RESUMO
Ie0 is the only gene of the baculovirus Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV) that is known to be spliced. In this study, cDNAs of ie0 were isolated, cloned, and sequenced. It was observed that IE0 contains 35 amino acids (aa) added to the N-terminus of IE1. In addition, it was found that the leader sequence of ie0 contains a 4-aa minicistron. To functionally characterize IE0, ie0 cDNAs were expressed under control of either the ie1 or the ie0 promoter. Unexpectedly, examination of ie0 translation products revealed that the predominant product from ie0 mRNAs was not IE0, but IE1. Mutation analysis showed that IE1 translation was preferentially initiated from either of two AUGs found in the first 15 nucleotides (nt) of the ie1 ORF that are internal to the ie0 ORF. It is unknown whether the internal translation initiation occurs via a leaky scanning mechanism or by an internal ribosomal entry site. Transactivation analysis with constructs that had point mutations in the ie1 AUGs and were translated only as IE0 revealed that OpMNPV IE0 is a 14- to 15-fold stronger transactivator than IE1. IE0 was also shown to be autoregulatory and to transactivate early genes in an enhancer-independent or -dependent manner. These results suggest that differential expression of baculovirus early genes can be obtained by coexpression of IE0 and IE1 in infected cells, which may permit subtle regulation of specific sets of viral genes.
Assuntos
Proteínas de Ligação a DNA , Proteínas Imediatamente Precoces/genética , Nucleopoliedrovírus/genética , Biossíntese de Proteínas , Transativadores/genética , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , DNA Bacteriano , Proteínas Imediatamente Precoces/biossíntese , Dados de Sequência Molecular , Mariposas , Transativadores/biossínteseRESUMO
This study presents a detailed analysis of the acidic N-terminal region of the Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV) transactivator IE1. The N-terminal region of IE1 is rich in acidic amino acids and has been hypothesized to be an acidic activation domain. Removal of the N-terminal 126 amino acids containing the acidic domain of IE1 resulted in complete loss of transactivation activity, indicating that this region is essential for transactivation. The OpMNPV acidic domain was replaced with the archetype acidic activation domain from VP16 and the acid-rich region of Autographa californica multicapsid NPV (AcMNPV) IE1. These chimeric constructs were fully capable of transactivation in transient assays. The chimeric OpMNPV IE1s containing the herpes simplex virus VP16 and AcMNPV IE1 acidic activation domains consistently transactivated a reporter gene to higher levels than the OpMNPV IE1 acidic activation domain. Transactivation by the chimeric constructs is enhanced synergistically when cotransfected with IE2 into Lymantria dispar and Spodoptera frugiperda cells. Both N- to C-terminal and C- to N-terminal deletions of the OpMNPV acidic activation domain were constructed to define functional domains within the OpMNPV IE1 acidic activation domain. At least two potential activation domains were identified. Within each of these domains, two core regions at amino acids 28-43 and amino acids 113-124 were identified that were similar to core regions of VP16 and GAL4, which contain predominately acidic and bulky hydrophobic amino acids.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas Imediatamente Precoces/química , Nucleopoliedrovírus/metabolismo , Transativadores/química , Sequência de Aminoácidos , Animais , Linhagem Celular , Proteínas de Ligação a DNA/genética , Proteína Vmw65 do Vírus do Herpes Simples/química , Proteína Vmw65 do Vírus do Herpes Simples/genética , Proteínas Imediatamente Precoces/genética , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/química , Alinhamento de Sequência , Spodoptera , Transativadores/genéticaRESUMO
Approximately 1% to 2% of persons in the general population are homozygous for a lipoprotein receptor-binding defective form of apoE (apoE2/2). However, only a small percentage (2% to 5%) of all apoE2/2 homozygotes develop type III hyperlipoproteinemia. Interaction with other genetic and environmental factors are required for the expression of this lipid abnormality. We sought to investigate the possible role of LPL gene mutations in the development of hyperlipoproteinemia in apoE2/2 homozygotes and in apoE2 heterozygotes. As a first step, we performed DNA sequence analysis of all 10 LPL coding exons in 2 patients with the apoE2/2 genotype who had type III hyperlipoproteinemia and identified a single missense mutation (Asn 291-->Ser) in exon 6 of the LPL gene. The mutation was then found in 5 of 18 patients with type III hyperlipoproteinemia who had the apoE2/2 genotype (allele frequency = 13.9%; P < or = 7.4 x 10(-5)) and 6 of 22 hyperlipidemic E2 heterozygous patients with the apoE3/2 and E4/2 genotype (allele frequency = 13.6%; P = 2.2 x 10(-5)). In contrast, this mutation was found in only 3 of 230 normolipidemic controls (allele frequency = 0.7%). In vitro mutagenesis studies revealed that the Asn 291-->Ser mutant LPL had approximately 60% of LPL catalytic activity and approximately 70% of specific activity compared with wild-type LPL. The heparin-binding affinity of the mutant LPL was not impaired. Our data suggest that the Asn 291-->Ser substitution is likely to be a significant predisposing factor contributing to the expression of different forms of hyperlipidemia when associated with other genetic factors such as the presence of apoE2.
Assuntos
Apolipoproteínas E/deficiência , Hiperlipidemias/genética , Hiperlipidemias/metabolismo , Lipase Lipoproteica/genética , Adolescente , Adulto , Idoso , Alelos , Apolipoproteína E3 , Sequência de Bases , Feminino , Técnicas de Transferência de Genes , Humanos , Lipase Lipoproteica/metabolismo , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação PuntualRESUMO
Lipoprotein lipase (LPL) is an enzyme responsible for the hydrolysis of triglyceride-rich circulating lipoproteins. Humans with complete defects in LPL activity present from infancy with failure to thrive, eruptive xanthomas, pancreatitis, and lactescent plasma. In addition, heterozygous carriers for this disorder may be at increased risk for the development of coronary artery disease. In view of a potential strategy for correcting complete or partial LPL deficiency, a 1.56-kb human LPL cDNA was inserted into a series of recombinant myeloproliferative sarcoma virus (MPSV)-based retroviral vectors under transcriptional control of the constitutive MPSV long terminal repeat (LTR). Stable gene transfer and enhanced expression of human LPL was observed at both the RNA and protein level in a variety of somatic cell types in vitro. Genetically modified cell populations included mouse NIH-3T3 fibroblasts and C2C12 myoblasts, primary human fibroblasts, and established human hematopoietic cell lines of erythroid (K562), myelocytic (HL60), and monocytic (U937,THP-1) type. The achieved levels of bioactive human LPL were found to vary widely between the different transduced cell lines, which may be critical to an approach to gene therapy. Transduced primary human fibroblasts yielded maximal elevation of LPL immunoreactive mass and activity of at least 24- and 50-fold, respectively, above constitutively expressed levels for this cell type. Human fibroblasts, therefore, appear to accommodate in vitro the complex processes readily leading to the maturation and secretion of bioactive human LPL and may serve as an effective cellular vehicle for LPL gene delivery and expression in human LPL deficiency.
Assuntos
Técnicas de Transferência de Genes , Vetores Genéticos , Lipase Lipoproteica/genética , Retroviridae/genética , Células 3T3 , Animais , Linhagem Celular , Regulação Enzimológica da Expressão Gênica , Humanos , Lipase Lipoproteica/metabolismo , CamundongosRESUMO
Lipoprotein lipase (LPL) interaction with membrane-associated polyanions is a critical component of normal catalytic function. Two strong candidate binding regions, rich in arginine and lysine residues, have been defined in the N-terminal domain (aa279-282 and aa292-304) that show homology to the heparin-binding consensus sequences -X-B-B-X-B-X- and -X-B-B-B-X-X-B-X-, respectively. Additional candidate regions appear in the C-terminal domain, (residues 390-393), which are homologous to the thrombospondin heparin-binding repeat, and the positively charged terminal decapeptide (residues 439-448). To determine residues and domains critical to heparin binding, we have generated different LPL mutants that have alanine substitutions of single arginine and lysine residues and sequence interchanges with the homologous hepatic (HL) and pancreatic (PL) lipases. The mutant cDNAs were expressed in COS-1 cells and catalytically active mutants were assessed for binding to heparin-Sepharose. All the alanine substitutions within the two regions homologous to the heparin-binding consensus sequences in the N-terminal domain either abolished activity or produced a lowering of heparin binding affinity. None of the mutants in the C-terminal domain of LPL showed a loss of activity or a reduction in heparin binding affinity. These data demonstrate that charged residues at positions 279-282 and 292-304 of LPL are important for heparin binding affinity whereas the residues 390-393 and 439-448 in the C-terminal domain are not involved in heparin binding.
Assuntos
Heparina/metabolismo , Lipase Lipoproteica/química , Lipase Lipoproteica/metabolismo , Mutagênese Sítio-Dirigida , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular , Simulação por Computador , Sequência Consenso , Humanos , Lipase Lipoproteica/genética , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Homologia de Sequência , Relação Estrutura-Atividade , TransfecçãoRESUMO
Partial deficiency in lipolysis usually results in only mild disturbances of lipid levels. However, when this is associated with impairment of the uptake of remnant particles and increased production of triglyceride-rich lipoproteins stimulated by environmental factors such as during normal pregnancy, chylomicronemia may ensue. We have previously reported a patient who had approximately 12% of normal LPL activity and developed severe chylomicronemia during pregnancy (Ma et al. 1993. J. Clin. Invest. 91: 1953-1958). Here we report four new patients with pregnancy-induced chylomicronemia. In the nonpregnant state, these patients had mild to modest elevation of triglyceride levels ranging from 80 to 623 mg/dl (0.9-7.0 mmol/l) but during the third trimester they became severely chylomicronemic with triglyceride levels ranging from 2314 to 14,596 mg/dl (26 to 164 mmol/l). Three of these four patients had partial lipoprotein lipase (LPL) deficiency. The molecular characterization of the LPL gene in these three patients with partial LPL deficiency revealed four novel unpublished mutations. Patient #1 is a compound heterozygote for Leu252Arg and Ala261Thr mutations which are associated with 25% of normal LPL activity. In addition, she has an apoE3/2 genotype. Patient #2 is a heterozygote for a Asn291Ser substitution with 69% of LPL activity and also has an apoE3/2 genotype, while patient #3 is a heterozygote for a Trp382Stop mutation with 54% of normal LPL activity and has an apoE4/2 genotype. The fourth patient (#4) with pregnancy-induced chylomicronemia does not have LPL deficiency and has an apoE3/3 genotype. The previously reported patient (#5) who had 12% of normal LPL activity due to homozygosity for a Ser172Cys mutation also has an E3/3 genotype. Our data suggest that mutations in the LPL gene that cause partial LPL deficiency might be a frequent factor in the pathogenesis of pregnancy-induced chylomicronemia.
Assuntos
Apolipoproteínas E/genética , Quilomícrons/sangue , Lipase Lipoproteica/genética , Mutação , Complicações na Gravidez , Adulto , Apolipoproteína E2 , Sequência de Bases , Linhagem Celular , DNA/análise , DNA/química , Feminino , Expressão Gênica , Genótipo , Humanos , Hipertrigliceridemia/complicações , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Pancreatite/complicações , GravidezRESUMO
Lipoprotein lipase (LPL) is a key enzyme required for the hydrolysis of triglyceride-rich particles. To assess the effects of increased plasma LPL on lipoprotein levels, transgenic mice expressing human LPL (hLPL) were produced. Abundant hLPL transcripts were detected in RNA from different tissues of transgenic mice which resulted in an increase in post-heparin plasma LPL activity of approximately 154%. On rodent chow (p = 0.01) and after a 16-h fast (p = 0.001), plasma triglycerides in transgenic mice were decreased by approximately 50% as compared to littermate controls. Gel filtration chromatography showed a 2-3-fold decrease in very low density lipoprotein triglycerides and cholesterol enrichment of low density lipoprotein. Transgenic mice maintained on a high carbohydrate diet exhibited a 78% (p = 0.03) lowering of low density lipoprotein and very low density lipoprotein cholesterol levels, in addition to a 68% (p = 0.01) lowering of total to high density lipoprotein cholesterol (TC/HDL-C) ratios compared to controls. The distribution of apoA-I and A-II were similar in the transgenics and their non-transgenic littermates, while the apoE distribution was mildly altered in the plasma from the transgenic mice. These data demonstrate that moderate increases in total LPL activity are associated with significant changes in lipoprotein levels and altered composition of lipoprotein particles.
Assuntos
Lipídeos/sangue , Lipase Lipoproteica/sangue , Lipase Lipoproteica/genética , Lipoproteínas/sangue , Animais , Northern Blotting , Colesterol/sangue , HDL-Colesterol/sangue , Citomegalovirus , Feminino , Genes Precoces , Humanos , Lipase Lipoproteica/biossíntese , Lipoproteínas LDL/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Transgênicos , Especificidade de Órgãos , Regiões Promotoras Genéticas , Valores de Referência , Fatores Sexuais , Triglicerídeos/sangueRESUMO
Cellulomonas fimi endo-beta-1,4-glucanase A (CenA) contains a discrete N-terminal cellulose-binding domain (CBDCenA). Related CBDs occur in at least 16 bacterial glycanases and are characterized by four highly conserved Trp residues, two of which correspond to W14 and W68 of CBDCenA. The adsorption of CBDCenA to crystalline cellulose was compared with that of two Trp mutants (W14A and W68A). The affinities of the mutant CBDs for cellulose were reduced by approximately 50- and 30-fold, respectively, relative to the wild type. Physical measurements indicated that the mutant CBDs fold normally. Fluorescence data indicated that W14 and W68 were exposed on the CBD, consistent with their participation in binding to cellobiosyl residues on the cellulose surface.
Assuntos
Proteínas de Bactérias/metabolismo , Celulase/metabolismo , Celulose/metabolismo , Bacilos Gram-Positivos/genética , Triptofano/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Alinhamento de Sequência , Homologia de Sequência de AminoácidosAssuntos
Hiperlipoproteinemia Tipo I/genética , Lipase Lipoproteica/genética , Deleção de Sequência , Sequência de Aminoácidos , Sequência de Bases , Pré-Escolar , DNA/genética , Éxons , Feminino , Humanos , Hiperlipoproteinemia Tipo I/enzimologia , Masculino , Dados de Sequência Molecular , LinhagemRESUMO
The cDNA for the alpha-isoenzyme from rhesus monkey (Macaca mulatta) liver was cloned and expressed in yeast. The alpha-isoenzymes of human and monkey liver alcohol dehydrogenase differ from the other human and horse liver enzymes in having Met57, Ala93, and Val116 instead of Leu57, Phe93, and Leu116 in the substrate binding pocket and Gly47 instead of Arg47 near the pyrophosphate moiety of the coenzyme. The effects of these differences on the kinetic mechanism, substrate specificity, and coenzyme binding were studied with the purified, recombinant monkey alpha-isoenzyme (MmADH alpha) and mutated enzymes with Gly47 substituted with His or Arg. The mechanism appears to be random for the binding of NAD+ and ethanol and ordered for NADH and acetaldehyde, with formation of a dead-end enzyme-NADH-ethanol complex. MmADH alpha reacts 130-fold slower (V/K) with ethanol and 3-25-fold slower with 2-methyl alcohols but 20-fold faster with cyclohexanol, as compared with horse (Equus caballus) liver EE isoenzyme (EqADH). MmADH alpha is stereoselective for the R isomer of 2-butanol, whereas EqADH favors the S isomer. Both enzymes have comparable reactivity with larger primary alcohols. MmADH alpha is more reactive with secondary alcohols and has highest activity with cyclohexanol. However, it does not react with steroids such as 5 beta-androstane-17 beta-ol-3-one. Molecular modeling suggests that the differences between MmADH alpha and EqADH are a result of the substitution of Ala for Phe93 and Thr for Ser48. MmADH alpha binds NAD+ most rapidly when a group with a pK of 7.4 is unprotonated, implicating His51 in this reaction. The G47R substitution decreased the dissociation constants for NAD+ and NADH and turnover numbers only about 2-fold, whereas the G47H substitution increased dissociation constants 7-14-fold and turnover numbers 4-fold. A basic residue at position 47 is not crucial for activity, as multiple interactions determine coenzyme affinity.