RESUMO
Children's language skills develop rapidly with increasing age, and several studies indicate that they use language- and age-specific strategies to understand complex sentences. In the present experiment, functional magnetic resonance imaging (fMRI) and behavioral measures were used to investigate the acquisition of case-marking cues for sentence interpretation in the developing brain of German preschool children with a mean age of 6 years. Short sentences were presented auditorily, consisting of a transitive verb and two case-marked arguments with canonical subject-initial or non canonical object-initial word order. Overall group results revealed mainly left hemispheric activation in the perisylvian cortex with increased activation in the inferior parietal cortex (IPC), and the anterior cingulate cortex (ACC) for object-initial compared to subject-initial sentences. However, single-subject analysis suggested two distinct activation patterns within the group which allowed a classification into two subgroups. One subgroup showed the predicted activation increase in the left inferior frontal gyrus (IFG) for the more difficult object-initial compared to subject-initial sentences, while the other group showed the reverse effect. This activation in the left IFG can be taken to reflect the degree to which adult-like sentence processing strategies, necessary to integrate case-marking information, are applied. Additional behavioral data on language development tests show that these two subgroups differ in their grammatical knowledge. Together with these behavioral findings, the results indicate that the use of a particular processing strategy is not dependent on age as such, but rather on the child's individual grammatical knowledge and the ability to use specific language cues for successful sentence comprehension.
Assuntos
Compreensão/fisiologia , Lateralidade Funcional/fisiologia , Idioma , Linguística , Córtex Pré-Frontal/fisiologia , Percepção da Fala/fisiologia , Mapeamento Encefálico , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Estatística como AssuntoRESUMO
Interpreting other people's intentions during communication represents a remarkable challenge for children. Although many studies have examined children's understanding of, for example, sarcasm, less is known about their interpretation. Using realistic audiovisual scenes, we invited 124 children between 8 and 12 years old to watch video clips of young adults using different speaker intentions. After watching each video clip, children answered questions about the characters and their beliefs, and the perceived friendliness of the speaker. Children's responses reveal age and gender differences in the ability to interpret speaker belief and social intentions, especially for scenarios conveying teasing and prosocial lies. We found that the ability to infer speaker belief of prosocial lies and to interpret social intentions increases with age. Our results suggest that children at the age of 8 years already show adult-like abilities to understand literal statements, whereas the ability to infer specific social intentions, such as teasing and prosocial lies, is still developing between the age of 8 and 12 years. Moreover, girls performed better in classifying prosocial lies and sarcasm as insincere than boys. The outcomes expand our understanding of how children observe speaker intentions and suggest further research into the development of teasing and prosocial lie interpretation.
Assuntos
Fatores Etários , Linguagem Infantil , Fatores Sexuais , Habilidades Sociais , Percepção da Fala , Criança , Comunicação , Compreensão , Feminino , Humanos , Intenção , Masculino , Gravação em VídeoRESUMO
Studies have shown that adolescents are more likely than adults to take risks in the presence of peers than when alone, and that young adolescents' risk perception is more influenced by other teenagers than by adults. The current fMRI study investigated the effect of social influence on risk perception in female adolescents (aged 12-14) and adults (aged 23-29). Participants rated the riskiness of everyday situations and were then informed about the (alleged) risk ratings of a social influence group (teenagers or adults), before rating each situation again. The results showed that adolescents adjusted their ratings to conform with others more than adults did, and both age groups were influenced more by adults than by teenagers. When there was a conflict between the participants' own risk ratings and the ratings of the social influence group, activation was increased in the posterior medial frontal cortex, dorsal cingulate cortex and inferior frontal gyrus in both age groups. In addition, there was greater activation during no-conflict situations in the right middle frontal gyrus and bilateral parietal cortex in adults compared with adolescents. These results suggest that there are behavioral and neural differences between adolescents and adults in conflict and no-conflict social situations.
Assuntos
Encéfalo/fisiologia , Percepção/fisiologia , Assunção de Riscos , Comportamento Social , Adolescente , Adulto , Mapeamento Encefálico , Feminino , Humanos , Imageamento por Ressonância Magnética , Adulto JovemRESUMO
Regulation of myristoylCoA pools in Saccharomyces cerevisiae plays an important role in modulating the activity of myristoylCoA:protein N-myristoyltransferase (NMT), an essential enzyme with an ordered Bi Bi reaction that catalyzes the transfer of myristate from myristoylCoA to greater than or equal to 12 cellular proteins. At least two pathways are available for generating myristoylCoA: de novo synthesis by the multifunctional, multisubunit fatty acid synthetase complex (FAS) and activation of exogenous myristate by acylCoA synthetase. The FAA1 (fatty acid activation) gene has been isolated by genetic complementation of a faal mutant. This single copy gene, which maps to the right arm of chromosome XV, specifies a long chain acylCoA synthetase of 700 amino acids. Analyses of strains containing NMT1 and a faal null mutation indicated that FAA1 is not essential for vegetative growth when an active de novo pathway for fatty acid synthesis is present. The role of FAA1 in cellular lipid metabolism and protein N-myristoylation was therefore assessed in strains subjected to biochemical or genetic blockade of FAS. At 36 degrees C, FAA1 is required for the utilization of exogenous myristate by NMT and for the synthesis of several phospholipid species. This requirement is not apparent at 24 or 30 degrees C, suggesting that S. cerevisiae contains another acylCoA synthetase activity whose chain length and/or temperature optima may differ from Faalp.
Assuntos
Acil Coenzima A/metabolismo , Coenzima A Ligases/genética , Ácidos Mirísticos/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Repressoras , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Fúngicos , Clonagem Molecular , Coenzima A Ligases/metabolismo , Ácido Graxo Sintases/metabolismo , Teste de Complementação Genética , Isoenzimas/genética , Dados de Sequência Molecular , Mutação/genética , Ácido Mirístico , Fenótipo , RNA Mensageiro/biossíntese , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência do Ácido NucleicoRESUMO
Saccharomyces cerevisiae has been used as a model for studying the regulation of protein N-myristoylation. MyristoylCoA:protein N-myristoyl-transferase (Nmt1p), is essential for vegetative growth and uses myristoylCoA as its substrate. MyristoylCoA is produced by the fatty acid synthetase (Fas) complex and by cellular acylCoA synthetases. We have recently isolated three unlinked Fatty Acid Activation (FAA) genes encoding long chain acylCoA synthetases and have now recovered a fourth by genetic complementation. When Fas is active and NMT1 cells are grown on media containing a fermentable carbon source, none of the FAA genes is required for vegetative growth. When Fas is inactivated by a specific inhibitor (cerulenin), NMT1 cells are not viable unless the media is supplemented with long chain fatty acids. Supplementation of cellular myristoylCoA pools through activation of imported myristate (C14:0) is predominantly a function of Faa1p, although Faa4p contributes to this process. Cells with nmt181p need larger pools of myristoylCoA because of the mutant enzyme's reduced affinity for this substrate. Faa1p and Faa4p are required for maintaining the viability of nmt1-181 strains even when Fas is active. Overexpression of Faa2p can rescue nmt1-181 cells due to activation of an endogenous pool of C14:0. This pool appears to be derived in part from membrane phospholipids since overexpression of Plb1p, a nonessential lysophospholipase/phospholipase B, suppresses the temperature-sensitive growth arrest and C14:0 auxotrophy produced by nmt1-181. None of the four known FAAs is exclusively responsible for targeting imported fatty acids to peroxisomal beta-oxidation pathways. Introduction of a peroxisomal assembly mutation, pas1 delta, into isogenic NMT1 and nmt1-181 strains with wild type FAA alleles revealed that when Fas is inhibited, peroxisomes contribute to myristoylCoA pools used by Nmt1p. When Fas is active, a fraction of cellular myristoylCoA is targeted to peroxisomes. A NMT1 strain with deletions of all four FAAs is still viable at 30 degrees C on media containing myristate, palmitate, or oleate as the sole carbon source--indicating that S. cerevisiae contains at least one other FAA which directs fatty acids to beta-oxidation pathways.
Assuntos
Ácidos Graxos/metabolismo , Proteínas Fúngicas/genética , Genes Fúngicos , Ácidos Mirísticos/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Acil Coenzima A/metabolismo , Sequência de Aminoácidos , Animais , Bactérias/genética , Sequência de Bases , Ácido Graxo Sintases/metabolismo , Proteínas Fúngicas/metabolismo , Humanos , Metabolismo dos Lipídeos , Mamíferos , Dados de Sequência Molecular , Ácido Mirístico , Oligodesoxirribonucleotídeos , Filogenia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Homologia de Sequência de AminoácidosRESUMO
Within its intermediate host, Toxoplasma gondii switches between two forms: a rapidly replicating tachyzoite and an encysted bradyzoite. Bradyzoites persist within the host throughout its life, hidden from antimicrobial agents and the immune system. The signals that mediate switching are poorly understood. A gene trap was employed to isolate genes whose expression is up-regulated early in the switching of bradyzoites via the negative and positive selectable marker hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT). T. gondii was transfected with promoterless HXGPRT and negatively selected with 6-thioxanthine to inhibit the growth of tachyzoites expressing HXGPRT. The surviving tachyzoites were then induced for in vitro bradyzoite formation and treated with mycophenolic acid and xanthine to positively select for parasites in which the construct had integrated downstream of a bradyzoite-specific gene. Strains were checked for their ability to differentiate by using Dolichos biflorus agglutinin (a bradyzoite-specific lectin) and a monoclonal antibody against P36 (a bradyzoite-specific surface antigen). After differentiation, all gene-trapped clones had Dolichos immunofluorescence and all but one expressed P36. The sequences flanking the insertion site of this P36-negative strain were homologous to the Toxoplasma family of surface antigens, strongly suggesting that P36 is encoded by the disruptive gene. Genetic mapping and complementation of the P36-negative strain further indicated that the disrupted gene is P36. Reverse transcriptase PCR and S1 nuclease digestion were used to compare mRNA levels during the tachyzoite and bradyzoite stages. The presumptive P36 gene does not appear to regulate its mRNA levels between the two stages, indicating a posttranscriptional mechanism of regulation for early bradyzoite-specific genes.
Assuntos
DNA de Protozoário/isolamento & purificação , Regulação da Expressão Gênica no Desenvolvimento , Genes de Protozoários , Pentosiltransferases/metabolismo , Toxoplasma/genética , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/genética , Antígenos de Superfície/genética , Glicoproteínas de Membrana/genética , Dados de Sequência Molecular , Mutagênese Insercional , Proteínas de Protozoários/genética , RNA Mensageiro/análise , Alinhamento de Sequência , Regulação para CimaRESUMO
Most studies on the development of face cognition abilities have focussed on childhood, with early maturation accounts contending that face cognition abilities are mature by 3-5 years. Late maturation accounts, in contrast, propose that some aspects of face cognition are not mature until at least 10 years. Here, we measured face memory and face perception, two core face cognition abilities, in 661 participants (397 females) in four age groups (younger adolescents (11.27-13.38 years); mid-adolescents (13.39-15.89 years); older adolescents (15.90-18.00 years); and adults (18.01-33.15 years)) while controlling for differences in general cognitive ability. We showed that both face cognition abilities mature relatively late, at around 16 years, with a female advantage in face memory, but not in face perception, both in adolescence and adulthood. Late maturation in the face perception task was driven mainly by protracted development in identity perception, while gaze perception abilities were already comparatively mature in early adolescence. These improvements in the ability to memorize, recognize and perceive faces during adolescence may be related to increasing exploratory behaviour and exposure to novel faces during this period of life.
Assuntos
Reconhecimento Facial , Reconhecimento Psicológico , Adolescente , Adulto , Criança , Demografia , Feminino , Humanos , Masculino , Memória , Tempo de Reação , Caracteres Sexuais , Análise e Desempenho de Tarefas , Adulto JovemRESUMO
The obligate intracellular parasite Toxoplasma gondii can infect virtually any nucleated cell in any warm-blooded host. Through the effort of many researchers, we are beginning to learn what makes T. gondii such a successful protozoan parasite. A high throughput genetic screen that allows simultaneous examination of a large panel of mutants would greatly facilitate a global investigation of this parasite. Signature-tagged mutagenesis uses a unique DNA sequence to tag an individual mutant so that it can later be identified within a pool. This system allows the efficient identification of parasites carrying mutations in genes that are essential for growth in restrictive but not permissive conditions. We have generated a bank of approximately 4900 signature-tagged T. gondii tachyzoites represented in 89 pools, each of which contains 60 uniquely tagged mutant parasites. We have demonstrated the usefulness of this negative screening strategy with a tissue culture model for pyrimidine salvage using resistance to the pro-drug FUDR. Mutants that are defective for growth in any defined growth condition versus standard tissue culture conditions can now be identified (eg, sensitive to a specific drug, growth in a specialized cell line, or growth within animals).
Assuntos
Genes de Protozoários , Mutagênese/genética , Toxoplasma/genética , Animais , Mutagênese Insercional/métodos , Seleção GenéticaRESUMO
As for any intracellular parasite, the surface of the Apicomplexan parasite Toxoplasma gondii must fulfil many functions including a role in attachment, signalling, invasion, transport and interaction with the immune response of the host. In this review, we describe the current state of knowledge on the molecules that are found on the surface of the different developmental stages of this parasite and speculate as to how at least some of these multiple functions are fulfilled. Special emphasis is given to the growing family of surface antigens that are related to the tachyzoite-specific surface antigen 1. We conclude that the surface (of tachyzoites, at least) is both more and less complex than previously thought: there are more proteins present but their sequences suggest that the majority may share a similar overall structure typified by surface antigen 1.
Assuntos
Antígenos de Protozoários/análise , Antígenos de Superfície/análise , Toxoplasma/química , Animais , Estágios do Ciclo de Vida , Toxoplasma/crescimento & desenvolvimentoAssuntos
Aciltransferases/metabolismo , Ácidos Mirísticos/metabolismo , Fatores de Ribosilação do ADP , Acil Coenzima A/metabolismo , Aciltransferases/biossíntese , Aciltransferases/química , Sequência de Aminoácidos , Animais , Clonagem Molecular/métodos , Coenzima A Ligases/química , Coenzima A Ligases/metabolismo , Escherichia coli/genética , Proteínas de Ligação ao GTP/biossíntese , Proteínas de Ligação ao GTP/metabolismo , Genótipo , Humanos , Indicadores e Reagentes , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ácido Mirístico , Plasmídeos , Mutação Puntual , Técnica de Diluição de Radioisótopos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimologia , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Transfecção/métodos , TrítioRESUMO
A system is described for studying protein N-myristoylation, a eukaryotic protein modification, in Escherichia coli strains containing components of eukaryotic metabolic pathways that regulate metabolism of myristoyl-CoA:protein N-myristoyltransferase (Nmt1p) substrates. Three recombinant plasmids were used to simultaneously direct synthesis of Saccharomyces cerevisiae Nmt1p, a substrate protein (S. cerevisiae ADP-ribosylation factor 1, Arf1p), and one of the acyl-CoA synthetases produced by S. cerevisiae (Faa1p) in isogenic strains of bacteria with wild type or mutant alleles of genes comprising the regulon for fatty acid degradation (FadR, FadE, FadL and FadD). Incorporation of exogenous tritiated myristate into Arf1p and bacterial phospholipid biosynthetic pathways was analyzed. Removal of FadL, a 448-residue protein necessary for efficient transport of fatty acids across the outer membrane, had no detectable effect on Nmt1p-dependent N-myristoylation of Arf1p. This finding is consistent with the notion that permeation of C14:0 across the bacterial inner membrane can occur by simple diffusion. Studies of strains that contain a mutation in FadE which inhibits beta-oxidation of exogenous fatty acids, confirm that Nmt1p retains its specificity for myristoyl-CoA over palmitoyl-CoA in E. coli. A mutation that inactivates FadD, a 580-residue protein which is the only acyl-CoA synthetase produced by this bacterium, completely blocks incorporation of exogenous myristate into Arf1p. This failure to be incorporated indicates that myristoyl-acyl carrier protein, generated by inner membrane acyl-acyl carrier protein synthetase, is not a substrate for Nmt1p. S. cerevisiae Faa1p can partially complement this mutant fadD allele. It can fully "restore" N-myristoylation of Arf1p. Faa1p can also rescue growth at 37 degrees C of fadD- strains on minimal media supplemented with C12:0, although this rescue becomes less efficient as the chain length of the supplemental fatty acid increases. In addition, S. cerevisiae Faa1p is better able to direct myristoyl-CoA to the bacteria's phospholipid biosynthetic pathways than FadD, while FadD is more efficient at directing myristoyl-CoA to the genetically engineered protein N-myristoylation pathway. Since cellular acyl-CoA synthetase activity in S. cerevisiae has been distributed to at least two functionally differentiated proteins, this system should be useful for comparing their structure-activity relationships as well as their interactions with Nmt1p in an organelle-free environment.
Assuntos
Aciltransferases/metabolismo , Coenzima A Ligases/metabolismo , Proteínas de Escherichia coli , Escherichia coli/genética , Mutação , Ácidos Mirísticos/metabolismo , Plasmídeos , Proteínas Repressoras , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimologia , Proteínas da Membrana Bacteriana Externa/genética , Transporte Biológico , Clonagem Molecular , Escherichia coli/metabolismo , Proteínas de Transporte de Ácido Graxo , Deleção de Genes , Genes Bacterianos , Ácido MirísticoRESUMO
Within intermediate hosts (such as humans), the protozoan parasite Toxoplasma gondii has two life cycle stages: a rapidly replicating form called a tachyzoite and a slowly growing, quiescent form called a bradyzoite. Recently, molecular biology studies have shown that tachyzoites and bradyzoites express a number of homologs (ie. evolutionary related genes)expressed exclusively in one or the other stage. Here, Laura Knoll and John Boothroyd describe examples of how these stage-specific homologs were discovered, and speculate about their regulation and functional significance.
RESUMO
Four unlinked fatty acid activation (FAA) genes encoding acyl-CoA synthetases have been identified in Saccharomyces cerevisiae and characterized by noting the phenotypes of isogenic strains containing all possible combinations of faa null alleles. None of these genes is required for vegetative growth when acyl-CoA production by the fatty acid synthetase (Fas) complex is active. When Fas is inhibited by cerulenin, exponentially growing cells are not viable on media containing a fermentable carbon source unless supplemented with fatty acids such as myristate, palmitate, or oleate. The functionally interchangeable FAA1 and FAA4 genes are responsible for activation of these imported fatty acids. Analysis of lysates prepared from isogenic FAA1FAA4 and faa1 delta faa4 delta strains indicated that Faa1p and Faa4p together account for 99% of total cellular myristoyl-CoA and palmitoyl-CoA synthetase activities. Genetic complementation studies revealed that rat liver acyl-CoA synthetase (RLACS) rescues the viability of faa1 delta faa4 delta cells in media containing a fermentable carbon source, myristate or palmitate, plus cerulenin. Rescue is greater at 37 degrees C compared with 24 degrees C, paralleling the temperature-dependent changes in RLACS activity in vitro as well as the enzyme's ability to direct incorporation of tritiated myristate and palmitate into cellular phospholipids in vivo. Complementation by RLACS is blocked by treatment of cells with triacsin C (1-hydroxy-3-(E,E,E,2',4',7'- undecatrienylidine)triazene). Even though Faa1p, Faa4p, and RLACS are all able to activate imported myristate and palmitate in S. cerevisiae, the sensitivity of Faa4p and RLACS, but not Faa1p, to inhibition by triacsin C suggests that the rat liver enzyme is functionally more analogous to Faa4p than to Faa1p. Finally, an assessment of myristate and palmitate import into FAA1FAA4 and faa1 delta faa4 delta strains, with or without episomes that direct overexpression of Faa1p, Faa4p or RLACS, indicated that fatty acid uptake is not coupled to activation in S. cerevisiae.
Assuntos
Coenzima A Ligases/genética , Animais , Transporte Biológico , Coenzima A Ligases/antagonistas & inibidores , Ácidos Graxos/metabolismo , Genes Fúngicos , Teste de Complementação Genética , Fígado/enzimologia , Ratos , Saccharomyces cerevisiae/genética , Triazenos/farmacologiaRESUMO
The efficiency and specificity of protein N-myristoylation appear to be influenced by the availability of myristoyl-CoA and other potential acyl-CoA substrates of myristoyl-CoA:protein N-myristoyltransferase. Recent studies have revealed that Saccharomyces cerevisiae contains at least three acyl-CoA synthetase genes (FAA for fatty acid activation). We have expressed Faa1p, Faa2p, and Faa3p in a strain of Escherichia coli that lacks its own endogenous acyl-CoA synthetase (FadD). Each S. cerevisiae acyl-CoA synthetase contained a carboxyl-terminal His tag so that it could be purified to homogeneity in a single step using nickel chelate affinity chromatography. In vitro assays of C3:0-C24:0 fatty acids indicate that Faa1p prefers C12:0-C16:0, with myristic and pentadecanoic acid (C15:0) having the highest activities. Faa2p can accommodate a wider range of acyl chain lengths: C9:0-C13:0 are preferred and have equivalent activities, although C7:0-C17:0 fatty acids are tolerated as substrates with no greater than a 2-fold variation in specific activity. The myristoyl-CoA synthetase activities of Faa1p and Faa2p are 2 orders of magnitude greater than that of Faa3p in vitro. Faa3p has a preference for C16 and C18 fatty acids with a cis-double bond at C-9-C-10. The temperature optimum for Faa1p is 30 degrees C, while Faa2p and Faa3p have the greatest activities at 25 degrees C. These in vitro observations were confirmed using two in vivo assays: (i) measurement of the ability of each S. cerevisiae acyl-CoA synthetase to direct the incorporation of exogenously derived tritiated myristate, palmitate, or oleate into cellular phospholipids produced in a fadD- strain of E. coli during exponential growth at 24 or 37 degrees C and (ii) measurement of the incorporation of [3H]myristate into a yeast N-myristoylprotein coexpressed with Nmt1p and Faa1p, Faa2p, or Faa3p in the fadD- strain.
Assuntos
Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Genes Fúngicos/genética , Saccharomyces cerevisiae/genética , Sequência de Bases , Coenzima A Ligases/isolamento & purificação , Escherichia coli/genética , Ácidos Graxos Insaturados/metabolismo , Genes Bacterianos/genética , Temperatura Alta , Concentração de Íons de Hidrogênio , Isomerismo , Dados de Sequência Molecular , Miristatos/metabolismo , Ácido Oleico , Ácidos Oleicos/metabolismo , Palmitatos/metabolismo , Fosfatidilgliceróis/biossíntese , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
Myristoyl-CoA:protein N-myristoyltransferase (NMT) catalyzes the cotranslational, covalent attachment of a rare fatty acid, myristic acid (C14:0), to the amino-terminal glycine residue of a number of eukaryotic proteins involved in cellular growth and signal transduction as well as several viral proteins necessary for assembly-replication. NMT has become a target for both anti-viral and anti-fungal therapy. Analysis of purified Saccharomyces cerevisiae NMT plus yeast strains with conditional lethal nmt1 mutations have provided insights about how this process is regulated in vivo. We have now defined the location of NMT in two strains of S. cerevisiae to better understand the functional and spatial relationships between this enzyme and cellular systems that generate its acyl-CoA and peptide ligands. Western blot studies using an affinity purified antibody raised in rabbits against purified S. cerevisiae NMT indicate that the acyltransferase represents 0.06% of total cellular proteins in an exponentially growing haploid strain with a wild type NMT1 allele. Another strain containing a single, integrated copy of a GAL1/NMT1 fusion gene and a nmt1 null allele had 12-fold higher levels of NMT when grown on galactose-containing media. This increase in NMT production had no detectable effects on growth or cellular morphology. Cell fractionation studies, confocal fluorescence immunocytochemical analysis, and immunogold electron microscopic surveys of fixed, gelatin-embedded cryosections of both strains revealed that NMT is a cytosolic protein that is not associated with cellular membranes (including the endoplasmic reticulum and plasma membrane), the nucleus, mitochondria, Golgi apparatus, or vacuoles. This finding is discussed in light of what is known about the location and activities of enzymes involved in de novo fatty acid biosynthesis and in amino-terminal processing of nascent proteins.
Assuntos
Aciltransferases/metabolismo , Saccharomyces cerevisiae/enzimologia , Aciltransferases/análise , Animais , Anticorpos , Western Blotting , Imunofluorescência , Coelhos/imunologia , Saccharomyces cerevisiae/ultraestrutura , Especificidade da Espécie , Frações Subcelulares/enzimologia , Frações Subcelulares/ultraestruturaRESUMO
Saccharomyces cerevisiae contains at least five acyl-CoA synthetases (fatty acid activation proteins, or Faaps). Four FAA genes have been recovered to date. Recent genetic studies indicate that Faa1p and Faa4p are involved in the activation of imported fatty acids, while Faa2p activates endogenous pools of fatty acids. We have now purified Faa4p from S. cerevisiae and compared its fatty acid substrate specificity in vitro with the specificities of purified Faa1p, Faa2p, and Faa3p. Among C8-C18 saturated fatty acids, Faa4p and Faa1p both prefer C14:0. Surveys of C14 fatty acids with single cis-double bonds at C2-C12 indicated that Faa4p and Faa1p prefer Z9-tetradecenoic acid, although Faa4p's preference is much greater and also evident in C16 and C18 fatty acids. Faa4p's selectivity for fatty acids with a C9-C10 cis-double bond is a feature it shares with Faa3p and is notable since in yeast Ole1p, a microsomal cis-delta 9 desaturase, accounts for de novo production of monoenoic acyl-CoAs from saturated acyl-CoA substrates. Faa4p has no detectable acyl-CoA synthetase activity when incubated with tetradecenoic acids having a trans-double bond at C2-3, C4-5, C5-6, C6-7, C7-8, or C9-10. Faa3p can only use E9-tetradecenoic acid as a substrate, while E4-, E6- and E9-tetradecenoic acids can be used by Faa1p and Faa2p. E2-tetradecenoic acid is an Faap inhibitor, with Faa2p exhibiting the greatest sensitivity (IC50 = 2.6 +/- 0.2 microM). Triacsin C (1-hydroxy-3-(E,E,E,2',4',7'- undecatrienylidine)-1,2,3-triazene) has trans-double bonds at positions that correspond to those in E2-, E5-, and E7-tetradecenoic acids. This compound is a potent inhibitor of Faa2p (Ki = 15 +/- 1 nM; competitive with fatty acid), less potent against Faa4p (Ki = 2 microM), and not active against Faa1p or Faa3p (IC50 > 500 microM). Analysis of an n-tetradecanal plus a series of oximes (tridecanal oxime, 1-azadeca-1,3,5-trienol, and 1-azaundeca-1,3,5-trienol) indicated that the combination of an azenol moiety (R-CH = N-OH) plus adjacent unsaturation are critical for triacsin C's selective inhibition of Faa2p. Triacsin C and oxime derivatives appear to be very useful for defining differences in molecular recognition among S. cerevisiae acyl-CoA synthetases. The > 25,000-fold range in the inhibitory effects of triacsin C on these four Faaps suggests that it may be possible to develop other selective inhibitors of eukaryotic acyl-CoA synthetases.
Assuntos
Coenzima A Ligases/metabolismo , Ácidos Graxos/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Bases , Coenzima A Ligases/antagonistas & inibidores , Coenzima A Ligases/genética , Primers do DNA/genética , DNA Fúngico/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Ácidos Graxos/química , Ácidos Graxos Monoinsaturados/química , Ácidos Graxos Monoinsaturados/metabolismo , Genes Fúngicos , Dados de Sequência Molecular , Oximas/metabolismo , Saccharomyces cerevisiae/genética , Relação Estrutura-Atividade , Especificidade por Substrato , Triazenos/metabolismo , Triazenos/farmacologiaRESUMO
NMT1 is an essential Saccharomyces cerevisiae gene which encodes myristoyl-CoA:protein N-myristoyltransferase (Nmt1p). Nmt1p transfers myristate (C14:0), from myristoyl-CoA to the amino-terminal Gly residue of several essential cellular proteins. Little information is available about how myristoyl-CoA metabolism is regulated in eukaryotic cells. We have isolated and characterized three unlinked Fatty Acid Activation genes from S. cerevisiae, FAA1, FAA2, and FAA3. In vitro biochemical assays reveal that the myristoyl-CoA synthetase activity of purified Faa2p is approximately equal to that of Faa1p, and two orders of magnitude greater than that of Faa3p. Analysis of NMT1 strains containing faa1, faa2, and/or faa3 null alleles indicates that Faa1p, Faa2p, and Faa3p are not essential for vegetative growth when de novo acyl-CoA synthesis by fatty acid synthetase (Fas) is active. S. cerevisiae strains containing nmt1-181 exhibit temperature-sensitive growth arrest and myristic acid auxotrophy due to the reduced affinity of its mutant protein product (nmtGly451-->Asp) for myristoyl-CoA. Comparison of the growth characteristics of isogenic NMT1 and nmt1-181 strains with all possible combinations of faa1, faa2, and faa3 null alleles, in the presence or absence of an active Fas complex, indicates that (i) Faa1p is responsible for activation of imported fatty acids to their CoA derivatives; (ii) Faa2p and Faa3p are able to access endogenous but not imported fatty acid substrates; (iii) nmt181p requires myristoyl-CoA production from both Fas and Faas for cells to remain viable at nonpermissive temperatures; (iv) Faa2p is unique among the three Faas in its ability, when overproduced, to partially rescue growth of a nmt1-181 strain at nonpermissive temperatures on yeast/peptone/dextrose (YPD) media without C14:0 supplementation; (v) acyl-CoAs produced by Faa1p, Faa2p, or Faa3p are not specifically targeted for beta-oxidation; and (vi) the ability of NMT1, faa1 delta, faa2 delta, faa3 delta strains to remain viable in the absence of an active Fas complex on YPD plus C14:0, or on media that contains fatty acids as the sole carbon source, suggests that S. cerevisiae contains other acyl-CoA synthetases which can activate imported fatty acids.
Assuntos
Aciltransferases/genética , Coenzima A Ligases/genética , Proteínas Fúngicas/genética , Proteínas Repressoras , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Aciltransferases/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Coenzima A Ligases/metabolismo , DNA Fúngico , Ácidos Graxos/metabolismo , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Ligação Genética , Dados de Sequência Molecular , Saccharomyces cerevisiae/enzimologia , Homologia de Sequência de AminoácidosRESUMO
Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (Nmt1p) is a monomeric enzyme that is essential for vegetative growth. Nmt1p catalyzes the co-translational transfer of myristate from CoA to the amino-terminal Gly of cellular proteins in an ordered Bi Bi reaction mechanism that initially involves binding of myristoyl-CoA to the apoenzyme. Forty one fatty acid analogs were synthesized to define features in the acyl chain of myristoyl-CoA which are important determinants of its recognition by Nmt1p's acyl-CoA binding site as well as to help us deduce the structure of the binding site itself. These analogs included dicarboxylic acids, omega-nitrocarboxylic acids, analogs equivalent in length to C13:0-C15:0 which contain electronegative halogens at their omega-termini, hydroxytetradecanoic acids with hydrogen replaced by OH from C3 to C13, and azidophenyl-containing fatty acids with the linear azide unit attached either meta or para to phenyl and with variations in the length of their methylene chains. These compounds were converted to their CoA derivatives using Pseudomonas acyl-CoA synthetase and then surveyed as substrates for purified Nmt1p in an in vitro assay system that included an octapeptide derived from residues 1-8 of the human immunodeficiency virus Pr55gag polyprotein precursor. The results suggest that the myristoyl-CoA binding site contains a conical-shaped "receptor" that interacts with the omega-terminus of the bound acyl chain of acyl-CoAs. The acuteness of this cone determines the enzyme's capacity to accommodate steric bulk at the omega-terminus as well as Nmt1p's sensitivity to the distance between the eclipsed C5-C6 bond of a bound acyl chain and its omega-terminus. The activity profile of the various analog-CoAs also indicates that the enzyme's myristoyl-CoA binding site can accommodate fatty acid analogs with marked increases in polarity at their omega-terminus (compared to C14:0) as long as their chain length is equivalent to that of myristate.
Assuntos
Acil Coenzima A/metabolismo , Aciltransferases/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Ácidos Mirísticos/metabolismo , Proteínas Repressoras , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimologia , Sítios de Ligação , Coenzima A Ligases/metabolismo , Ácidos Graxos não Esterificados/síntese química , Ácidos Graxos não Esterificados/química , Indicadores e Reagentes , Cinética , Modelos Moleculares , Estrutura Molecular , Ácido Mirístico , Pseudomonas/enzimologia , Especificidade por SubstratoRESUMO
Toxoplasma gondii has recently come under intense study as a model for intracellular parasitism because it has a number of properties that facilitate experimental manipulation. Attention is now being turned towards understanding the developmental biology of this complex parasite. The differentiation between the two asexual stages, the rapidly growing tachyzoites and the more slowly dividing, encysted bradyzoites, is of particular interest. Progression from the former to the latter is influenced by the host's immune response. This paper describes current progress on a number of research fronts, all aimed at understanding the triggers that push the tachyzoite-bradyzoite equilibrium in one or other direction and the changes that occur in gene expression (and ultimately metabolism and function). Chief among the techniques used for these studies are genetics and molecular genetics. Recent progress in these areas is described.
Assuntos
Mapeamento Cromossômico , Hipoxantina Fosforribosiltransferase/genética , Toxoplasma/crescimento & desenvolvimento , Toxoplasma/genética , Acetilglucosamina/metabolismo , Hidrolases Anidrido Ácido/genética , Hidrolases Anidrido Ácido/metabolismo , Animais , Membrana Celular/química , Membrana Celular/metabolismo , Quitina/metabolismo , Cistos , Genes de Protozoários , Teste de Complementação Genética , Humanos , Hipoxantina Fosforribosiltransferase/efeitos dos fármacos , Nucleosídeo-Trifosfatase , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Seleção Genética , Sitios de Sequências Rotuladas , Toxoplasma/efeitos dos fármacos , Xantinas/farmacologiaRESUMO
We have explored the acyl-CoA substrate specificity of Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (NMT) by synthesizing 81 fatty acid analogs and surveying their activity in a coupled in vitro assay containing Pseudomonas acyl-CoA synthetase and Escherichia coli-derived yeast NMT. Single oxygen or sulfur substitution for C-3 through C-13 is well tolerated by both enzymes. Detailed kinetic analyses suggest that the acyl-CoA and peptide-binding sites of NMT are relatively insensitive to placement of single group 6B heteroatoms. By contrast, di-oxygen-substituted analogs were very poor substrates, producing dramatic reductions in the affinity of NMTs peptide-binding site for a synthetic octapeptide substrate derived from the NH2-terminal sequence of a known N-myristoylprotein, the gag poly-protein precursor of human immunodeficiency virus 1 (HIV-1). This observation provides an example of binding site cooperativity in NMT. Replacement of one oxygen with sulfur at either the 6, 9, or 12 position of dioxatetradecanoic acids results in a general increase in peptide catalytic efficiency (Vmax/Km). An analysis of five fatty acids from octanoic to dodecanoic having terminal phenyl groups indicated that the best substrate was 10-phenyldecanoic acid even though Corey-Pauling-Koltun molecular models indicate that it has a length equivalent to that of tridecanoic acid. Six analogs having an equivalent length of 13 carbon atoms were subsequently prepared in which the phenyl group was systematically moved one methylene group closer to carboxyl. Movement of the phenyl just one carbon closer to carboxyl (producing 9-(p-methylphenyl) nonanoic acid) decreases peptide catalytic efficiency (Vmax/Km) severalfold compared to 10-phenyldecanoic acid. 10-(4-Tolyl)decanoic acid has the same relative positions of phenyl and carboxyl as 10-phenyldecanoic acid even though a methyl group is present on the phenyl ring. It produces peptide Km and Vmax values that are the same as 10-phenyldecanoic acid. Substitution of either oxygen or sulfur for a methylene group fails to override the effects noted when the phenyl group position is altered in the C-14 equivalent fatty acid series. Several fatty acids of differing chain lengths with cyclohexyl-, 2-furyl, and 2-thienyl groups at their omega termnius had activity profiles that paralleled those of the comparable phenyl-substituted compounds. Myristic acid analogs with triple bonds (beginning at positions 2 through 13), cis-double bonds (positions 3 through 13) and trans-double bond isomers (E5, E6, and E7) were also tested.(ABSTRACT TRUNCATED AT 400 WORDS)