RESUMEN
The centrosome is the major microtubule-organizing center and plays important roles in intracellular transport, cellular morphology, and motility. In mitotic cells, centrosomes function as spindle poles to pull a set of chromosomes into daughter cells. In quiescent cells, primary cilia are originated from the centrosomes. Given its involvement in various cellular processes, it is little surprising that the organelle would also participate in apoptotic events. However, it remains elusive how the centrosome changes in structure and organization during apoptosis. Apoptosis, a programmed cell death, is required for homeostatic tissue maintenance, embryonic development, stress responses, etc. Activation of caspases generates a cascade of apoptotic pathways, explaining much of what happens during apoptosis. Here, we report the proteolytic cleavage of selected centrosomal proteins in apoptotic cells. SAS-6, a cartwheel component of centrioles, was specifically cleaved at the border of the coiled-coil domain and the disordered C-terminus. Pericentrin, a scaffold of pericentriolar material, was also cleaved during apoptosis. These cleavages were efficiently blocked by the caspase inhibitors. We propose that the caspase-dependent proteolysis of the centrosomal proteins may destabilize the configuration of a centrosome. Loss of centrosomes may be required for the formation of apoptotic microtubule networks, which are essential for apoptotic fragmentation. This work demonstrates the first centrosomal targets by caspases during apoptosis.
Asunto(s)
Apoptosis , Caspasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centriolos/metabolismo , Proteolisis , Caspasas/genética , Proteínas de Ciclo Celular/genética , Centriolos/genética , Células HeLa , HumanosRESUMEN
Centrioles are assembled during S phase and segregated into 2 daughter cells at the end of mitosis. The initiation of centriole assembly is regulated by polo-like kinase 4 (PLK4), the major serine/threonine kinase in centrioles. Despite its importance in centriole duplication, only a few substrates have been identified, and the detailed mechanism of PLK4 has not been fully elucidated. CP110 is a coiled-coil protein that plays roles in centriolar length control and ciliogenesis in mammals. Here, we revealed that PLK4 specifically phosphorylates CP110 at the S98 position. The phospho-resistant CP110 mutant inhibited centriole assembly, whereas the phospho-mimetic CP110 mutant induced centriole assembly, even in PLK4-limited conditions. This finding implies that PLK4 phosphorylation of CP110 is an essential step for centriole assembly. The phospho-mimetic form of CP110 augmented the centrosomal SAS6 level. Based on these results, we propose that the phosphorylated CP110 may be involved in the stabilization of cartwheel SAS6 during centriole assembly.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Centriolos/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Fosfoproteínas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Células HeLa , Humanos , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/genética , Mutación Missense , Fosfoproteínas/química , Fosfoproteínas/genética , Fosforilación , Proteínas Serina-Treonina Quinasas/química , Transporte de ProteínasRESUMEN
We prepared monoclonal antibodies (mAbs) against a recombinant tethered follicle-stimulating hormone (rec-FSH) from Japanese eel Anguilla japonica that was produced in Escherichia coli. Positive hybridomas (clones eFA-C5, eFA-C10, eFA-C11, eFA-C12, eFA-C13, and eFB-C14) were selected by using the eel FSH antigen in ELISA, and anti-eel FSH mAbs were purified from culture supernatants by performing affinity chromatography. Three of the 6mAbs were characterized and their isotypes were identified as IgG2b (eFA-C5 and eFA-C11) and IgG1 (eFB-C14). In western blotting assays, the mAbs recognized the antigen as a 24.3-kDa band, and further detected bands of 34 and 32kDa in the supernatants of CHO cells transfected with cDNA encoding tethered eel FSHß/α and LHß/α, respectively. PNase F-mediated deglycosylation of the recombinant proteins resulted in a drastic reduction in their molecular weight, to 7-9kDa. The mAbs eFA-C5 and eFA-C11 recognized the eel FSHα-subunit that is commonly encoded among glycoprotein hormones, whereas eFB-C14 recognized the eel FSHß-subunit, and immunohistochemical analysis revealed that the staining by these mAbs was specifically localized in the eel pituitary. We also established an ELISA system for detecting rec-tethered FSHß/α and LHß/α produced from CHO cell lines. Measurement of biological activities in vitro revealed that only weak activity of rec-FSHß/α was detected. The activity of rec-LHß/α was found to be increased in a dose-dependent manner for eel oocyte maturation.
Asunto(s)
Anguilla , Anticuerpos Monoclonales/análisis , Anticuerpos Monoclonales/inmunología , Hormona Folículo Estimulante/inmunología , Anguilla/inmunología , Anguilla/metabolismo , Animales , Anticuerpos Monoclonales/metabolismo , Células CHO , Cricetinae , Cricetulus , Femenino , Hormona Folículo Estimulante/metabolismo , Hormona Folículo Estimulante de Subunidad beta/metabolismo , Hormona Luteinizante de Subunidad beta/metabolismo , Oogénesis , Hipófisis/metabolismo , Unión Proteica , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismoRESUMEN
A procentriole is assembled next to the mother centriole during S phase and remains associated until M phase. After functioning as a spindle pole during mitosis, the mother centriole and procentriole are separated at the end of mitosis. A close association of the centriole pair is regarded as an intrinsic block to the centriole reduplication. Therefore, deregulation of this process may cause a problem in the centriole number control, resulting in increased genomic instability. Despite its importance for faithful centriole duplication, the mechanism of centriole separation is not fully understood yet. Here, we report that centriole pairs are prematurely separated in cells whose cell cycle is arrested at M phase by STLC. Dispersal of the pericentriolar material (PCM) was accompanied. This phenomenon was independent of the separase activity but needed the PLK1 activity. Nocodazole effectively inhibited centriole scattering in STLC-treated cells, possibly by reducing the microtubule pulling force around centrosomes. Inhibition of PLK1 also reduced the premature separation of centrioles and the PCM dispersal as well. These results revealed the importance of PCM integrity in centriole association. Therefore, we propose that PCM disassembly is one of the driving forces for centriole separation during mitotic exit.
Asunto(s)
División Celular , Centriolos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centriolos/efectos de los fármacos , Células HeLa , Humanos , Nocodazol/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Moduladores de Tubulina/farmacología , Quinasa Tipo Polo 1RESUMEN
We examined morphological changes and molecular mechanisms of ion regulation in mitochondrion-rich (MR) cells of Japanese eel acclimated to different environmental salinities. Electron microscopic observations revealed that the apical membrane of MR cells appeared as a flat or slightly projecting disk with a mesh-like structure on its surface in eel acclimated to freshwater (FW). In seawater (SW)-acclimated eel, in contrast, the apical membrane of MR cells showed a slightly concave surface without a mesh-like structure. The mRNA expression of Na(+)/H(+) exchanger-3 (NHE3) in deionized FW and normal SW was higher than that in normal FW and 30%-diluted SW. Expression of Na(+)/K(+)/2Cl(-) cotransporter-1a (NKCC1a) became higher with increasing environmental salinity. Immunofluorescence staining showed that the apical NHE3 immunoreaction was stronger in deionized FW and normal SW than in the other groups. Basolateral NKCC1 immunoreaction was most intense in normal SW. These results indicate that apical NHE3 is involved in ion uptake in fish acclimated to hypotonic environments, and that basolateral NKCC1 is important for acclimation to hypertonic environments. The relatively high expression of NHE3 in SW further indicates a possible role of NHE3 in acid-base regulation in the gills in SW-acclimated fish.
Asunto(s)
Aclimatación , Anguilla/metabolismo , Proteínas de Peces/metabolismo , Branquias/metabolismo , Animales , Clonación Molecular , Proteínas de Peces/genética , Expresión Génica , Regulación de la Expresión Génica , Branquias/ultraestructura , Transporte Iónico , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Especificidad de Órganos , Osmorregulación , Filogenia , Subunidades de Proteína/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Salinidad , Tolerancia a la Sal , Intercambiadores de Sodio-Hidrógeno/genética , Intercambiadores de Sodio-Hidrógeno/metabolismo , Miembro 2 de la Familia de Transportadores de Soluto 12/genética , Miembro 2 de la Familia de Transportadores de Soluto 12/metabolismo , ATPasas de Translocación de Protón Vacuolares/genética , ATPasas de Translocación de Protón Vacuolares/metabolismoRESUMEN
In a previous study, we showed that 1,3-propanediol (1,3-PD) was still produced from glycerol by the Klebsiella pneumoniae mutant strain defective in 1,3-PD oxidoreductase (DhaT), although the production level was lower compared to the parent strain. As a potential candidate for another putative 1,3-PD oxidoreductase, we identified and characterized a homolog of Escherichia coli yqhD (88% homology in amino acid sequence), which encodes an alcohol dehydrogenase and is well known to replace the function of DhaT in E. coli. Introduction of multiple copies of the yqhD homolog restored 1,3-PD production in the mutant K. pneumoniae strain defective in DhaT. In addition, by-product formation was still eliminated in the recombinant strain due to the elimination of the glycerol oxidative pathway. An increase in NADP-dependent 1,3-PD oxidoreductase activity was observed in the recombinant strain harboring multiple copies of the yqhD homolog. The level of 1,3-PD production during batch fermentation in the recombinant strain was comparable to that of the parent strain; further engineering can generate an industrial strain producing 1,3-propanediol.
Asunto(s)
Oxidorreductasas de Alcohol/genética , Oxidorreductasas de Alcohol/metabolismo , Glicerol/metabolismo , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Glicoles de Propileno/metabolismo , Alcohol Deshidrogenasa , Vías Biosintéticas/genética , Escherichia coli/genética , Fermentación , Eliminación de Gen , Dosificación de Gen , Expresión Génica , Prueba de Complementación Genética , Isoenzimas/genética , Isoenzimas/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
Although levan produced by Bacillus amyloliquefaciens is known to have efficient immunostimulant property which gives 100% survival of common carp when infected with Aeromonas hydrophila, no detailed reports are available describing kinetic studies of D: -glucose production and levan formation. In this study, we cloned and characterized the enzymatic kinetics using levansucrase expressed in Escherichia coli. Optimum pH for D: -glucose production and levan formation was 6.0 and 8.0, respectively, whereas optimum temperature was 30 degrees C and 4 degrees C, respectively. The K (m) and V (max) values for levansucrase were calculated to be 47.81 mM sucrose and 57.47 1mole/min mg protein, respectively. Prominent expression of levansucrase was obtained through xylose induction in Bacillus megaterium, where most of the His(6)-tagged protein was secreted into the culture broth, giving levansucrase activity of 12,906 U/l. Response-surface methodology (RSM) was further employed to optimize the fermentation conditions and improve the level of levansucrase production. Maximum levansucrase activity of 20,251 U/l was obtained in 12 h of fermentation carried out at 28 degrees C, starting induction with 0.735% xylose when A (600) was 1.2, which was 1.6- and 62-fold higher than those obtained in the nonoptimized conditions for the recombinant strain and the native strain, respectively.
Asunto(s)
Bacillus/enzimología , Hexosiltransferasas/genética , Bacillus/genética , Bacillus megaterium/genética , Bacillus megaterium/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Clonación Molecular , ADN Bacteriano/genética , Escherichia coli/genética , Fermentación , Fructanos/metabolismo , Genes Bacterianos , Hexosiltransferasas/biosíntesis , Hexosiltransferasas/aislamiento & purificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Análisis de Secuencia de ADNRESUMEN
The microbial production of 1,3-propanediol (1,3-PD) by Klebsiella pneumoniae involves the formation of various by-products, which are synthesized through the oxidative pathway. To eliminate the by-products synthesis, the oxidative branch of glycerol metabolism was inactivated by constructing two mutant strains. In one of the mutant strains, the structural genes encoding glycerol dehydrogenase and dihydroxyacetone kinase were deleted from the chromosomal DNA, whereas in the second mutant strain dhaR, which is a putative transcription factor that activates, gene expression was deleted from the chromosomal DNA. In the resultant mutant strains lacking the dhaT gene encoding 1,3-PD oxidoreductase, which was simultaneously deleted while replacing the native promoter with the lacZ promoter, the by-product formation except for acetate was eliminated, but it still produced 1,3-PD at a lower level, which might be due to a putative oxidoreductase that catalyzes the production of 1,3-PD. The recombinant strains in which the reductive pathway was recovered produced slightly lower amount of 1,3-PD as compared to the parent strain, which might be due to the reduced activity of DhaB caused by the substitution of the promoter. However, the production yield was higher in the recombinant strain (0.57 mol mol(-1)) than the wild type Cu strain (0.47 mol mol(-1)).
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Glicerol/metabolismo , Klebsiella pneumoniae/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Glicoles de Propileno/metabolismo , Deshidrogenasas del Alcohol de Azúcar/genética , Biotecnología/métodos , Eliminación de Gen , Ingeniería Genética , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/crecimiento & desarrollo , Mutación , Oxidación-Reducción , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Deshidrogenasas del Alcohol de Azúcar/metabolismoRESUMEN
Leuconostoc mesenteroides B-512 FMC produces dextran and levan using sucrose. Because of the industrial importance of dextrans and oligosaccharides synthesized by dextransucrase (one of glycansucrases from L. mesenteroides), much is known about the dextransucrase, including expression and regulation of gene. However, no detailed report about levansucrase, another industrially important glycansucrase from L. mesenteroides, and its gene was available. In this paper, we report the first-time isolation and molecular characterization of a L. mesenteroides levansucrase gene (m1ft). The gene m1ft is composed of 1272-bp nucleotides and codes for a protein of 424 amino acid residues with calculated molecular mass of 47.1 kDa. The purified protein was estimated to be about 51.7 kDa including a His-tag based on SDS-PAGE. It showed an activity band at 103 kDa on a non-denaturing SDS-PAGE, indicating a dimeric form of the active M1FT. M1FT levan structure was confirmed by NMR and dot blot analysis with an anti-levan-antibody. M1FT converted 150 mM sucrose to levan (18%), 1-kestose (17%), nystose (11%) and 1,1,1-kestopentaose (7%) with the liberation of glucose. The M1FT enzyme produced erlose [O-alpha-D-glucopyranosyl-(1-->4)-O-alpha-D-glucopyranosyl-(1-->2)-beta-D-fructofuranoside] as an acceptor product with maltose. The optimum temperature and pH of this enzyme for levan formation were 30 degrees C and pH 6.2, respectively. M1FT levansucrase activity was completely abolished by 1 mM Hg2+ or Ag2+. The Km and Vmax values for levansucrase were calculated to be 26.6 mM and 126.6 micromol min-1 mg-1.
Asunto(s)
Hexosiltransferasas/genética , Leuconostoc/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Escherichia coli/genética , Concentración de Iones de Hidrógeno , Cinética , Datos de Secuencia Molecular , TemperaturaRESUMEN
We have expressed and characterized the severe acute respiratory syndrome coronavirus (SARS-CoV) spike protein in cDNA-transfected mammalian cells. The full-length spike protein (S) was newly synthesized as an endoglycosidase H (endo H)-sensitive glycoprotein (gp170) that is further modified into an endo H-resistant glycoprotein (gp180) in the Golgi apparatus. No substantial proteolytic cleavage of S was observed, suggesting that S is not processed into head (S1) and stalk (S2) domains as observed for certain other coronaviruses. While the expressed full-length S glycoprotein was exclusively cell associated, a truncation of S by excluding the C-terminal transmembrane and cytoplasmic tail domains resulted in the expression of an endoplasmic reticulum-localized glycoprotein (gp160) as well as a Golgi-specific form (gp170) which was ultimately secreted into the cell culture medium. Chemical cross-linking, thermal denaturation, and size fractionation analyses suggested that the full-length S glycoprotein of SARS-CoV forms a higher order structure of approximately 500 kDa, which is consistent with it being an S homotrimer. The latter was also observed in purified virions. The intracellular form of the C-terminally truncated S protein (but not the secreted form) also forms trimers, but with much less efficiency than full-length S. Deglycosylation of the full-length homotrimer with peptide N-glycosidase-F under native conditions abolished recognition of the protein by virus-neutralizing antisera raised against purified virions, suggesting the importance of the carbohydrate in the correct folding of the S protein. These data should aid in the design of recombinant vaccine antigens to prevent the spread of this emerging pathogen.
Asunto(s)
Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/metabolismo , Animales , Antígenos Virales/química , Antígenos Virales/genética , Antígenos Virales/inmunología , Antígenos Virales/metabolismo , Células COS , Línea Celular , Chlorocebus aethiops , Cricetinae , Medios de Cultivo/química , ADN Complementario , ADN Viral/genética , ADN Viral/metabolismo , Retículo Endoplásmico/química , Glicósido Hidrolasas/metabolismo , Aparato de Golgi/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Peso Molecular , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/metabolismo , Pliegue de Proteína , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Subunidades de Proteína/análisis , Transporte de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Glicoproteína de la Espiga del Coronavirus , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
Hepatitis C virus (HCV) is a positive-strand RNA virus that encodes a helicase required for viral replication. Although HCV does not replicate through a DNA intermediate, HCV helicase unwinds both RNA and DNA duplexes. An X-ray crystal structure of the HCV helicase complexed with (dU)(8) has been solved, and the substrate-amino acids interactions within the catalytic pocket were shown. Among these, residues W501 and V432 were reported to have base stacking interactions and to be important for the unwinding function of HCV helicase. It has been hypothesized that specific interactions between the enzyme and substrate in the catalytic pocket are responsible for the substrate specificity phenotype. We therefore mutagenized W501 and V432 to investigate their role in substrate specificity in HCV helicase. Replacement of W501, but not V432, with nonaromatic residues resulted in complete loss of RNA unwinding activity, whereas DNA unwinding activity was largely unaffected. The loss of unwinding activity was fully restored in the W501F mutant, indicating that the aromatic ring is crucial for RNA helicase function. Analysis of ATPase and nucleic acid binding activities in the W501 mutant enzymes revealed that these activities are not directly responsible for the substrate specificity phenotype. Molecular modeling of the enzyme-substrate interaction at W501 revealed a putative pi-facial hydrogen bond between the 2'-OH of ribose and the aromatic tryptophan ring. This evidence correlates with biochemical results suggesting that the pi-facial bond may play an important role in the RNA unwinding activity of the HCV NS3 protein.