RESUMEN
CBP501 is a chemically modified peptide composed of twelve unnatural d-amino acids, which inhibits Chk kinase and abrogates G2 arrest induced by DNA-damaging agents. Here we identified an alphaC helix in 14-3-3 protein as a CBP501-binding site using T7 phage display technology. An affinity selection of T7 phage-displayed peptide using biotinylated CBP501 identified a 14-mer peptide NSDCIISRKIEQKE. This peptide sequence showed similarity to a portion of the alphaC helix of human 14-3-3ε, suggesting that CBP501 may bind to this region. Surface plasmon resonance (SPR) and ELISA demonstrated that CBP501 interacts with 14-3-3ε specifically at the screen-guided region. An avidin-agarose bead pull-down assay showed that CBP501 also binds to other 14-3-3 isoforms in Jurkat cells. Among the other known Chk kinase inhibitors tested, CBP501 showed the strongest affinity for 14-3-3ε. Thus, we conclude that in addition to the direct inhibition of Chk kinase activity, CBP501 directly binds to cellular 14-3-3 proteins through alphaC helix.
Asunto(s)
Proteínas 14-3-3/metabolismo , Bacteriófago T7/metabolismo , Fragmentos de Péptidos/metabolismo , Péptidos/metabolismo , Fosfatasas cdc25/metabolismo , Proteínas 14-3-3/genética , Secuencia de Aminoácidos , Bacteriófago T7/genética , Sitios de Unión , Dicroismo Circular , Humanos , Células Jurkat , Modelos Moleculares , Datos de Secuencia Molecular , Biblioteca de Péptidos , Péptidos/química , Péptidos/genética , Estructura Secundaria de ProteínaRESUMEN
The eukaryotic GINS complex is essential for the establishment of DNA replication forks and replisome progression. We report the crystal structure of the human GINS complex. The heterotetrameric complex adopts a pseudo symmetrical layered structure comprising two heterodimers, creating four subunit-subunit interfaces. The subunit structures of the heterodimers consist of two alternating domains. The C-terminal domains of the Sld5 and Psf1 subunits are connected by linker regions to the core complex, and the C-terminal domain of Sld5 is important for core complex assembly. In contrast, the C-terminal domain of Psf1 does not contribute to the stability of the complex but is crucial for chromatin binding and replication activity. These data suggest that the core complex ensures a stable platform for the C-terminal domain of Psf1 to act as a key interaction interface for other proteins in the replication-initiation process.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Proteínas Cromosómicas no Histona/química , Replicación del ADN , Complejos Multiproteicos/química , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Miembro 3 de la Subfamilia B de Transportadores de Casetes de Unión a ATP , Proteínas Portadoras/química , Cristalografía por Rayos X , Proteínas de Unión al ADN/química , Dimerización , Humanos , Subunidades de Proteína/químicaRESUMEN
This study investigates the separation of two types of marrow stromal cells, KUSA-A1 osteoblasts and H-1/A preadipocytes, by filtration through various porous polymeric membranes. It was found that KUSA-A1 permeates better than H-1/A cells through 12-microm polyurethane foaming membranes. This appears to be due to the relatively smaller cell size of KUSA-A1 cells. In addition, when feed solutions containing suspensions of either cell type or a mixture of the two were used, the permeation ratio was relatively low (< 6%) through polyurethane and surface-modified polyurethane foaming membranes. It was also found that there was some degree of separation between KUSA-A1 and H-1/A cells (separation factor = 1.8) with nylon-net filter membranes, but no separation was obtained when filters made of nonwoven fabrics or silk screens were used. This ability of the nylon-net filter membranes to separate the two cell types was due to a sieving effect that results from an optimal pore size. Finally, permeation of a solution of human serum albumin through the membrane following filtration of the cells did not result in a separation of cells in the recovery solution.
Asunto(s)
Materiales Biocompatibles/química , Separación Celular/instrumentación , Separación Celular/métodos , Membranas Artificiales , Mesodermo/patología , Polímeros/química , Células Madre/citología , Adipocitos/citología , Animales , Células de la Médula Ósea/citología , Membrana Celular , Citometría de Flujo , Mesodermo/ultraestructura , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Microscopía Electrónica de Rastreo , Modelos Químicos , Osteoblastos/metabolismo , Poliuretanos/química , Dispersión de Radiación , Células Madre/ultraestructura , Células del Estroma/citología , Propiedades de SuperficieRESUMEN
Spiropyran is a photoresponsive molecule, and nonionic spiropyran is reversibly changed by UV irradiation to a hydrophilic polar, zwitterionic merocyanine isomer, and back again by visible light irradiation. A copolymer of nitrobenzospiropyran and methyl methacrylate, poly(NSP-co-MMA) was used as a material with a photosensitive surface. UV irradiation of the photosensitive surface of poly(NSP-co-MMA)-coated glass plates decreased the water contact angles (11 +/- 1 degrees ) and increased diameter of a water drop relative to the unexposed surface. Light-induced detachment of platelets and mesenchymal stem (KUSA-A1) cells on poly(NSP-co-MMA)-coated glass plates was observed upon simple- and patterned-light irradiation, whereas no light-induced detachment of platelets and mesenchymal stem cells was observed on poly(methyl methacrylate)-coated glass plates. This is a result of the change from a closed nonpolar spiropyran to the polar zwitterionic merocyanine isomer induced by UV irradiation. Light-induced detachment of fibrinogen adsorbed on poly(NSP-co-MMA) coated glass plates was also observed in this investigation.
