RESUMO
Cellular prion protein (PrP) copurifies with neuregulin type I-ß1 (NRG I-ß1), but no interaction has been detected by a general immunoprecipitation study. We speculate that PrP interacts with NRG I-ß1. Here, the interaction of PrP with NRG I-ß1 was detected by measuring fluorescence resonance energy transfer (FRET) between enhanced blue (EBFP) and enhanced green (EGFP) fluorescent protein-fusion proteins. Full-length PrP interacted with EGFP in addition to NRG I-ß1. From this result, we deduced that PrP interacts with EGFP through its unstructured N-terminal domain. We therefore detected FRET between PrP deleting the N-terminal domain and NRG I-ß1. In contrast, the C-terminal domain of PrP interacted with NRG I-ß1 and the proteins dissociated completely in the presence of sodium chloride. This interaction occurs at the nanomolar level, which is important for the reaction to be functional in organisms. We concluded that PrP interacted with NRG I-ß1 through its C-terminal domain.
Assuntos
Neuregulina-1/metabolismo , Proteínas Priônicas/metabolismo , Transferência Ressonante de Energia de Fluorescência , Ligação ProteicaRESUMO
Mucosal vaccines can induce mucosal immunity, including antigen-specific secretory IgA production, to protect from invasion by pathogens and to neutralize toxins at mucosal surfaces. We established an effective antigen-delivering fusion protein, anti-GP2-SA, as a mucosal vaccine. The anti-GP2-SA consists of streptavidin (SA) fused to the antigen-binding fragment region from a mAb against glycoprotein 2 (GP2), an antigen-uptake receptor specifically expressed on M cells. Anti-GP2-SA targets antigen-sampling M cells in the follicle-associated epithelium covering Peyer's patches. Immunofluorescence showed that anti-GP2-SA specifically bound to M cells. Orally administered biotinylated ovalbumin peptide (bOVA) conjugated with anti-GP2-SA more efficiently induced OVA-specific fecal IgA secretion compared with bOVA alone or bOVA conjugated with SA. Furthermore, mice immunized by oral administration of the biotinylated Salmonella enterica serovar Typhimurium (S. Typhimurium) lysate conjugated with anti-GP2-SA were significantly better protected from subsequent infection by virulent S. Typhimurium than mice treated with the bacterial lysate alone or conjugated with SA. These results suggest that anti-GP2-SA-based M-cell-targeting vaccines are a novel strategy for inducing efficient mucosal immunity.
Assuntos
Antígenos/imunologia , Imunidade nas Mucosas , Imunoglobulina A/imunologia , Mucosa/imunologia , Nódulos Linfáticos Agregados/imunologia , Vacinas/imunologia , Animais , Especificidade de Anticorpos/imunologia , Antígenos de Bactérias/imunologia , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/imunologia , Imunoglobulina A Secretora/imunologia , Masculino , Camundongos , Camundongos Knockout , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Infecções por Salmonella/prevenção & controle , Salmonella typhimurium/imunologia , Vacinas/administração & dosagemRESUMO
The mucosal immune system forms the largest part of the entire immune system, containing about three-quarters of all lymphocytes and producing grams of secretory IgA daily to protect the mucosal surface from pathogens. To evoke the mucosal immune response, antigens on the mucosal surface must be transported across the epithelial barrier into organized lymphoid structures such as Peyer's patches. This function, called antigen transcytosis, is mediated by specialized epithelial M cells. The molecular mechanisms promoting this antigen uptake, however, are largely unknown. Here we report that glycoprotein 2 (GP2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for mucosal antigens. Recombinant GP2 protein selectively bound a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane. Consistently, these bacteria were colocalized with endogenous GP2 on the apical plasma membrane as well as in cytoplasmic vesicles in M cells. Moreover, deficiency of bacterial FimH or host GP2 led to defects in transcytosis of type-I-piliated bacteria through M cells, resulting in an attenuation of antigen-specific immune responses in Peyer's patches. GP2 is therefore a previously unrecognized transcytotic receptor on M cells for type-I-piliated bacteria and is a prerequisite for the mucosal immune response to these bacteria. Given that M cells are considered a promising target for oral vaccination against various infectious diseases, the GP2-dependent transcytotic pathway could provide a new target for the development of M-cell-targeted mucosal vaccines.
Assuntos
Adesinas de Escherichia coli/metabolismo , Antígenos de Bactérias/metabolismo , Células Epiteliais/imunologia , Proteínas de Fímbrias/metabolismo , Imunidade nas Mucosas/imunologia , Glicoproteínas de Membrana/metabolismo , Nódulos Linfáticos Agregados/citologia , Adesinas de Escherichia coli/genética , Adesinas de Escherichia coli/imunologia , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Linhagem Celular , Células Epiteliais/metabolismo , Escherichia coli/imunologia , Escherichia coli/metabolismo , Proteínas de Fímbrias/genética , Proteínas de Fímbrias/imunologia , Proteínas Ligadas por GPI , Glicoproteínas , Células HeLa , Humanos , Intestinos/citologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Nódulos Linfáticos Agregados/imunologia , Salmonella typhimurium/genética , Salmonella typhimurium/imunologia , Salmonella typhimurium/metabolismo , Especificidade por SubstratoRESUMO
Annexin A4 (Anx4) is a cytosolic calcium-binding protein with four repeat domains, each containing one calcium-binding site (CBS). The protein interacts with the phospholipid membrane through the CBS-coordinated calcium ion, although the role of each CBS in the calcium-dependent association is unclear. To determine the role of each CBS, 15 CBS-abolished variants were produced in various combinations by substitution of a calcium-liganding residue on each CBS by Ala. Various mutant combinations produced different influences on calcium-dependent membrane-binding behavior and on the sodium-dependent dissociation of membrane-bound Anx4. Our data suggest the interaction of Anx4 with the lipid membrane consists of strong and weak interactions. CBSs I and IV mediate formation of strong interactions, while CBSs II and III are important for weak interactions. We also suggest Anx4 binds the lipid membrane through CBSs I and IV in the cytoplasmic fluids.
Assuntos
Anexina A4/química , Anexina A4/metabolismo , Cálcio/metabolismo , Membrana Celular/metabolismo , Substituição de Aminoácidos , Animais , Anexina A4/genética , Sítios de Ligação , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , RatosRESUMO
Pyridine nucleotide coenzymes are involved in >500 enzyme reactions and are biosynthesized from the amino acid L-tryptophan (L-Trp) as well as the vitamin niacin. Hence, "true" niacin-deficient animals cannot be "created" using nutritional techniques. We wanted to establish a truly niacin-deficient model animal using a protocol that did not involve manipulating dietary L-Trp. We generated mice that are missing the quinolinic acid (QA) phosphoribosyltransferase (QPRT) gene. QPRT activity was not detected in qprt(-/-)mice. The qprt(+/+), qprt(+/-), or qprt(-/-) mice (8 wk old) were fed a complete diet containing 30 mg nicotinic acid (NiA) and 2.3 g L-Trp/kg diet or an NiA-free diet containing 2.3 g L-Trp/kg diet for 23 d. When qprt(-/-)mice were fed a complete diet, food intake and body weight gain did not differ from those of the qprt(+/+) and qprt(+/-) mice. On the contrary, in the qprt(-/-) mice fed the NiA-free diet, food intake and body weight were reduced to 60% (P < 0.01) and 70% (P < 0.05) of the corresponding values for the qprt(-/-) mice fed the complete diet at d 23, respectively. The nutritional levels of niacin, such as blood and liver NAD concentrations, were also lower in the qprt(-/-) mice than in the qprt(+/+) and the qprt(+/-) mice. Urinary excretion of QA was greater in the qprt(-/-) mice than in the qprt(+/+) and qprt(+/-) mice (P < 0.01). These data suggest that we generated truly niacin-deficient mice.
Assuntos
Modelos Animais de Doenças , Niacina/deficiência , Pentosiltransferases/deficiência , Animais , Peso Corporal , Ingestão de Alimentos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NAD/metabolismo , Niacinamida/urina , Pentosiltransferases/metabolismo , Ácido Quinolínico/urinaRESUMO
Quinolinate phosphoribosyltransferase (QAPRTase) is a key enzyme in NAD biosynthesis; it catalyzes the formation of nicotinate mononucleotide (NAMN) from quinolinate and 5-phosphoribosyl-1-pyrophosphate. In order to elucidate the mechanism of NAMN biosynthesis, crystals of Sus scrofa QAPRTase (Ss-QAPRTase) purified from porcine kidney in complex with NAMN were obtained and diffraction data were collected and processed to 2.1â Å resolution. The Ss-QAPRTase-NAMN cocrystals belonged to space group P321, with unit-cell parameters a=119.1, b=119.1, c=93.7â Å, γ=120.0°. The Matthews coefficient and the solvent content were estimated as 3.10â Å3 Da(-1) and 60.3%, respectively, assuming the presence of two molecules in the asymmetric unit.
Assuntos
Rim/enzimologia , Mononucleotídeo de Nicotinamida/análogos & derivados , Pentosiltransferases/química , Animais , Cristalografia por Raios X , Modelos Moleculares , Mononucleotídeo de Nicotinamida/química , Mononucleotídeo de Nicotinamida/metabolismo , Pentosiltransferases/metabolismo , Conformação Proteica , Suínos/metabolismoRESUMO
Quinolinate phosphoribosyltransferase (QPRTase) is a key NAD-biosynthetic enzyme which catalyzes the transfer of quinolinic acid to 5-phosphoribosyl-1-pyrophosphate, yielding nicotinic acid mononucleotide. Homo sapiens QPRTase (Hs-QPRTase) appeared as a hexamer during purification and the protein was crystallized. Diffraction data were collected and processed at 2.8â Å resolution. Native Hs-QPRTase crystals belonged to space group P2(1), with unit-cell parameters a=76.2, b=137.1, c=92.7â Å, ß=103.8°. Assuming the presence of six molecules in the asymmetric unit, the calculated Matthews coefficient is 2.46â Å3â Da(-1), which corresponds to a solvent content of 49.9%.
Assuntos
Pentosiltransferases/química , Estrutura Quaternária de Proteína , Animais , Cristalização , Cristalografia por Raios X , Humanos , Dados de Sequência Molecular , NAD/biossíntese , Pentosiltransferases/metabolismoRESUMO
Recombinant prion protein has been produced in insoluble form and refolded following solubilization with denaturants. It is, however, preferable to use a soluble recombinant protein prepared without artificial solubilization. In this study, a soluble recombinant prion protein was produced in Escherichia coli cells by coexpression of neuregulin I-ß1 and purified to high purity.
Assuntos
Expressão Gênica , Neuregulina-1/genética , Príons , Proteínas Recombinantes , Animais , Western Blotting , Escherichia coli , Humanos , Camundongos , Neuregulina-1/metabolismo , Plasmídeos , Doenças Priônicas/metabolismo , Doenças Priônicas/fisiopatologia , Príons/genética , Príons/isolamento & purificação , Príons/metabolismo , Dobramento de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Solubilidade , Espectrometria de Fluorescência , Transformação BacterianaRESUMO
Annexin A4 (Anx4) possesses four repeat domains with one Ca(2+)-binding site (CBS) in each domain. In this study, we resolved two crystal structures of the Na(+)-bound form at high resolution (1.58 and 1.35 A). This is the first report that Anx4 binds the Na(+) ion in CBSs. Electron density maps, valence screening, and atomic absorbance spectrometry confirmed that Anx4 bound the Na(+) ion. One structure (1.58 A) bound the Na(+) ion in CBS I, whereas another structure (1.35 A) bound the Na(+) ion in CBS II and CBS III. We compared the two Na(+)-bound forms by superimposing their C(alpha) traces. The C(alpha) atoms of CBS III largely moved by coordination of the Na(+) ion. In the C(alpha) atoms of CBS I, however, little change resulted from Na(+)-coordination. Only the side chain of Glu71 was moved by Na(+)-coordination in CBS I. These results indicate that Anx4 also binds not only Ca(2+) but also Na(+) ion in the CBS.
Assuntos
Anexina A4/química , Anexina A4/metabolismo , Sódio/metabolismo , Absorção , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cálcio/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Movimento , Estrutura Secundária de Proteína , RatosRESUMO
Rat P23 is an isoform of trypsin (ogens) synthesized by rat acinar cells. Expression of P23 is stimulated strongly by caerulein, an analogue of cholecystokinin (CCK). However, the physiological relevance of rat P23 in healthy and pathological conditions such as caerulein-induced pancreatitis is largely unknown. Using recombinant P23 trypsinogen and reconstitution analysis of zymogen autoactivation, unique inhibitor-resistance characteristics of P23 were elucidated. P23 cDNA was expressed in Escherichia coli periplasm, yielding recombinant P23 trypsinogen. Autoactivation of zymogen granule contents from caerulein-induced rat pancreas was also studied. Activation kinetics of P23 by enterokinase was similar to those of rat anionic trypsinogen, which is a major isoform of trypsinogen. Interestingly, rat pancreatic secretory trypsin inhibitor (PSTI), which protects against deleterious activation of trypsinogens in zymogen granules, failed to inhibit P23 trypsin even with four-fold molar excess, at which concentration it effectively inhibited rat anionic trypsin to almost 100%. P23 trypsin also showed marked resistance to proteinaceous trypsin inhibitors such as soybean trypsin inhibitor and aprotinin. P23 trypsin activated by enterokinase dramatically accelerated the cascade of autoactivation of anionic trypsinogen even in the presence of PSTI. Taken together with a previous observation that P23 is specifically upregulated 14-fold by 24-h caerulein infusion, these results suggest that elevated levels of P23 should be taken into consideration in the mechanism of trypsinogens within the pancreas in pathological conditions.
Assuntos
Tripsinogênio/biossíntese , Sequência de Aminoácidos , Animais , Ceruletídeo , Clonagem Molecular , Enteropeptidase/metabolismo , Ativação Enzimática/efeitos dos fármacos , Escherichia coli/metabolismo , Pâncreas/metabolismo , Pancreatite/induzido quimicamente , Pancreatite/metabolismo , Isoformas de Proteínas/química , Ratos , Inibidores da Tripsina/farmacologia , Tripsinogênio/química , Tripsinogênio/genéticaRESUMO
ZAP36, a zymogen granule membrane associated protein with 36 kDa, was cloned from both canine and rat pancreas. ZAP36 is found to be a novel member of annexin IV, and showed an apical localization in exocrine pancreas and an ubiquitous expression in epithelial tissues. ZAP36 may be involved in exocytosis in apical regions of polarized cells.
Assuntos
Anexina A4/genética , Anexinas/genética , Pâncreas/metabolismo , Sequência de Aminoácidos , Animais , Anexina A4/metabolismo , Anexinas/metabolismo , Sequência de Bases , Grânulos Citoplasmáticos/metabolismo , Cães , Dados de Sequência Molecular , Pâncreas/ultraestrutura , Ratos , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
To cause food-borne botulism, botulinum neurotoxin (BoNT) in the gastrointestinal lumen must traverse the intestinal epithelial barrier. However, the mechanism by which BoNT crosses the intestinal epithelial barrier remains unclear. BoNTs are produced along with one or more non-toxic components, with which they form progenitor toxin complexes (PTCs). Here we show that serotype A1 L-PTC, which has high oral toxicity and makes the predominant contribution to causing illness, breaches the intestinal epithelial barrier from microfold (M) cells via an interaction between haemagglutinin (HA), one of the non-toxic components, and glycoprotein 2 (GP2). HA strongly binds to GP2 expressed on M cells, which do not have thick mucus layers. Susceptibility to orally administered L-PTC is dramatically reduced in M-cell-depleted mice and GP2-deficient (Gp2(-/-)) mice. Our finding provides the basis for the development of novel antitoxin therapeutics and delivery systems for oral biologics.
Assuntos
Toxinas Botulínicas Tipo A/química , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Intestinos/citologia , Animais , Carboidratos/química , Clostridium botulinum , Células Dendríticas/citologia , Cães , Endocitose , Feminino , Proteínas Ligadas por GPI/metabolismo , Glutationa Transferase/metabolismo , Hemaglutininas/química , Humanos , Mucosa Intestinal/metabolismo , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Neurônios/metabolismo , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/química , Ligação Proteica , Proteínas Recombinantes de Fusão/químicaRESUMO
Rabphilin is a synaptic vesicle-associated protein proposed to play a role in regulating neurotransmitter release. Here we report the isolation and identification of a novel protein complex containing rabphilin, annexin A4 and synaptotagmin 1. We show that the rabphilin C2B domain interacts directly with the N-terminus of annexin A4 and mediates the co-complexing of these two proteins in PC12 cells. Analyzing the cellular localisation of these co-complexing proteins we find that annexin A4 is located on synaptic membranes and co-localises with rabphilin at the plasma membrane in PC12 cells. Given that rabphilin and synaptotagmin are synaptic vesicle proteins involved in neurotransmitter release, the identification of this complex suggests that annexin A4 may play a role in synaptic exocytosis.
Assuntos
Anexina A4/metabolismo , Proteínas de Ligação ao Cálcio , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Anexina A4/fisiologia , Células Cultivadas , Exocitose , Substâncias Macromoleculares , Neurônios/química , Neurônios/citologia , Células PC12 , Ligação Proteica , Mapeamento de Interação de Proteínas , Ratos , Ratos Sprague-Dawley , Membranas Sinápticas/química , Vesículas Sinápticas/química , Sinaptotagmina I , Sinaptotagminas , Proteínas de Transporte Vesicular , Rabfilina-3ARESUMO
Tryptophan is metabolized to alpha-amino-beta-carboxymuconate-epsilon-semialdehyde (ACMS) via 3-hydroxyanthranilate (3-HA). ACMS decarboxylase (ACMSD) directs ACMS to acetyl CoA; otherwise ACMS is non-enzymatically converted to quinolinate (QA), leading to the formation of NAD and its degradation products. Thus, ACMSD is a critical enzyme for tryptophan metabolism. Phthalate esters have been suspected of being environmental endocrine disrupters. Because of the structural similarity of phthalate esters with tryptophan metabolites, we examined the effects of phthalate esters on tryptophan metabolism. Phthalate esters containing diets were orally given to rats and the urinary excreted tryptophan metabolites were quantified. Of the phthalate esters with different side chains tested, di(2-ethylhexyl)phthalate (DEHP) and its metabolite, mono(2-ethylhexyl)phthalate (MEHP), most strongly enhanced the production of QA and degradation products of nicotinamide, while 3-HA was unchanged. This pattern of metabolic change led us to assume that these esters lowered ACMSD protein or its activity. Although DEHP could not be tested because of its low solubility, MEHP reversibly inhibited ACMSD from rat liver and mouse kidney, and also the recombinant human enzyme. Correlation between inhibition of ACMSD by phthalate esters with different side chains and urinary excretion of QA supports the notion that phthalate esters perturb tryptophan metabolism by inhibiting ACMSD. Quinolinate is a potential endogenous toxin and has been implicated in the pathogenesis of various disorders. Although toxicity of phthalate esters through accumulation of QA remains to be investigated, they may be detrimental by acting as metabolic disrupters when intake of a tryptophan-rich diet and exposure to phthalate esters occur coincidentally.
Assuntos
Carboxiliases/antagonistas & inibidores , Dietilexilftalato/análogos & derivados , Inibidores Enzimáticos/farmacologia , Ácidos Ftálicos/toxicidade , Ácido Quinolínico/metabolismo , Triptofano/metabolismo , Animais , Peso Corporal/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Dieta , Dietilexilftalato/toxicidade , Ingestão de Alimentos/efeitos dos fármacos , Ésteres/toxicidade , Humanos , Fígado/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos ICR , Tamanho do Órgão/efeitos dos fármacos , Ácido Quinolínico/urina , Ratos , Ratos WistarRESUMO
Quinolinate (quinolinic acid) is a potent endogenous excitotoxin of neuronal cells. Elevation of quinolinate levels in the brain has been implicated in the pathogenesis of various neurodegenerative disorders, the so-called "quinolinate hypothesis." Quinolinate is non-enzymatically derived from 2-amino-3-carboxymuconate-6-semialdehyde (ACMS). 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase (ACMSD) is the only known enzyme which can process ACMS to a benign catabolite and thus prevent the accumulation of quinolinate from ACMS. ACMSD seems to be regulated by nutritional and hormonal signals, but its molecular mechanism has, to date, been largely unknown. Utilizing partial amino acid sequences obtained from highly purified porcine kidney ACMSD, a cDNA encoding human ACMSD was cloned and characterized. The cDNA encodes a unique open reading frame of 336 amino acids and displays little homology to any known enzymes or motifs in mammalian databases, suggesting that ACMSD may contain a new kind of protein fold. Real-time PCR-based quantification of ACMSD revealed very low but significant levels of the expression in the brain. Brain ACMSD messages was down- and up-regulated in response to low protein diet and streptozocin-induced diabetes, respectively. Expression of QPRT, another enzyme which catabolizes quinolinate, was also found in the brain. This suggests that a pathway does exist by which the levels of quinolinate in the brain are regulated. In this report, we address the molecular basis underlying quinolinate metabolism and the regulation of ACMSD expression.
Assuntos
Carboxiliases/genética , Carboxiliases/metabolismo , DNA Complementar/genética , Niacina/metabolismo , Ácido Quinolínico/metabolismo , Triptofano/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Clonagem Molecular , Expressão Gênica , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , TransfecçãoRESUMO
Since kale (Brassica oleracea var. acephala), a cruciferous vegetable with a high level of vitamins and functional compounds beneficial to health and wellness, has become widely used in the juice industry, a precise method for quality control of vegetable species is necessary. We describe here a DNA-based identification method to distinguish kale from cabbage (Brassica oleracea var. capitata), a closely related species, which can be inadvertently mixed with kale during the manufacturing process. Using genomic DNA from these vegetables and combinatory sets of nucleotide primers, we screened for random amplified polymorphic DNA (RAPD) fragments and found three cabbage-specific fragments. These RAPD fragments, with lengths of 1.4, 0.5, and 1.5 kb, were purified, subcloned, and sequenced. Based on sequence-tagged sites (STS), we designed sets of primers to detect cabbage-specific identification (CAI) DNA markers. Utilizing the CAI markers, we successfully distinguished more than 10 different local cabbage accessions from 20 kale accessions, and identified kale juices experimentally spiked with different amounts of cabbage.
Assuntos
Bebidas/análise , Bebidas/classificação , Brassica/genética , DNA de Plantas/análise , Sequência de Bases , DNA de Plantas/química , Manipulação de Alimentos , Indústria Alimentícia , Dados de Sequência Molecular , Técnica de Amplificação ao Acaso de DNA PolimórficoRESUMO
We have determined the crystal structure of porcine quinolinate phosphoribosyltransferase (QAPRTase) in complex with nicotinate mononucleotide (NAMN), which is the first crystal structure of a mammalian QAPRTase with its reaction product. The structure was determined from protein obtained from the porcine kidney. Because the full protein sequence of porcine QAPRTase was not available in either protein or nucleotide databases, cDNA was synthesized using reverse transcriptase-polymerase chain reaction to determine the porcine QAPRTase amino acid sequence. The crystal structure revealed that porcine QAPRTases have a hexameric structure that is similar to other eukaryotic QAPRTases, such as the human and yeast enzymes. However, the interaction between NAMN and porcine QAPRTase was different from the interaction found in prokaryotic enzymes, such as those of Helicobacter pylori and Mycobacterium tuberculosis. The crystal structure of porcine QAPRTase in complex with NAMN provides a structural framework for understanding the unique properties of the mammalian QAPRTase active site and designing new antibiotics that are selective for the QAPRTases of pathogenic bacteria, such as H. pylori and M. tuberculosis.
Assuntos
Rim/química , Mononucleotídeo de Nicotinamida/análogos & derivados , Pentosiltransferases/química , Animais , Domínio Catalítico , Cristalografia por Raios X , DNA Complementar/genética , Helicobacter pylori/química , Helicobacter pylori/enzimologia , Humanos , Rim/enzimologia , Modelos Moleculares , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/enzimologia , Mononucleotídeo de Nicotinamida/química , Pentosiltransferases/genética , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Especificidade da Espécie , Homologia Estrutural de Proteína , SuínosRESUMO
We developed a novel method for enhancing light-microscopic visualization of pancreatic zymogen granules in a selective manner on hematoxylin and eosin-stained sections. By using an absorption filter that transmits light with wavelength from 510 to 550 nm, corresponding to the narrow absorption spectrum of eosin, only eosinophilic tissue and cellular components were remarkably highlighted as distinct shadows against lighter background consisting of basophilic components. Using a pair of mirror sections of the pancreas, immunocytochemistry with anti-amylase antibody confirmed that the shadows observed through the filter represented zymogen granules. Immersion in formalin for 36 h at room temperature was the optimal fixation condition. Here we designate the procedure as the "eosin-shadow method" and propose that this technique is convenient and useful to help investigators identify zymogen granules more easily in routine pathological examination and histological studies.
Assuntos
Amarelo de Eosina-(YS) , Hematoxilina , Técnicas Histológicas/métodos , Microscopia/métodos , Pâncreas/citologia , Vesículas Secretórias/ultraestrutura , Coloração e Rotulagem/métodos , Animais , Camundongos , Camundongos Endogâmicos ICR , Fixação de Tecidos/métodosRESUMO
The physiological function of prion proteins (PrP) remains unclear. To investigate the physiological relevance of PrP, we constructed a fusion protein of PrP with enhanced blue fluorescent protein (PrP-EBFP) to quantify the interaction of PrP with other molecules. Production of soluble PrP-EBFP was achieved by lowering the expression temperature in Escherichia coli (E. coli) cells to 15 degrees C. Soluble PrP-EBFP was purified on cation exchange and heparin-affinity columns to yield high purity protein. This is the first report of the preparation of soluble recombinant PrP without refolding following solubilization using denaturants or disruption using detergents. To confirm the integrity of PrP-EBFP, anisotropy was estimated under physiological conditions in the presence of heparin, which interacts with PrP. The dissociation constant was determined to be 0.88+/-0.07 microM. PrP-EBFP should be useful in the quantification of PrP interactions with other molecules.
Assuntos
Detergentes/farmacologia , Proteínas PrPSc/química , Dobramento de Proteína , Escherichia coli/genética , Polarização de Fluorescência , Corantes Fluorescentes/metabolismo , Heparina/metabolismo , Proteínas PrPSc/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Solubilidade , Espectrometria de Fluorescência , TemperaturaRESUMO
To investigate the role of phospholipids in exocytotic secretory events, we utilized rat pancreatic acinar AR42J cells that secreted amylase in response to cholecystokinin octapeptide (CCK-8). Wortmannin, an inhibitor of phosphoinositide 3-kinase (PI3K), was found to inhibit the secretion in a dose-dependent manner. When changes in cell membrane phospholipids were investigated before and after CCK-8 stimulation using [32P]orthophosphoric acid-labeled AR42J cells, we observed a rapid increase in phosphatidic acid (PtdOH) levels right after stimulation, which was not observed in non-stimulated cells. The increase, however, was suppressed by wortmannin pre-treatment, which also inhibited amylase secretion. Changes in other major phospholipids were not significant. These results indicate that CCK-8 induces amylase secretion through PI3K-regulated production of PtdOH in cell membranes.