREs of elements B, G, or I4 or in the SP1-binding site of element H, which abolished the binding of nuclear hormone receptors or SP1 to their cognate site, reduced the promoter strength and exhibited different responses to the ligand-dependent nuclear receptors. The findings suggest that modulation of the apoCIII promoter activity by orphan and ligand-dependent nuclear receptors involves complex interactions among nuclear receptors, SP1 and possibly other factors bound to the enhancer and the proximal promoter region.
Assuntos
Apolipoproteínas C/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Regiões Promotoras Genéticas , Receptores Citoplasmáticos e Nucleares/fisiologia , Receptores de Esteroides , Animais , Apolipoproteína C-III , Apolipoproteínas C/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Células COS , Fator II de Transcrição COUP , Fatores de Transcrição COUP , Proteínas de Ligação a DNA/genética , Dimerização , Fator 4 Nuclear de Hepatócito , Humanos , Ligantes , Fígado , Fosfoproteínas/metabolismo , Ratos , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores do Ácido Retinoico/metabolismo , Receptor alfa de Ácido Retinoico , Receptores X de Retinoides , Fatores de Transcrição/metabolismo , Células Tumorais CultivadasRESUMO
It was shown previously that cytokines such as tumor necrosis factor-alpha that stimulate signal transduction pathways involving transcription factors ATF-2 and Jun repress apoCIII promoter activity in HepG2 cells. In the present study, DNase I footprinting analysis established that ATF-2 protected three regions in the apoCIII promoter. One region (-747/-726) present in the apoCIII enhancer is within the previously identified footprint I and has overlapping boundaries with the binding sites of Sp1 (-764/-742) and HNF-4 (-736/-714). The other two regions represent new footprints and have been designated D/E (-219/-199) and B/C (-102/-75). The B/C region overlaps with the previously identified footprint B which contains an HNF-4 binding site (-87/-63). Cotransfection experiments in HepG2 cells showed that ATF-2 transactivated the -890/+24 apoCIII promoter 1.6-fold. In addition, mutations in the proximal D/E (-219/-199) and distal I (-747/-726) ATF-2-binding sites reduced the apoCIII promoter strength to 33 and 9% of control, respectively, indicating that ATF-2 is a positive regulator of apoCIII gene transcription. Cotransfections with ATF-2 and HNF-4 expression plasmids resulted in additive transactivation of the apoCIII promoter. Furthermore, apoCIII promoter constructs bearing mutations in the D/E and I ATF-2 binding sites were efficiently transactivated by HNF-4, suggesting that these two factors contribute independently to the apoCIII promoter strength. Members of the Jun family (c-Jun, JunB, and JunD) caused a dose-dependent inhibition of the -890/+24 apoCIII promoter activity. A synthetic promoter containing the apoCIII enhancer in front of the minimal AdML promoter was also repressed by Jun. In contrast, apoCIII promoter segments lacking the enhancer region were transactivated by Jun. The findings suggest that homodimers of Jun or heterodimers of Jun with other AP-1 subunits could be responsible for the observed repression by interfering with the function(s) of the apoCIII enhancer. Repression by Jun could be reversed in the presence of ATF-2 and HNF-4, suggesting that ATF2 and possibly Jun/ATF-2 heterodimers exert a positive effect on apoCIII gene transcription, as opposed to Jun homodimers or heterodimers with other AP-1 members. These findings suggest a role for members of the Jun family and ATF-2 that participate in signal transduction pathways in basal or induced apoCIII promoter activity in cells of hepatic origin.
Assuntos
Apolipoproteínas C/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-jun/fisiologia , Proteínas Repressoras/fisiologia , Fatores de Transcrição/fisiologia , Ativação Transcricional , Fator 2 Ativador da Transcrição , Apolipoproteína C-III , Apolipoproteínas C/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Carcinoma Hepatocelular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Regulação para Baixo , Elementos Facilitadores Genéticos/fisiologia , Humanos , Dados de Sequência Molecular , Fatores de Transcrição/genética , Células Tumorais CultivadasRESUMO
The regulatory elements CIIC (-159/-116) and CIIB (-102/-81) of the apolipoprotein CII (apoCII) promoter have distinct specificities for orphan nuclear receptors (Vorgia, P., Zannis, V. I., and Kardassis, D. (1998) J. Biol. Chem. 273, 4188-4199). In this communication we investigated the contribution of ligand-dependent and orphan nuclear receptors on the transcriptional regulation of the human apoCII gene. It was found that element CIIC in addition to ARP-1 and EAR-2 binds RXRalpha/T3Rbeta heterodimers strongly, whereas element CIIB binds hepatic nuclear factor 4 (HNF-4) exclusively. Binding is abolished by mutations that alter the HRE binding motifs. Transient cotransfection experiments showed that in the presence of T3, RXRalpha/T3Rbeta heterodimers transactivated the -205/+18 apoCII promoter 1.6- and 11-fold in HepG2 and COS-1 respectively. No transactivation was observed in the presence of 9-cis-retinoic acid. Transactivation requires the regulatory element CIIC, suggesting that this element contains a thyroid hormone response element. HNF-4 did not affect the apoCII promoter activity in HepG2 cells. However, mutations in the HNF-4 binding site on element CIIB and inhibition of HNF-4 synthesis in HepG2 cells by antisense HNF-4 constructs decreased the apoCII promoter activity to 25-40% of the control, indicating that HNF-4 is a positive regulator of the apoCII gene. ARP-1 repressed the -205/+18 but not the -104/+18 apoCII promoter activity in HepG2 cells, indicating that the repression depends on the regulatory element CIIC. In contrast, combination of ARP-1 and HNF-4 transactivated different apoCII promoter segments as well as a minimal adenovirus major late promoter driven by the regulatory element CIIB. Mutagenesis or deletion of elements CIIB or CIIC established that the observed transactivation requires DNA binding of one of the two factors and may result from HNF-4-ARP-1 interactions that elicit the transactivation functions of HNF-4. The combined data indicate that RXRalpha/T3Rbeta in the presence of T3 and HNF-4 can upregulate the apoCII promoter activity by binding to the regulatory elements CIIC and CIIB, respectively. In addition, ARP-1 can either have inhibitory or stimulatory effects on the apoCII promoter activity via different mechanisms.
Assuntos
Apolipoproteínas C/genética , Regiões Promotoras Genéticas , Receptores do Ácido Retinoico/metabolismo , Receptores de Esteroides , Receptores dos Hormônios Tireóideos/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Animais , Apolipoproteína C-II , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Células COS , Fator II de Transcrição COUP , Fatores de Transcrição COUP , Linhagem Celular , Primers do DNA , Proteínas de Ligação a DNA/metabolismo , Dimerização , Fator 4 Nuclear de Hepatócito , Humanos , Fosfoproteínas/metabolismo , Sequências Reguladoras de Ácido Nucleico , Receptores X de RetinoidesRESUMO
Footprinting analysis of the human apoCIII promoter identified a set of four proximal (A-D) and six distal (E-J) regulatory elements between nucleotides -792 and -25 [Ogami, K., et al. (1990) J. Biol. Chem. 265, 9808-9815]. The distal regulatory elements of the apoCIII gene increase by 10-fold the strength of the homologous as well as of heterologous proximal promoters. Required for such transcriptional enhancement is the presence of an intact hormone response element (HRE) on the proximal promoter which binds a variety of nuclear hormone receptors. To understand the mechanism of this transcriptional activation, we identified the nature and the importance of the factors which bind to the upstream regulatory elements of the apoCIII promoter by DNA binding, competition, supershift, and transient transfection assays. These analyses showed that the upstream apoCIII promoter contains multiple binding sites for the ubiquitous transcription factor SP1, which recognizes the regulatory elements F, H, and I. The regulatory element G represents a specialized HRE which is recognized by the orphan receptors ARP-1 and EAR-3 but not by HNF-4. A single activity designated CIII J1 binds to the regulatory element J. The same or a similar activity binds as a minor component to the regulatory elements F and I where SP1 is the predominant binding activity. Finally, a minor activity designated CIII 15 binds to the regulatory element I.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Apolipoproteínas C/genética , Fígado/metabolismo , Fosfoproteínas , Regiões Promotoras Genéticas , Fator de Transcrição Sp1/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Apolipoproteína C-III , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , DNA , Proteínas de Ligação a DNA/metabolismo , Fator 4 Nuclear de Hepatócito , Humanos , Dados de Sequência Molecular , Ligação Proteica , Células Tumorais CultivadasRESUMO
We have previously shown that transcription of the human apolipoprotein A-II (apoA-II) gene is controlled by a complex set of regulatory elements [Cardot et al. (1993) Biochemistry 32, 9080-9093]. We have also identified previously described, as well as new activities which bind to these elements and influence to varying degrees the transcription of the apoA-II gene. DNA binding and competition assays indicated that element D binds three new activities, designated AIID1, AIID2, and AIID4, as well as an activity related to C/EBP. Activities AIID1, AIID2, and AIID4 were purified and characterized further in order to determine their function on the transcriptional regulation of human apoA-II gene. SDS-PAGE analysis as well as photoaffinity cross-linking of the affinity-purified AIID2 showed that it consists of three proteins with molecular masses ranging between 54 and 63 kDa. The amino acid sequence of tryptic peptides obtained from AIID2 protein bands revealed that it is homologous to GABP, an Ets-related protein. Similar analysis showed that affinity-purified AIID4 has an apparent molecular mass of 130 kDa. AIID1 activity was purified partially; in addition to binding to the apoA-II promoter, AIID1 also binds to the regulatory element C of apoCIII and may play a role in the transcriptional regulation of both genes. Methylation interference of G residues and permanganate modification of T residues indicated that the binding sites of AIID2 and AIID4 were contiguous on element D. However, the binding site of AIID1 overlaps with the binding sites of both AIID2 and AIID4. This suggests that the binding of AIID1 and AIID2 or of AIID1 and AIID4 may be mutually exclusive, whereas AIID2 and AIID4 may bind simultaneously. Transcription from a minimal promoter containing elements AB, C, and D of apoA-II increased 1.5- to 1.6-fold when element D is deleted, as well as by promoter mutations which eliminated the binding of both AIID1 and/or AIID4 to element D, but permitted the binding of AIID2/GABP. The findings suggest that element D has a negative regulatory role on apoA-II gene transcription when it is occupied by protein AIID1 and/or AIID4. This negative effect is reversed when element D is occupied only by the regulatory factor AIID2/GABP.
Assuntos
Apolipoproteína A-II/genética , Proteínas de Ligação a DNA/química , Proteínas Nucleares/metabolismo , Oligonucleotídeos/síntese química , Proteínas Oncogênicas , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/química , Fatores de Transcrição/farmacologia , Sequência de Aminoácidos , Animais , Apolipoproteína A-II/química , Apolipoproteína A-II/metabolismo , Sequência de Bases , Sítios de Ligação , Ligação Competitiva , Cloranfenicol O-Acetiltransferase/metabolismo , Reagentes de Ligações Cruzadas , Eletroforese em Gel de Poliacrilamida , Fator de Transcrição de Proteínas de Ligação GA , Humanos , Fígado/metabolismo , Metilação , Camundongos , Dados de Sequência Molecular , Peso Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/isolamento & purificação , Oligonucleotídeos/química , Oligonucleotídeos/genética , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-ets , Ratos , Fatores de Transcrição/metabolismo , Transcrição Gênica/genéticaRESUMO
Transfected mouse mammary-derived cells (C127) expressing human apolipoprotein (apo) E (C127E) were used a) to determine whether the lipid-binding character of apoE is sufficient to promote its assembly with lipid to form lipoprotein-like particles when expressed by cells not normally expressing apolipoproteins; b) to characterize the secreted complexes in terms of morphology, size, and composition; and finally c) to determine whether apoE or apoA-I gene expression by these transfected cells has any effect on the levels and the profiles of the synthesized and secreted lipids. The findings of the present study demonstrate that: a) as determined by density gradient ultracentrifugation and gel filtration chromatography, about 20% of the secreted [35S]methionine-labeled apoE expressed by C127E cells is lipid-associated. b) Negative-stain electron microscopic analysis of the lipid-protein complexes recovered in the lipoprotein fractions (d less than 1.21 g/ml) revealed that approximately 13% of the total population of particles were discs (16 +/- 5 nm mean diameter and 4-6 nm thick), resembling nascent high density lipoproteins (HDL). The majority of the particles however (greater than 82%) appeared vesicular with varying diameters (48 +/- 40 nm mean diameter). The discoidal and the vesicular appearance of the particles secreted by C127E cells is consistent with the composition of lipids. These consisted mostly of surface lipids, phospholipids (45 +/- 18%), diacylglycerols (36 +/- 17%), and free cholesterol (17 +/- 7%) (by weight). c) Expression of apoE by C127E cells was associated with an increased release of [35S]methionine-labeled protein and [3H]glycerol-labeled lipid (3- to 5- and 4- to 8-fold, respectively) compared to nontransfected C127 cells. Expression of mutant apoE or normal apoA-I, however, was not associated with increased release of the major lipid classes compared to the parent C127 cells, strongly suggesting that this character of C127E cells is specific to apoE expression. The release of lipids by C127E cells could be reduced considerably by the addition of the metabolic inhibitors, colchicine or cycloheximide (10 and 1 microM, respectively), suggesting that lipid release by C127E cells is an active process requiring both protein synthesis and functional secretory mechanisms. Taken together the findings suggest that apoE expression by C127 cells promotes the formation of nascent discoidal lipoprotein-like particles and enhances the release of vesicular lipids, possibly by promoting shedding of cell plasma membrane fragments.
Assuntos
Apolipoproteína A-I/metabolismo , Apolipoproteínas E/metabolismo , Metabolismo dos Lipídeos , Animais , Apolipoproteína A-I/biossíntese , Apolipoproteínas E/biossíntese , Fracionamento Celular , Linhagem Celular , Centrifugação com Gradiente de Concentração , Cromatografia em Gel , Células Clonais , Meios de Cultura/química , Humanos , Bicamadas Lipídicas , Substâncias Macromoleculares , Neoplasias Mamárias Experimentais , Camundongos , Transfecção , Células Tumorais CultivadasRESUMO
The apolipoprotein A-II (apoA-II) gene regulatory region -911 to +29 strongly promotes the transcription of the promotorless chloramphenicol acetyltransferase (CAT) gene in cells of hepatic (HepG2) and intestinal (CaCo2) origin but not in HeLa cells. Deletion of nucleotides -911 to -860 decreased the hepatic and intestinal transcription to 7% and 18% of control, respectively. Further progressive deletions extending to nucleotides -614, -440, -230, and -80 abolished both hepatic and intestinal transcription, indicating that the distal promoter region -911 to -614 contains regulatory elements that are essential for intestinal and hepatic transcription. An internal deletion of the -614 to -230 region decreased hepatic transcription 60% while it increased intestinal transcription 140% of control indicating that the elements which control hepatic and intestinal transcription of the apoA-II gene may be different within this regulatory region. DNase I footprinting analysis with rat liver nuclear extracts identified 14 protected regions: A, -40 to -33; B, -65 to -42; C, -126 to -110; D, -276 to -255; E, -377 to -364; F, -404 to -384; G, -468 to -455; H, -573 to -554; I, -706 to -680; J, -734 to -716; K, -760 to -743; L, -803 to -773; M, -853 to -829, and N, -903 to -879, as the DNA binding sites for nuclear factors. Five of the regions (B, C, G, H, and K) bind to heat-stable factors. DNA binding gel electrophoretic assays indicated that region N (-903 to -879), which is essential for efficient transcription, binds predominantly a nuclear activity designated AIIN3. This activity is present in cells of hepatic and intestinal origin but absent in HeLa cells. Similar analysis showed that region H (-573 to -554) binds to the liver-specific factor HNF1/LFB1. Deletion of this region decreased hepatic and intestinal transcription 80 and 64% of control, respectively, suggesting that HNF1/LFB1 or a related activity contributes to optimal transcription but is not essential for the tissue-specific expression of the human apoA-II gene.