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2.
Mol Genet Genomics ; 269(4): 562-73, 2003 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12827498

RESUMO

The GAS1-related genes of fungi encode GPI-anchored proteins with beta-1,3-glucanosyltransferase activity. Loss of this activity results in defects in the assembly of the cell wall. We isolated mutants that show a synthetic defect when combined with a gas1Delta allele in Saccharomyces cerevisiae, and identified nine wild-type genes that rescue this defect. The indispensability of BIG1 and KRE6 for the viability of gas1Delta cells confirmed the important role of beta-1,6-glucan in cells that are defective in the processing of beta-1,3-glucan. The identification of the Wsc1p hypo-osmotic stress sensor and components of the PKC signal transduction pathway in our screen also confirmed that the cell wall integrity response attenuates the otherwise lethal gas1Delta defect. Unexpectedly, we found that the KEX2 gene is also required for the viability of the gas1Delta mutant. Kex2p is a Golgi/endosome-membrane-anchored protease that processes secretory preproteins. A cell wall defect was also found in the kex2Delta mutant, which was suppressible by multiple copies of the MKC7 or YAP3 gene, both of which encode other GPI-anchored proteases. Therefore, normal cell wall assembly requires proteolytic processing of secretory preproteins. Furthermore, the genes CSG2 and IPT1 were found to be required for normal growth of gas1Delta cells in the presence of 1 M sorbitol. This finding suggests that complex sphingolipids play a role in the hyper-osmotic response.


Assuntos
Genes Letais , Glicoproteínas de Membrana/genética , Pró-Proteína Convertases , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Parede Celular/genética , Parede Celular/metabolismo , Mutação , Saccharomyces cerevisiae/metabolismo , Subtilisinas/genética , Subtilisinas/metabolismo
3.
Biosci Biotechnol Biochem ; 65(8): 1812-23, 2001 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-11577722

RESUMO

Clofibrate-induced retrograde Golgi membrane movement was blocked or retarded when NRK cells were treated with sodium azide/2-deoxyglucose, nocodazole, taxol, and destruxin B, indicating that it depends on energy, and the dynamic state of microtubules, and being acidic or vacuolar-type ATPase function. PDMP and phospholipase A2 inhibitors also blocked it. These characteristics are similar to those of brefeldin A (BFA) and nordihydroguaiaretic acid (NDGA), inducers of retrograde Golgi membrane movement. However, clofibrate was distinguished from BFA in that BFA action was insensitive to phospholipase A2 inhibitors and from NDGA in that NDGA stabilized microtubules against nocodazole and its action was almost insensitive to taxol. The trans Golgi network (TGN) was resistant to clofibrate, while BFA and NDGA dispersed it. To our knowledge, clofibrate is the first drug to show such different effects on the Golgi and TGN and, therefore, is expected to be a useful tool to distinguish their architecture and/or membrane dynamics.


Assuntos
Anticolesterolemiantes/farmacologia , Clofibrato/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Complexo de Golgi/efeitos dos fármacos , Adenosina Trifosfatases/antagonistas & inibidores , Trifosfato de Adenosina/fisiologia , Animais , Anticorpos Monoclonais/farmacologia , Brefeldina A/farmacologia , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Corantes Fluorescentes , Membranas Intracelulares/efeitos dos fármacos , Masoprocol/farmacologia , Microscopia de Fluorescência , Fosfolipases A/antagonistas & inibidores , Fosfolipases A2 , Ratos
4.
J Antibiot (Tokyo) ; 54(6): 489-93, 2001 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-11513037

RESUMO

In the course of screening for inhibitors of intracellular trafficking of glycoprotein, a new inhibitor, F13459 was isolated from the culture broth of a Penicillium sp. It was purified using solvent extraction, silica gel, Sephadex LH-20 and ODS column chromatography. From structural analysis, F13459 was a derivative of mycophenolic acid, an inhibitor of inosine 5'-monophosphate dehydrogenase. F13459 inhibited hemagglutinin synthesis of NDV at concentrations more than 25 microg/ml. However, syncytium formation as a result of cell surface expression of F-glycoprotein of NDV was inhibited at concentrations of F13459 lower than those required for appreciable inhibition of glycoprotein synthesis.


Assuntos
Antivirais/isolamento & purificação , Ácido Micofenólico/análogos & derivados , Ácido Micofenólico/isolamento & purificação , Ácido Micofenólico/farmacologia , Penicillium/química , Animais , Antivirais/farmacologia , Células Cultivadas , Cricetinae , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Células Gigantes/efeitos dos fármacos , Hemaglutininas/biossíntese , Rim/citologia , Microscopia Eletrônica de Varredura , Vírus da Doença de Newcastle/efeitos dos fármacos , Vírus da Doença de Newcastle/metabolismo , Penicillium/classificação , Proteínas Virais de Fusão/antagonistas & inibidores , Proteínas Virais de Fusão/biossíntese
5.
J Antibiot (Tokyo) ; 54(6): 494-500, 2001 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-11513038

RESUMO

F13459 is a new inhibitor of synthesis and trafficking of virus glycoprotein isolated from the culture broth of a Penicillium sp. The molecular formula of F13459 was determined to be C27H28O11 by HRFAB-MS and NMR spectral analyses. The structure of F13459 was elucidated to be 3,4-dihydro-3,4,6,8-tetrahydroxy-3-methyl-1H-2-benzopyran-1-one 4-O-mycophenolate, an ester derivative of mycophenolic acid. F13459 was isolated as the optically inactive form. F13459 exists in epimeric mixtures at C-3' through relatively fast hemiacetal-ketone tautomerism and at C-4' through slow keto-enol tautomerism. Those epimerizations were confirmed by NOE differential experiments for fast chemical exchange and equilibrium and by deuteration experiments in NMR for slow chemical exchange.


Assuntos
Antivirais/química , Ácido Micofenólico/química , Penicillium/química , Fenômenos Químicos , Físico-Química , Espectroscopia de Ressonância Magnética , Ácido Micofenólico/análogos & derivados , Espectrometria de Massas de Bombardeamento Rápido de Átomos , Estereoisomerismo
6.
Yeast ; 18(11): 1003-14, 2001 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-11481671

RESUMO

Saccharomyces cerevisiae Sly1 protein is a member of the Sec1/Munc18-family proteins, which are essential for vesicular trafficking, but their exact biological roles are yet to be determined. A temperature-sensitive sly1 mutant arrests the vesicular transport from the ER to Golgi compartments at 37 degrees C. We screened for multicopy suppressor genes that restore the colony formation of the sly1(ts) mutant to discover functionally interacting components. The suppressor genes obtained were classified as: (1) those that encode a multifunctional suppressor, SSD1; (2) heat shock proteins, SSB1 and SSB2; (3) cell surface proteins, WSC1, WSC2 and MID2; (4) ER-Golgi transport proteins, USO1 and BET1; and (5) an as-yet-uncharacterized protein, HSD1 (high-copy suppressor of SLY1 defect 1). By epitope tagging of the gene product, we found that Hsd1 protein is an ER-resident membrane protein. Its overproduction induced enlargement of ER-like membrane structures.


Assuntos
Proteínas de Transporte/genética , Retículo Endoplasmático/metabolismo , Proteínas Fúngicas/genética , Genes Supressores , Complexo de Golgi/metabolismo , Proteínas de Membrana Transportadoras , Proteínas Repressoras/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Proteínas de Transporte Vesicular , Sequência de Aminoácidos , Proteínas de Transporte/metabolismo , Mapeamento de Epitopos , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Dosagem de Genes , Genes Fúngicos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Proteínas Munc18 , Mutação , Transporte Proteico , Proteínas Qc-SNARE , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Proteínas SNARE , Saccharomyces cerevisiae/metabolismo , Temperatura
7.
Biosci Biotechnol Biochem ; 65(6): 1369-78, 2001 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-11471737

RESUMO

PDMP (D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol) and PPMP (D,L-threo-1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol), inhibitors of glucosylceramide synthesis, blocked brefeldin A (BFA)- and nordihydroguaiaretic acid-induced dispersal of the Golgi and trans Golgi network, and Golgi-derived vesicles were retained in the juxtanuclear region. PDMP and PPMP did not stabilize microtubules but blocked nocodazole-induced extensive fragmentation and dispersal of the Golgi, and large Golgi vesicles were retained in the juxtanuclear region. PPMP is a stronger inhibitor of glucosylceramide synthesis than PDMP, but PDMP showed a stronger activity against BFA-induced retrograde membrane flow. However, PPMP showed a stronger activity for Golgi disruption and inhibition of anterograde trafficking from the endoplasmic reticulum, and rebuilding of the Golgi architecture. Cumulatively, these results suggest that sphingolipid metabolism is implicated in maintenance of the Golgi architecture and anterograde membrane flow from the endoplasmic reticulum but not in Golgi dispersal induced by BFA.


Assuntos
Membrana Celular/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Glucosilceramidas/antagonistas & inibidores , Glucosilceramidas/biossíntese , Complexo de Golgi/efeitos dos fármacos , Morfolinas/farmacologia , Esfingolipídeos/metabolismo , Esfingolipídeos/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Antineoplásicos/antagonistas & inibidores , Antineoplásicos/farmacologia , Brefeldina A/antagonistas & inibidores , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/ultraestrutura , Retículo Endoplasmático/ultraestrutura , Corantes Fluorescentes , Complexo de Golgi/ultraestrutura , Inibidores de Lipoxigenase/farmacologia , Masoprocol/farmacologia , Microscopia de Fluorescência , Microtúbulos/efeitos dos fármacos , Microtúbulos/ultraestrutura , Nocodazol/antagonistas & inibidores , Nocodazol/farmacologia , Ratos
8.
Biosci Biotechnol Biochem ; 65(4): 996-8, 2001 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11388491

RESUMO

Induction of retrograde trafficking of mannosidase II and TGN38 in NRK cells and inhibition of alpha-glucosidase in vitro by nordihydroguaiaretic acid (NDGA) were strongly interfered with by serum, serum albumin, or other unrelated proteins added to the medium or incubation mixture. These observations indicate that NDGA interacts with diverse kinds of proteins, and therefore, pharmacological effects of NDGA at cellular levels should be carefully interpreted.


Assuntos
Retículo Endoplasmático/metabolismo , Glucosidases/metabolismo , Complexo de Golgi/metabolismo , Masoprocol/farmacologia , Proteínas/metabolismo , Animais , Linhagem Celular , Neoplasias do Colo/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Complexo de Golgi/efeitos dos fármacos , Células Tumorais Cultivadas
9.
Nature ; 409(6816): 113-5, 2001 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-11343125

RESUMO

Helical filaments driven by linear molecular motors are anticipated to rotate around their axis, but rotation consistent with the helical pitch has not been observed. 14S dynein and non-claret disjunctional protein (ncd) rotated a microtubule more efficiently than expected for its helical pitch, and myosin rotated an actin filament only poorly. For DNA-based motors such as RNA polymerase, transcription-induced supercoiling of DNA supports the general picture of tracking along the DNA helix. Here we report direct and real-time optical microscopy measurements of rotation rate that are consistent with high-fidelity tracking. Single RNA polymerase molecules attached to a glass surface rotated DNA for >100 revolutions around the right-handed screw axis of the double helix with a rotary torque of >5 pN nm. This real-time observation of rotation opens the possibility of resolving individual transcription steps.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , DNA/metabolismo , Transcrição Gênica , DNA/química , RNA Polimerases Dirigidas por DNA/química , Escherichia coli/enzimologia , Corantes Fluorescentes , Magnetismo , Microscopia , Microesferas , Proteínas Motores Moleculares , Conformação de Ácido Nucleico , RNA/metabolismo , Rotação
10.
Biochem Biophys Res Commun ; 281(2): 582-8, 2001 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-11181087

RESUMO

Arachidonyltrifluoromethy ketone (AACOCF(3)), a phospholipase A(2) antagonist, reversibly induced dispersal of Golgi stack- and trans Golgi network (TGN)-resident proteins throughout the cytoplasm in NRK cells as followed by immunocytochemical staining of ManII and TGN38, respectively. The action of AACOCF(3) was partly blocked by other PLA(2) antagonists, suggesting it be not caused by a general inhibition of phospholipase A(2). AACOCF(3) neither dissociated beta-COP from membranes nor prevented brefeldin A-induced beta-COP release. Action of AACOCF(3) on the Golgi stack and TGN is different from that of brefeldin A and nordihydroguaiaretic acid. The most prominent difference is that the Golgi stack and TGN showed a similar sensitivity to AACOCF(3), while the TGN was dispersed more slowly than the Golgi stack in brefeldin A- or nordihydroguaiaretic acid-treated NRK cells. This novel action of AACOCF(3) may be used as pharmacological tool and give new insights into vesicle-mediated traffic and Golgi membrane dynamics.


Assuntos
Ácidos Araquidônicos/farmacologia , Citoplasma/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Complexo de Golgi/efeitos dos fármacos , Proteínas de Membrana/efeitos dos fármacos , Aminobenzoatos/farmacologia , Animais , Transporte Biológico/efeitos dos fármacos , Brefeldina A/farmacologia , Linhagem Celular , Clorobenzoatos , Cinamatos/farmacologia , Proteína Coatomer/efeitos dos fármacos , Proteína Coatomer/metabolismo , Citoplasma/metabolismo , Relação Dose-Resposta a Droga , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Masoprocol/farmacologia , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência , Naftalenos/farmacologia , Fosfolipases A/antagonistas & inibidores , Fosfolipases A/metabolismo , Pironas/farmacologia , Fatores de Tempo , ortoaminobenzoatos/farmacologia
11.
Biosci Biotechnol Biochem ; 64(5): 1103-5, 2000 May.
Artigo em Inglês | MEDLINE | ID: mdl-10879494

RESUMO

Natural azasugars have the ring oxygen substituted by nitrogen. They show potent inhibitory activity against glycosidases. The effect of substituting the ring carbon with nitrogen was examined with 1-azafagomine. 1-Azafagomine exhibited similar activity against processing glucosidase to that of fagomine.


Assuntos
Inibidores Enzimáticos/farmacologia , Glucosidases/antagonistas & inibidores , Indolizinas/farmacologia , 1-Desoxinojirimicina/análogos & derivados , 1-Desoxinojirimicina/farmacologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Glucosidases/metabolismo , Glucosídeos/metabolismo , Humanos , Imino Piranoses , Piperidinas/farmacologia , Processamento de Proteína Pós-Traducional , Especificidade por Substrato
12.
J Histochem Cytochem ; 48(2): 251-8, 2000 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-10639491

RESUMO

The autofluorescent substance monodansylcadaverine has recently been reported as a specific in vivo marker for autophagic vacuoles. However, the mechanism for this specific labeling remained unclear. Our results reveal that the common model of ion trapping in acidic compartments cannot completely account for the observed autophagic vacuole staining. Because autophagic vacuoles are characterized by myelin-like membrane inclusions, we tested whether this lipid-rich environment is responsible for the staining properties of monodansylcadaverine. In in vitro experiments using either liposomes or solvents of different polarity, monodansylcadaverine showed an increased relative fluorescence intensity in a hydrophobic environment as well as a Stokes shift dependent on the solvent polarity. To test the effect of autophagic vacuoles or autophagic vacuole lipids on monodansylcadaverine fluorescence, we isolated autophagic vacuoles and purified autophagic vacuole lipids depleted of proteins. Entire autophagic vacuoles and autophagic vacuole lipids had the same effect on monodansylcadaverine fluorescence properties, suggesting lipids as the responsible component. Our results suggest that the in vivo fluorescence properties of monodansylcadaverine do not depend exclusively on accumulation in acidic compartments by ion trapping but also on an effective interaction of this molecule with autophagic vacuole membrane lipids. (J Histochem Cytochem 48:251-258, 2000)


Assuntos
Cadaverina/análogos & derivados , Depsipeptídeos , Corantes Fluorescentes/química , Vacúolos/metabolismo , 2-Naftilamina/análogos & derivados , 2-Naftilamina/química , Cadaverina/química , Cadaverina/metabolismo , Caseínas/química , Corantes Fluorescentes/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Lipídeos/química , Lipossomos/efeitos dos fármacos , Lipossomos/metabolismo , Peptídeos Cíclicos/farmacologia , Soroalbumina Bovina/química , Células Tumorais Cultivadas , Vacúolos/efeitos dos fármacos
13.
J Bacteriol ; 180(7): 1700-8, 1998 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-9537365

RESUMO

The Saccharomyces cerevisiae SCS2 gene has been cloned as a suppressor of inositol auxotrophy of CSE1 and hac1/ire15 mutants (J. Nikawa, A. Murakami, E. Esumi, and K. Hosaka, J. Biochem. 118:39-45, 1995) and has homology with a synaptobrevin/VAMP-associated protein, VAP-33, cloned from Aplysia californica (P. A. Skehel, K. C. Martin, E. R. Kandel, and D. Bartsch, Science 269:1580-1583, 1995). In this study we have characterized an SCS2 gene product (Scs2p). The product has a molecular mass of 35 kDa and is C-terminally anchored to the endoplasmic reticulum, with the bulk of the protein located in the cytosol. The disruption of the SCS2 gene causes yeast cells to exhibit inositol auxotrophy at temperatures of above 34 degrees C. Genetic studies reveal that the overexpression of the INO1 gene rescues the inositol auxotrophy of the SCS2 disruption strain. The significant primary structural feature of Scs2p is that the protein contains the 16-amino-acid sequence conserved in yeast and mammalian cells. The sequence is required for normal Scs2p function, because a mutant Scs2p that lacks the sequence does not complement the inositol auxotrophy of the SCS2 disruption strain. Therefore, the Scs2p function might be conserved among eukaryotic cells.


Assuntos
Retículo Endoplasmático/química , Proteínas Fúngicas/análise , Inositol/metabolismo , Proteínas de Membrana/análise , Saccharomyces cerevisiae/química , Sequência de Aminoácidos , Sequência Conservada , Proteínas Fúngicas/fisiologia , Proteínas de Membrana/fisiologia , Dados de Sequência Molecular , Peso Molecular , Proteínas R-SNARE , Saccharomyces cerevisiae/metabolismo
14.
Biosci Biotechnol Biochem ; 62(2): 390-2, 1998 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-9532803

RESUMO

We investigated the mechanism of multidrug resistance conferred by overexpression of bfr2+/pad1+/sks1+ or pap1+ genes of Schizosaccharomyces pombe. Overexpression of bfr2+ did not confer multidrug resistance on a pap1-disrupted strain. In a mutant with bfr1+ (a putative membrane transporter which belongs to the ATP-binding cassette superfamily) disrupted, overexpression of either bfr2+ or pap1+ did not confer multidrug resistance. These findings suggest that bfr1+ acts as the most downstream effector of the multidrug resistance conferred by bfr2+ and pap1+ genes.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Antifúngicos/farmacologia , Ciclopentanos/farmacologia , Resistência a Múltiplos Medicamentos/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/genética , Proteínas Repressoras/genética , Proteínas de Saccharomyces cerevisiae , Schizosaccharomyces/efeitos dos fármacos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/fisiologia , Antibacterianos/farmacologia , Brefeldina A , Carbazóis/farmacologia , Citocalasina B/farmacologia , Dactinomicina/farmacologia , Sinergismo Farmacológico , Inibidores Enzimáticos/farmacologia , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/fisiologia , Alcaloides Indólicos , Macrolídeos , Micotoxinas/farmacologia , Proteínas Associadas a Pancreatite , Fenótipo , Proteína Quinase C/antagonistas & inibidores , Inibidores da Síntese de Proteínas/farmacologia , Proteínas Repressoras/biossíntese , Proteínas Repressoras/fisiologia , Schizosaccharomyces/genética , Schizosaccharomyces/fisiologia , Análise de Sequência de DNA , Estaurosporina/farmacologia
15.
J Biol Chem ; 273(3): 1654-61, 1998 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-9430709

RESUMO

Trichothecene mycotoxins such as deoxynivalenol, 4,15-diacetoxyscirpenol, and T-2 toxin, are potent protein synthesis inhibitors for eukaryotic organisms. The 3-O-acetyl derivatives of these toxins were shown to reduce their in vitro activity significantly as assessed by assays using a rabbit reticulocyte translation system. The results suggested that the introduction of an O-acetyl group at the C-3 position in the biosynthetic pathway works as a resistance mechanism for Fusarium species that produce t-type trichothecenes (trichothecenes synthesized via the precursor trichotriol). A gene responsible for the 3-O-acetylation reaction, Tri101, has been successfully cloned from a Fusarium graminearum cDNA library that was designed to be expressed in Schizosaccharomyces pombe. Fission yeast transformants were selected for their ability to grow in the presence of T-2 toxin, and this strategy allowed isolation of 25 resistant clones, all of which contained a cDNA for Tri101. This is the first drug-inactivating O-acetyltransferase gene derived from antibiotic-producing organisms. The open reading frame of Tri101 codes for a polypeptide of 451 amino acid residues, which shows no similarity to any other proteins reported so far. TRI101 from recombinant Escherichia coli catalyzes O-acetylation of the trichothecene ring specifically at the C-3 position in an acetyl-CoA-dependent manner. By using the Tri101 cDNA as a probe, two least overlapping cosmid clones that cover a region of 70 kilobase pairs have been isolated from the genome of F. graminearum. Other trichothecene biosynthetic genes, Tri4, Tri5, and Tri6, were not clustered in the region covered by these cosmid clones. These new cosmid clones are considered to be located in other parts of the large biosynthetic gene cluster and might be useful for the study of trichothecene biosynthesis.


Assuntos
Acetiltransferases/genética , Micotoxinas/farmacologia , Tricotecenos/biossíntese , Acetilação , Acetiltransferases/química , Acetiltransferases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Cricetinae , Modelos Químicos , Dados de Sequência Molecular , Plasmídeos/efeitos dos fármacos , Coelhos , Toxina T-2/farmacologia
16.
J Biol Chem ; 272(8): 4795-803, 1997 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-9030535

RESUMO

The vacuolar membrane H+-ATPase (V-ATPase) of the yeast Saccharomyces cerevisiae is composed of peripheral catalytic (V1) and integral membrane (V0) domains. The 17-kDa proteolipid subunit (VMA3 gene product; Vma3p) is predicted to constitute at least part of the proton translocating pore of V0. Recently, two VMA3 homologues, VMA11 and VMA16 (PPA1), have been identified in yeast, and VMA11 has been shown to be required for the V-ATPase activity. Cells disrupted for the VMA16 gene displayed the same phenotypes as those lacking either Vma3p or Vma11p; the mutant cells lost V-ATPase activity and failed to assemble V-ATPase subunits onto the vacuolar membrane. Epitope-tagged Vma11p and Vma16p were detected on the vacuolar membrane by immunofluorescence microscopy. Density gradient fractionation of the solubilized vacuolar proteins demonstrated that the tagged proteins copurified with the V-ATPase complex. We conclude that Vma11p and Vma16p are essential subunits of the V-ATPase. Vma3p contains a conserved glutamic acid residue (Glu137) whose carboxyl side chain is predicted to be important for proton transport activity. Mutational analysis of Vma11p and Vma16p revealed that both proteins contain a glutamic acid residue (Vma11p Glu145 and Vma16p Glu108) functionally similar to Vma3p Glu137. These residues could only be functionally substituted by an aspartic acid residue, because other mutations we examined inactivated the enzyme activity. Assembly and vacuolar targeting of the enzyme complex was not inhibited by these mutations. These results suggest that the three proteolipid subunits have similar but not redundant functions, each of which is most likely involved in proton transport activity of the enzyme complex. Yeast cells contain V0 and V1 subcomplexes in the vacuolar membrane and in the cytosol, respectively, that can be assembled into the active V0V1 complex in vivo. Surprisingly, loss-of-function mutations of either Vma11p Glu145 or Vma16p Glu108 resulted in a higher degree of assembly of the V1 subunits onto the V0 subcomplex in the vacuolar membrane.


Assuntos
Proteínas Fúngicas/metabolismo , Proteolipídeos/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimologia , ATPases Vacuolares Próton-Translocadoras , Sequência de Aminoácidos , Membrana Celular , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Dados de Sequência Molecular , Mutação , Proteolipídeos/genética , Análise de Sequência
17.
Microbiol Immunol ; 41(7): 563-70, 1997.
Artigo em Inglês | MEDLINE | ID: mdl-9272702

RESUMO

Peptides derived from endogenous proteins are presented by MHC class I molecules, whereas those derived from exogenous proteins are presented by MHC class II molecules. This strict segregation has been reconsidered in recent reports in which exogenous antigens are shown to be presented by MHC class I molecules in the phagocytic pathway. In this report, the presentation pathway of an exogenously added highly antigenic polypeptide encoded by the murine AIDS (MAIDS) defective virus gag p12 gene is investigated. A 25-mer polypeptide (P12-25) encoded within the gag p12 region of the MAIDS defective virus was found to be effective in stimulating unprimed B6 (H-2b) CD8+ T cells in vitro. The presentation of P12-25 is sensitive to cytochalasin B and D, brefeldin A and gelonin, a ribosome-inactivating protein synthesis inhibitor, but less sensitive or resistant to lactacystin, a highly specific inhibitor of the proteasome. Interestingly, CA-074, a selective inhibitor of cathepsin B, inhibited presentation of the polypeptide, indicating its involvement in the degradation of the P12-25 polypeptide. In fact, when P12-25 was digested with purified cathepsin B in vitro, a highly antigenic 11-mer peptide containing the class I (H-2Db)-binding motif was obtained. Our results favor the phagosome/macropinosome-to-cytosol-to-endoplasmic reticulum (ER)-to-cell surface pathway for exogenous antigens presented by MHC class I molecules. These findings may be relevant to exploiting peptide vaccines that specifically elicit CD8+ T cell immunity in vivo.


Assuntos
Produtos do Gene gag/imunologia , Vírus da Leucemia Murina/genética , Vírus da Leucemia Murina/imunologia , Complexo Principal de Histocompatibilidade/imunologia , Síndrome de Imunodeficiência Adquirida Murina/imunologia , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacologia , Sequência de Aminoácidos , Animais , Apresentação de Antígeno , Antivirais/farmacologia , Brefeldina A , Linfócitos T CD8-Positivos/imunologia , Catepsina B/metabolismo , Ciclopentanos/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Citocalasina B/farmacologia , Citocalasina D/farmacologia , Dipeptídeos/farmacologia , Feminino , Produtos do Gene gag/genética , Antígenos H-2/imunologia , Interleucina-2/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Síndrome de Imunodeficiência Adquirida Murina/genética , Inibidores da Síntese de Ácido Nucleico/farmacologia , Proteínas de Plantas/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , Proteínas Inativadoras de Ribossomos Tipo 1
19.
J Antibiot (Tokyo) ; 49(11): 1119-26, 1996 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-8982341

RESUMO

The nonapeptide leucinostatin A (LSA) inhibited syncytium formation without profoundly affecting HN glycoprotein synthesis in Newcastle disease virus (NDV)-infected BHK cells. At similar doses of LSA, cytopathic effect and infectious virus production were suppressed in vesicular stomatitis virus (VSV)-infected BHK cells. Blockade by LSA of cell surface expression of NDV-HN and VSV-G glycoproteins was demonstrated, accompanied by intracellular accumulation of these virus glycoproteins. LSA acts as an inhibitor of mitochondrial F-type H(+)-translocating ATPase, a key enzyme in the generation of ATP, but its action against cell surface expression of virus glycoproteins was independent of the depletion of intracellular ATP. LSA also acts as an ionophore, but its action on intoxication by ricin and diphtheria toxin was different from that of monensin. This novel action of LSA is expected to be useful in investigation of the mechanism of intracellular trafficking of proteins.


Assuntos
Antibacterianos/farmacologia , Antibióticos Antineoplásicos/farmacologia , Rim/efeitos dos fármacos , Rim/virologia , Glicoproteínas de Membrana/biossíntese , Vírus da Doença de Newcastle/efeitos dos fármacos , Peptídeos , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacos , Proteínas Virais/efeitos dos fármacos , Animais , Peptídeos Catiônicos Antimicrobianos , Células Cultivadas , Cricetinae , Ionóforos/farmacologia , Glicoproteínas de Membrana/efeitos dos fármacos , Vírus da Doença de Newcastle/metabolismo , Vírus da Estomatite Vesicular Indiana/metabolismo , Proteínas Virais/biossíntese
20.
Biochem Biophys Res Commun ; 227(3): 800-9, 1996 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-8886013

RESUMO

During the course of screening studies to identify inhibitors of intracellular protein trafficking, we isolated efrapeptins as active principles. These compounds arrested syncytium formation (SF) and cytopathic effect (CPE) in Newcastle disease virus (NDV)- and vesicular stomatitis virus (VSV)-infected BHK cells, respectively, without profoundly affecting glycoprotein synthesis. Efrapeptins blocked cell surface expression of NDV-HN and VSV-G glycoproteins, but did not suppress intoxication by ricin or diphtheria toxin even after prolonged pretreatment. Efrapeptins are inhibitors of F-ATPase, or ATP synthase, but their inhibitory effect on SF and CPE was independent of the amount of intracellular ATP.


Assuntos
Antibacterianos/farmacologia , Endocitose/efeitos dos fármacos , Exocitose/efeitos dos fármacos , Peptídeos , Trifosfato de Adenosina/metabolismo , Animais , Transporte Biológico , Linhagem Celular , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Cricetinae , Efeito Citopatogênico Viral/efeitos dos fármacos , Toxina Diftérica/toxicidade , Células Gigantes , Microscopia de Fluorescência , Vírus da Doença de Newcastle/efeitos dos fármacos , Vírus da Doença de Newcastle/fisiologia , ATPases Translocadoras de Prótons/antagonistas & inibidores , ATPases Translocadoras de Prótons/metabolismo , Ricina/antagonistas & inibidores , Ricina/toxicidade , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacos , Vírus da Estomatite Vesicular Indiana/fisiologia , Proteínas Virais/biossíntese
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