RESUMO
AIMS/HYPOTHESIS: Inflammation contributes to both insulin resistance and pancreatic beta cell failure in human type 2 diabetes. Toll-like receptors (TLRs) are highly conserved pattern recognition receptors that coordinate the innate inflammatory response to numerous substances, including NEFAs. Here we investigated a potential contribution of TLR2 to the metabolic dysregulation induced by high-fat diet (HFD) feeding in mice. METHODS: Male and female littermate Tlr2(+/+) and Tlr2(-/-) mice were analysed with respect to glucose tolerance, insulin sensitivity, insulin secretion and energy metabolism on chow and HFD. Adipose, liver, muscle and islet pathology and inflammation were examined using molecular approaches. Macrophages and dendritic immune cells, in addition to pancreatic islets were investigated in vitro with respect to NEFA-induced cytokine production. RESULTS: While not showing any differences in glucose homeostasis on chow diet, both male and female Tlr2(-/-) mice were protected from the adverse effects of HFD compared with Tlr2(+/+) littermate controls. Female Tlr2(-/-) mice showed pronounced improvements in glucose tolerance, insulin sensitivity, and insulin secretion following 20 weeks of HFD feeding. These effects were associated with an increased capacity of Tlr2(-/-) mice to preferentially burn fat, combined with reduced tissue inflammation. Bone-marrow-derived dendritic cells and pancreatic islets from Tlr2(-/-) mice did not increase IL-1beta expression in response to a NEFA mixture, whereas Tlr2(+/+) control tissues did. CONCLUSION/INTERPRETATION: These data suggest that TLR2 is a molecular link between increased dietary lipid intake and the regulation of glucose homeostasis, via regulation of energy substrate utilisation and tissue inflammation.
Assuntos
Gorduras na Dieta/metabolismo , Resistência à Insulina/genética , Células Secretoras de Insulina/metabolismo , Receptor 2 Toll-Like/metabolismo , Análise de Variância , Animais , Glicemia/metabolismo , Calorimetria Indireta , Células Cultivadas , Feminino , Inflamação/genética , Inflamação/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/citologia , Masculino , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptor 2 Toll-Like/genéticaRESUMO
AIMS: Statins - inhibitors of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase - are known to reduce blood cholesterol levels. In this paper, we present a Saccharomyces cerevisiae expression system, which enables quick evaluation of the sensitivity of the wild-type and/or mutant forms of human HMG-CoA reductase towards statins or other drugs. METHODS AND RESULTS: We analysed the sequence of the HMG-CoA reductase gene in DNA extracted from blood samples of 16 patients with cardiovascular disorders. We applied the yeast system to examine the sensitivity of the wild-type and mutated versions of the hHMG-CoA reductase to different types of statins. CONCLUSION: The yeast and mammalian HMG-CoA reductases demonstrate structural and functional conservation, and expression of human HMG-CoA reductase in yeast complements the lethal phenotype of strains lacking the HMG1 and HMG2 genes. SIGNIFICANCE AND IMPACT OF THE STUDY: These data indicate that a yeast expression system can serve to study the influence of selected mutations in human HMG-CoA reductase on the sensitivity of the enzyme to commonly prescribed statins. Our results suggest that this model system is suitable for the development and selection of lipid-lowering drugs as well as for the examination of DNA sequence variations in the context of statin therapy.
Assuntos
Doenças Cardiovasculares/tratamento farmacológico , Hidroximetilglutaril-CoA Redutases/genética , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Saccharomyces cerevisiae/enzimologia , Primers do DNA/genética , Éxons , Proteína HMGB1/genética , Proteína HMGB2/genética , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Mutagênese Sítio-Dirigida , Reação em Cadeia da Polimerase , Saccharomyces cerevisiae/efeitos dos fármacosRESUMO
AIMS/HYPOTHESIS: Adipocytes in obesity are characterised by increased cell size and insulin resistance compared with adipocytes isolated from lean patients. However, it is not clear at present whether hypertrophy actually does drive adipocyte insulin resistance. Thus, the aim of the present study was to metabolically characterise small and large adipocytes isolated from epididymal fat pads of mice fed a high-fat diet (HFD). METHODS: C57BL/6J mice were fed normal chow or HFD for 8 weeks. Adipocytes from epididymal fat pads were isolated by collagenase digestion and, in HFD-fed mice, separated into two fractions according to their size by filtration through a nylon mesh. Viability was assessed by lactate dehydrogenase and 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium assays. Basal and insulin-stimulated D-[U-(14)C]glucose incorporation and lipolysis were measured. Protein levels and mRNA expression were determined by western blot and real-time RT-PCR, respectively. RESULTS: Insulin-stimulated D-[U-(14)C]glucose incorporation into adipocytes isolated from HFD-fed mice was reduced by 50% compared with adipocytes from chow-fed mice. However, it was similar between small (average diameter 60.9 +/- 3.1 microm) and large (average diameter 83.0 +/- 6.6 microm) adipocytes. Similarly, insulin-stimulated phosphorylation of protein kinase B and AS160 were reduced to the same extent in small and large adipocytes isolated from HFD-mice. In addition, insulin failed to inhibit lipolysis in both adipocyte fractions, whereas it decreased lipolysis by 30% in adipocytes of chow-fed mice. In contrast, large and small adipocytes differed in basal lipolysis rate, which was twofold higher in the larger cells. The latter finding was associated with higher mRNA expression levels of Atgl (also known as Pnpla2) and Hsl (also known as Lipe) in larger adipocytes. Viability was not different between small and large adipocytes. CONCLUSIONS/INTERPRETATION: Rate of basal lipolysis but not insulin responsiveness is different between small and large adipocytes isolated from epididymal fat pads of HFD-fed mice.
Assuntos
Adipócitos/citologia , Gorduras na Dieta/farmacologia , Resistência à Insulina/fisiologia , Insulina/farmacologia , Lipólise/fisiologia , Adipócitos/efeitos dos fármacos , Adipócitos/fisiologia , Animais , Hidrolases de Éster Carboxílico/genética , Separação Celular , Tamanho Celular , Sobrevivência Celular , Glucose/metabolismo , Teste de Tolerância a Glucose , Lipase , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genéticaRESUMO
In this study, we show that the Saccharomyces cerevisiae ORF YBR142w, which encodes a putative DEAD-box RNA helicase, corresponds to MAK5. The mak5-1 allele is deficient in the maintenance of the M1 dsRNA virus, resulting in a killer minus phenotype. This allele carries two mutations, G218D in the conserved ATPase A-motif and P618S in a non-conserved region. We have separated these mutations and shown that it is the G218D mutation that is responsible for the killer minus phenotype. Mak5p is an essential nucleolar protein; depletion of the protein leads to a reduction in the level of 60S ribosomal subunits, the appearance of half-mer polysomes, and a delay in production of the mature 25S and 5.8S rRNAs. Thus, Mak5p is involved in the biogenesis of 60S ribosomal subunits.
Assuntos
Fases de Leitura Aberta/genética , RNA Helicases/genética , RNA de Cadeia Dupla/genética , Ribossomos/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Substituição de Aminoácidos , Sequência de Bases , RNA Helicases DEAD-box , Primers do DNA , Deleção de Genes , Teste de Complementação Genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fenótipo , Plasmídeos/genética , Reação em Cadeia da Polimerase , RNA Helicases/metabolismo , Proteínas Recombinantes/metabolismo , Ribossomos/ultraestrutura , Transcrição GênicaRESUMO
Previously we have shown that the Saccharomyces cerevisiae CCZ1 (YBR131w) gene encodes a protein involved in protein trafficking. Deletion of this gene leads to fragmentation of the vacuole typical of the class B vps mutants. Genetic and biochemical data indicated that Ccz1p is required for fusion of various transport intermediates with the vacuole. Here we present data indicating that CCZ1 is a close partner of the YPT7 gene, which encodes Rab GTPase and is required for fusion of transport vesicles to vacuole and homotypic vacuole fusion. We isolated extragenic suppressors of CCZ1 deletion. All these suppressors belong to one complementation group and correspond to mutated alleles of the YPT7 gene. The mutated residues are located in two Ypt7p domains responsible for guanine binding. These data suggest that Ccz1p and Ypt7p interact physically. Coimmunoprecipitation experiments provide direct evidence that this indeed is the case. A possible mechanism of Ccz1p action is discussed.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Fatores de Troca do Nucleotídeo Guanina , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , Proteínas de Transporte Vesicular , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Alanina/química , Alelos , Sequência de Aminoácidos , Ácido Aspártico/química , Transporte Biológico , Cafeína/farmacologia , Cálcio/metabolismo , Proteínas de Transporte/química , Estimulantes do Sistema Nervoso Central/farmacologia , DNA/metabolismo , Epitopos , Deleção de Genes , Biblioteca Gênica , Teste de Complementação Genética , Guanina/química , Hemaglutininas/química , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Fenótipo , Reação em Cadeia da Polimerase , Testes de Precipitina , Ligação Proteica , Proteínas de Saccharomyces cerevisiae/química , Homologia de Sequência de Aminoácidos , Zinco/metabolismoRESUMO
The role of the yeast vacuole, a functional analogue of the mammalian lysosome, in the turnover of proteins and organelles has been well documented. This review provides an overview of the current knowledge of vesicle mediated vacuolar transport in the yeast Saccharomyces cerevisiae cells. Due to the conservation of the molecular transport machinery S. cerevisiae has become an important model system of vacuolar trafficking because of the facile application of genetics, molecular biology and biochemistry.
Assuntos
Lisossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular , Sequência de Aminoácidos , Transporte Biológico , Membrana Celular/metabolismo , Complexo de Golgi/metabolismo , Metabolismo dos Lipídeos , Proteínas de Membrana/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Proteínas SNARE , Homologia de Sequência de Aminoácidos , Vacúolos/química , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Saccharomyces cerevisiae homologues of phosphotyrosyl phosphatase activator (PTPA) are encoded byRRD1 and RRD2, genes whose combined deletion is synthetic lethal. Previously we have shown that the lethality of rrd1,2delta can be suppressed by increasing the osmolarity of the medium. Here we show that the lethality of rrd1,2delta is also suppressed under oxygen-limited conditions. The absence of respiration per se is not responsible for the suppression since elimination of the mitochondrial genome or a block in heme biosynthesis fail to rescue the rrd1,2delta double mutation.
Assuntos
Saccharomyces cerevisiae/metabolismo , Cafeína/farmacologia , Diploide , Ativação Enzimática , Deleção de Genes , Heme/biossíntese , Mitocôndrias/metabolismo , Mutação , Oxigênio/metabolismo , Fenótipo , Plasmídeos/metabolismo , Sirolimo/farmacologia , TemperaturaRESUMO
CCZ1 was previously identified by the sensitivity of ccz1(delta) mutants to high concentrations of Caffeine and the divalent ions Ca(2+ )and Zn(2+). In this paper we show that deletion of CCZ1 leads to aberrant vacuole morphology, similar to the one reported for the family of vacuolar protein sorting (vps) mutants of class B. The ccz1(&Dgr;) cells display severe vacuolar protein sorting defects for both the soluble carboxipeptidase Y and the membrane-bound alkaline phosphatase, which are delivered to the vacuole by distinct routes. Ccz1p is a membranous protein and the vast majority of Ccz1p resides in late endosomes. These results, along with a functional linkage found between the CCZ1 and YPT7 genes, indicate that the site of Ccz1p function is at the last step of fusion of multiple transport intermediates with the vacuole.
Assuntos
Proteínas de Transporte de Cátions , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Trocadores de Sódio-Hidrogênio , Vacúolos/metabolismo , Proteínas de Transporte Vesicular , Proteínas rab de Ligação ao GTP/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Cálcio/metabolismo , ATPases Transportadoras de Cálcio/genética , ATPases Transportadoras de Cálcio/metabolismo , Proteínas de Transporte/análise , Proteínas de Transporte/metabolismo , Compartimento Celular/fisiologia , Endocitose/fisiologia , Proteínas Fúngicas/análise , Deleção de Genes , Dosagem de Genes , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Chaperonas Moleculares , Mutagênese/fisiologia , Fenótipo , ATPases Transportadoras de Cálcio da Membrana Plasmática , Plasmídeos , Receptores de Superfície Celular/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Vacúolos/química , Zinco/metabolismo , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Saccharomyces cerevisiae genes related to respiration are typically controlled by oxygen and haem. Usually the regulation by these factors is co-ordinated; haem is indicated as the oxygen sensor. However, the responsiveness of peroxisome functions to these regulatory factors is poorly understood. The expression of CTA1, POX1 and PEX1 genes encoding the peroxisomal proteins catalase A, acyl-CoA oxidase and Pex1p peroxin respectively was studied under various conditions: in anaerobiosis, in the absence of haem and in respiratory incompetence caused by the lack of a mitochondrial genome (rho(0)). The influence of haem deficiency or rho(0) on peroxisomal morphology was also investigated. Respiratory incompetence has no effect on the expression of CTA1 and POX1, whereas in the absence of haem their expression is markedly decreased. The synthesis of Pex1p is decreased in rho(0) cells and is decreased even more in haem-deficient cells. Nevertheless, peroxisomal morphology in both these types of cell does not differ significantly from the morphology of peroxisomes in wild-type cells. The down-regulating effect of anoxia on the expression of CTA1 and POX1 is even stronger than the effect of haem deficiency and is not reversed by the addition of exogenous haem or the presence of endogenous haem. Moreover, neither of these genes responds to the known haem-controlled transcriptional factor Hap1p. In contrast with the other two genes studied, PEX1 is up-regulated in anaerobiosis. The existence of one or more novel mechanisms of regulation of peroxisomal genes by haem and oxygen, different from those already known in S. cerevisiae, is postulated.
Assuntos
Catalase/biossíntese , Proteínas Fúngicas/biossíntese , Glicoproteínas/biossíntese , Heme/metabolismo , Proteínas de Membrana , Oxirredutases/biossíntese , Oxigênio/metabolismo , Peroxissomos/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Acil-CoA Oxidase , Adenosina Trifosfatases , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Saccharomyces cerevisiae/enzimologiaRESUMO
In the context of the cooperative project for functional analysis of novel genes uncovered during the systematic sequencing of the Saccharomyces cerevisiae genome, we deleted two paralogous ORFs: YIL153w and YPL152w. Based on the resulting phenotypes, the corresponding genes were named RRD1 and RRD2, respectively. Rrd proteins show significant similarity to the human phosphotyrosyl phosphatase activator (PTPA). Both single mutants, rrd1delta and rrd2delta, were viable. Deletion of RRD1 caused pleiotropic phenotypes under a wide range of conditions, including sensitivity to Ca2+, vanadate, ketoconazole, cycloheximide and Calcofluor white, and resistance to caffeine and rapamycin. The only phenotypes found for rrd2delta - resistance to caffeine and rapamycin - were weaker than the corresponding phenotypes of rrd1delta. The double mutant rrd1,2delta was inviable on rich glucose medium, but could grow in the presence of an osmotic stabilizer. The rrd1,2delta mutant was partially rescued by inactivation of HOG1 or PBS2, suggesting an interaction between the RRD genes and the Hog1p signal transduction pathway. Introduction of slt2delta into the rrd1,2delta background improved the growth of rrd1,2delta on sorbitol-containing medium, indicating that the Rrd proteins also interact with the Slt2p/Mpk1p signaling pathway. Suppression of the lethal phenotype of the rrd1,2delta mutant by overexpression of PPH22 suggested that the products of the RRD genes function positively with catalytic subunits of PP2A. The synthetic lethality was also suppressed by the "viable" allele (SSD1-v1) of the SSD1 gene.
Assuntos
Genes Fúngicos , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Alelos , Meios de Cultura , Ativação Enzimática/genética , Deleção de Genes , Glucose , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Mutação , Concentração Osmolar , Consumo de Oxigênio , Peptidilprolil Isomerase , Fenótipo , Proteínas/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Transdução de Sinais , SorbitolRESUMO
We reported previously that the product of the DNA damage-inducible gene of Saccharomyces cerevisiae, DIN7, belongs to a family of proteins that are involved in DNA repair and replication. The family includes S. cerevisiae proteins Rad2p and its human homolog XPGC, Rad27p and its mammalian homolog FEN-1, and Exonuclease I (Exo I). Here, we report that Din7p specifically affects metabolism of mitochondrial DNA (mtDNA). We have found that dun1 strains, defective in the transcriptional activation of the DNA damage-inducible genes RNR1, RNR2, and RNR3, exhibit an increased frequency in the formation of the mitochondrial petite (rho(-)) mutants. This high frequency of petites arising in the dun1 strains is significantly reduced by the din7::URA3 allele. On the other hand, overproduction of Din7p from the DIN7 gene placed under control of the GAL1 promoter dramatically increases the frequency of petite formation and the frequency of mitochondrial mutations conferring resistance to erythromycin (E(r)). The frequencies of chromosomal mutations conferring resistance to canavanine (Can(r)) or adenine prototrophy (Ade(+)) are not affected by enhanced synthesis of Din7p. Experiments using Din7p fused to the green fluorescent protein (GFP) and cell fractionation experiments indicate that the protein is located in mitochondria. A possible mechanism that may be responsible for the decreased stability of the mitochondrial genome in S. cerevisiae cells with elevated levels of Din7p is discussed.
Assuntos
Dano ao DNA/genética , Exodesoxirribonucleases , Proteínas Fúngicas/genética , Mitocôndrias/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Sequência de Bases , Primers do DNA , Reparo do DNA/genética , DNA Mitocondrial/metabolismo , Exonucleases/metabolismo , Proteínas Fúngicas/metabolismo , Humanos , MutaçãoRESUMO
The newly discovered Saccharomyces cerevisiae gene KRR1 (YCL059c) encodes a protein essential for cell viability. Krr1p contains a motif of clustered basic amino acids highly conserved in the evolutionarly distant species from yeast to human. We demonstrate that Krr1p is localized in the nucleolus. The KRR1 gene is highly expressed in dividing cells and its expression ceases almost completely when cells enter the stationary phase. In vivo depletion of Krr1p leads to drastic reduction of 40S ribosomal subunits due to defective 18S rRNA synthesis. We propose that Krr1p is required for proper processing of pre-rRNA and the assembly of preribosomal 40S subunits.
Assuntos
Proteínas Fúngicas/fisiologia , RNA Ribossômico 18S/metabolismo , RNA Ribossômico/metabolismo , RNA Ribossômico/fisiologia , Proteínas de Ligação a RNA , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Sítios de Ligação , Northern Blotting , Western Blotting , Divisão Celular , Epitopos , Proteínas Fúngicas/genética , Modelos Genéticos , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase , Polirribossomos/metabolismo , RNA/metabolismo , Fatores de TempoRESUMO
A PCR-based method for targeted gene deletion by kanMX4 module was used to construct complete deletion mutants of six individual open reading frames from chromosome II: YBR128c, YBR131w, YBR133c, YBR137w, YBR138c and YBR142w. The ORFs were deleted in two diploid strains, FY1679 and W303. Sporulation and tetrad analysis revealed that only one ORF, YBR142w, encoding a putative DEAD-box RNA helicase, is an essential gene. A systematic phenotypic analysis of the deleted mutants was carried out. Homozygous diploids ybr128cDelta/ybr128cDelta and ybr131wDelta/ybr131wDelta did not sporulate. The ybr131cDelta mutant whether haploid or homozygous diploid, in addition displayed an increased sensitivity to Caffeine, Calcium and Zinc, and to emphasize this phenotype we named the gene CCZ1. ORF YBR133c was independently reported by others as Histone Synthetic Lethal (HSL7) (Ma et al., 1996). We found that the aberrant morphology characteristic for ybr133cDelta (hsl7Delta) cells was observed in W303 but not in FY1679 genetic background. Furthermore, we observed that deletion of YBR133c had a pleiotropic effect under a wide range of conditions, including increased sensitivity to calcium, caffeine, calcofluor white, vanadate and verapamil. The effects of the deletion were reinforced in W303 background. We found no phenotypic effects of the two remaining deletions, ybr137wDelta and ybr138cDelta.
Assuntos
Cromossomos Fúngicos/genética , Genes Fúngicos , Saccharomyces cerevisiae/genética , Antifúngicos/farmacologia , Cafeína/farmacologia , Cátions/farmacologia , Clonagem Molecular , Primers do DNA , Deleção de Genes , Genes Essenciais , Fenótipo , Reação em Cadeia da Polimerase , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/fisiologia , Esporos Fúngicos/fisiologia , Transformação Genética , Verapamil/farmacologiaRESUMO
The ability of a microorganism to adhere to a solid support and to initiate a colony is often the first stage of microbial infections. To date, studies on S. cerevisiae cell-cell and cell-solid support interactions concerned only cell agglutination during mating and flocculation. Colony formation has not been studied before probably because this species is not pathogenic. However, S. cerevisiae can be a convenient model to study this process, thanks to well-developed genetics and the full knowledge of its nucleotide sequence. A preliminary characterization of the recently cloned essential IRR1 gene indicated that it may participate in cell-cell/substrate interactions. Here we show that lowering the level of expression of IRR1 (after fusion with a regulatory catalase A gene promoter) affects colony formation and disturbs zygote formation and spore germination. All these processes involve cell-cell or cell-solid support contacts. The IRR1 protein is localized in the cytosol as verified by immunofluorescence microscopy, and confirmed by cell fractionation and Western blotting. This indicates that Irr1p is not directly involved in the cell-solid support adhesion, but may be an element of a communication pathway between the cell and its surroundings.
Assuntos
Proteínas Fúngicas/fisiologia , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiologia , Western Blotting , Adesão Celular , Proteínas de Ciclo Celular , Fracionamento Celular , Citosol/química , Imunofluorescência , Proteínas Fúngicas/análise , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Poliestirenos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Esporos Fúngicos/fisiologia , Transcrição GênicaRESUMO
We have constructed a series of chimeric yeast/mouse and yeast/Bacillus subtilis ferrochelatase genes in order to investigate domains of the ferrochelatase that are important for activity and/or association with the membrane. These genes were expressed in a Saccharomyces cerevisiae mutant in which the endogenous ferrochelatase gene (HEM15) had been deleted, and the phenotypes of the transformants were characterized. Exchanging the approximately 40-amino-acid C-terminus between the yeast and mouse ferrochelatases caused a total loss of activity and the hybrid proteins were unstable when overproduced in Escherichia coli. The water-soluble ferrochelatase of B. subtilis did not complement the yeast mutant, although a large amount of active protein accumulated in the cytosol. Addition of the N-terminal leader sequence of yeast ferrochelatase to the B. subtilis enzyme targeted the fusion protein to mitochondria, but both the precursor and the mature forms of the enzyme were inactive in vivo and had residual activity when measured in vitro. An internal approximately 45-amino-acid segment located at the N-terminus of yeast ferrochelatase was identified, which, when replaced with the corresponding 30-amino-acid segment of the B. subtilis enzyme, caused the yeast enzyme to be located in the mitochondrial matrix as a soluble protein. The fusion protein was inactive in vivo and had residual activity in vitro. We speculate that this segment, which shows the greatest variability between species, is responsible for the association of the enzyme with the membrane.
Assuntos
Bacillus subtilis/enzimologia , Ferroquelatase/genética , Ferroquelatase/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Animais , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Membrana Celular/enzimologia , Ativação Enzimática/genética , Ferroquelatase/biossíntese , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/metabolismo , Fenótipo , Sinais Direcionadores de Proteínas/síntese química , Sinais Direcionadores de Proteínas/genética , Proteínas Recombinantes de Fusão/síntese química , Proteínas Recombinantes de Fusão/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Deleção de Sequência , Homologia de Sequência de AminoácidosRESUMO
We have deleted the yeast gene YJR025c and shown that this leads to an auxotrophy for nicotinic acid. The deduced protein sequence of the gene product is homologous to the human 3-hydroxyanthranilic acid dioxygenase (EC 1.13.11.6) which is part of the kynurenine pathway for the degradation of tryptophan and the biosynthesis of nicotinic acid. In cell-free extracts the 3-hydroxyanthranilic acid dioxygenase activity is proportional to the copy number of the YJR025c gene. As YJR025c encodes the yeast 3-hydroxyanthranilic acid dioxygenase, we have named this gene BNA1 for biosynthesis of nicotinic acid.
Assuntos
Dioxigenases , Niacina/biossíntese , Oxigenases/genética , Oxigenases/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , 3-Hidroxiantranilato 3,4-Dioxigenase , Sequência de Bases , Deleção de Genes , Genes Fúngicos , Teste de Complementação Genética , Cinurenina/metabolismo , Dados de Sequência Molecular , Niacina/metabolismo , Proteínas de Saccharomyces cerevisiae , Triptofano/metabolismoRESUMO
The genome of the yeast Saccharomyces cerevisiae was sequenced by an international consortium of laboratories from Europe, Canada, the U.S.A. and Japan. This project is now finished and the complete sequence of the first eukaryotic genome was released to the public data bases in April 1996. An overview and preliminary analysis of the entire genome sequence was presented in a special issue of Nature in May 1997, entitled "The yeast genome directory". At its origin the Yeast Genome Sequencing Project provoked much debate and controversy; however, the final results obtained and the insights this has given us into the organisation and content of a eukaryotic genome have more than justified the expectations of the supporters of the project. The importance of genomic sequencing and analysis, especially of model organisms, is now widely accepted and this has resulted in the birth of the new science of genomics (Botstein & Cherry, 1997, Proc. Natl. Acad. Sci. U.S.A. 94, 5506). The information from gene and protein sequences ultimately lead to functional description of all genes. The main strategies describing possible ways to analyse the function of new genes that have been identified by systematic sequencing of Saccharomyces cerevisiae genome are described.
Assuntos
Genoma Fúngico , Saccharomyces cerevisiae/genética , Genes SupressoresRESUMO
The yeast Saccharomyces cerevisiae was used as a model eukaryotic organism to study the uptake of diamino acid derivatives of porphyrins and their phototoxicity with particular emphasis on possible mutagenic effects. The water-soluble hematoporphyrin derivatives diarginate (HpD[Arg]2) and 1-arginin di(N-amino acid)-protoporphyrinate used in this study are effective photosensitizers in tumor photodynamic therapy. Depending on the amino acid substituent, the porphyrin derivatives differ in their affinity for yeast cells. It is shown that HpD(Arg)2 and PP(Met)2 (Arg)2 penetrate into the yeast cell and are metabolized. Both compounds sensitize yeast cells to photodamage but have no mutagenic effect on nuclear or mitochondrial genomes.
Assuntos
Diamino Aminoácidos/metabolismo , Hematoporfirinas/metabolismo , Fármacos Fotossensibilizantes/metabolismo , Protoporfirinas/metabolismo , Saccharomyces cerevisiae/metabolismo , Diamino Aminoácidos/farmacologia , Hematoporfirinas/química , Hematoporfirinas/farmacologia , Mutagênese , Fotoquímica , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacologia , Protoporfirinas/química , Protoporfirinas/farmacologia , Saccharomyces cerevisiae/genéticaRESUMO
Biosynthesis of polyprenols was followed in the erg mutants of Saccharomyces cerevisiae impaired in various steps of the mevalonate pathway. The end products of the enzymatic reaction carried out in vitro, in the wild type yeast and all mutants tested, were identified as dehydrodolichols (alpha-unsaturated polyprenols) whereas in vivo, yeast synthesize dolichols (alpha-saturated polyprenols) (Biochimie, 1996.78:111-112.) The strain defective in the farnesyl diphosphate (FPP) synthase, (coded by the erg20-2 gene) required the presence of exogenous FPP for synthesis of dehydrodolichols to occur in vitro. Overexpression of the ERG20 gene restored synthesis of polyprenols in vitro indicating that FPP is the allylic "starter" for cis-prenyltransferase in yeast. Overexpression of the ERG20 gene in the erg 9 mutant, defective in squalene synthase activity, not only restored synthesis of dehydrodolichols in vitro, but also increased the synthesis of dolichols in vivo, almost 10-fold in comparison with wild type yeast. On the other hand overexpression of the mutated FPP synthase, coded by the gene erg20-2 in the same genetic background, resulted in a 100-fold increase of the amount of dehydrodolichols. Interestingly, in addition to the family of typical for yeast C60-C80 compounds, dehydrodolichols of chain length up to C135 were synthesized both in vitro and in vivo.
Assuntos
Alquil e Aril Transferases , Dolicóis/biossíntese , Saccharomyces cerevisiae/metabolismo , Transferases/biossíntese , Cromatografia Líquida de Alta Pressão , Regulação Fúngica da Expressão Gênica , Geraniltranstransferase , Transferases/genéticaRESUMO
Mutants of Saccharomyces cerevisiae accumulating uroporphyrin (UP) or protoporphyrin (PP) were used as a model for the in vivo phototoxic effect of porphyrins observed in the human skin photosensitivity associated with porphyrias (porphyria cutanea tarda and erythropoietic protoporphyria). We have found that UP is localized in vacuoles and PP is present in all compartments except vacuoles in yeast cells. Endogenous PP is much more effective as a photosensitizer of yeast cells than UP. Protoporphyrin action is strictly dependent on the presence of oxygen. In contrast, UP displays a phototoxic effect even if oxygen is not present in the suspension, implicating a free radical mechanism that operates in anaerobiosis upon photosensitization by UP. Catalase or superoxide dismutase deficiency affects photosensitization by UP. A possible mechanism of UP photosensitizing activity is discussed.