RESUMO
Wild-type and deglycosylated forms of human prostate-specific antigen were expressed in Chinese hamster ovary (CHO) cells as zymogens. ProPSA was collected from conditioned medium and purified using a single cation-exchange chromatographic step for the deglycosylated form and cation-exchange followed by gel filtration chromatography for the wild-type form. Recombinant wild-type proPSA produced in CHO cells has an average MW of 34.5 kDa, whereas the deglycosylated proPSA has a MW of 32.4 kDa. Both forms of proPSA were activated in vitro and the kinetic properties measured for the deglycosylated PSA are very similar to those of the wild-type recombinant PSA and the native PSA isolated from seminal fluid. These results suggest that deglycosylated PSA is likely to be very similar to native PSA with respect to its three-dimensional structure and will provide a homogeneous protein preparation necessary for X-ray crystallographic analysis.
Assuntos
Antígeno Prostático Específico/genética , Animais , Células CHO , Bovinos , Clonagem Molecular , Cricetinae , Glicosilação , Humanos , Mutagênese , Antígeno Prostático Específico/isolamento & purificação , Antígeno Prostático Específico/metabolismo , Precursores de Proteínas/genética , Precursores de Proteínas/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
Cathepsin V is a lysosomal cysteine protease that is expressed in the thymus, testis and corneal epithelium. We have determined the 1.6 A resolution crystal structure of human cathepsin V associated with an irreversible vinyl sulfone inhibitor. The fold of this enzyme is similar to the fold adopted by other members of the papain superfamily of cysteine proteases. This study provides a framework for understanding the structural basis for cathepsin V's activity and will aid in the design of inhibitors of this enzyme. A comparison of cathepsin V's active site with the active sites of related proteases revealed a number of differences, especially in the S2 and S3 subsites, that could be exploited in identifying specific cathepsin V inhibitors or in identifying inhibitors of other cysteine proteases that would be selective against cathepsin V.
Assuntos
Catepsinas/química , Cisteína Endopeptidases/química , Sítios de Ligação , Domínio Catalítico , Catepsinas/antagonistas & inibidores , Catepsinas/genética , Catepsinas/isolamento & purificação , Simulação por Computador , Cristalografia por Raios X , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/isolamento & purificação , Inibidores de Cisteína Proteinase/síntese química , Inibidores de Cisteína Proteinase/química , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Fenilalanina/análogos & derivados , Piperazinas/síntese química , Piperazinas/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Especificidade por Substrato , Sulfonas/síntese química , Sulfonas/química , Compostos de TosilRESUMO
The p53 tumor-suppressor gene has been implicated in the inducible activation of excision repair of ultraviolet (UV)-induced cyclobutane pyrimidine dimers (CPDs) in human cells. Because the large T antigen (LTAg) of the simian virus 40 (SV40) binds p53 protein and can interfere with its function, it was of interest to study DNA repair in normal human fibroblasts that had been transformed by SV40 compared with that in their nontransformed parental counterparts and to determine whether such transformation attenuated global genomic repair (GGR) of CPDs. Three methods were used to measure GGR in UV-irradiated cells: (i) an immunoassay using monoclonal antibodies specific for CPDs or 6-4 photoproducts (6-4PPs), (ii) zone sedimentation in alkaline sucrose gradients to measure the average DNA strand size after specific nicking at CPD sites in duplex DNA with T4 endonuclease V (TEV), and (iii) Southern hybridization of TEV-treated DNA with strand-specific mRNA probes to assess removal of CPDs from either strand of a defined genetic sequence in an expressed gene. Whereas repair of 6-4PPs was very similar in paired SV40-transformed and primary fibroblasts, GGR of CPDs was significantly reduced in the SV40-transformed cells. In contrast, SV40 transformation did not appreciably affect the efficiency of transcription-coupled repair. These data support the hypothesis that SV40 transformation can result in reduced levels of GGR, most likely because of the inhibition of normal p53 function by LTAg.
Assuntos
Reparo do DNA , Dímeros de Pirimidina/genética , Raios Ultravioleta , Linhagem Celular Transformada , Humanos , Vírus 40 dos Símios , Tetra-Hidrofolato Desidrogenase/genéticaRESUMO
We have used the buoyant density shift method to measure excision-repair patch lengths in UV-irradiated repair-proficient human cells and in primary fibroblasts belonging to xeroderma pigmentosum complementation group C (XP-C), in which excision repair of UV-induced photoproducts is dependent upon transcription. The patch size was found to be about 30 nucleotides for both cell types. This agrees with the size of the DNA fragments excised in vitro by the dual incisions of the structure-specific nucleases XPG and ERCC1-XPF. We conclude that the XPC protein is not required to target the excision nucleases to sites of DNA cleavage in transcribed strands of expressed genes or to protect the newly incised DNA from further processing by exonucleases.
Assuntos
Reparo do DNA/genética , DNA/genética , Linhagem Celular , DNA/química , Fibroblastos , Fibrossarcoma , Humanos , Peso Molecular , Dímeros de Pirimidina , Tetra-Hidrofolato Desidrogenase/genética , Transcrição Gênica , Células Tumorais Cultivadas , Raios Ultravioleta , Xeroderma Pigmentoso/genéticaRESUMO
We have discovered a new DNA endonuclease in the fission yeast Schizosaccharomyces pombe which recognizes cyclobutane pyrimidine dimers and (6-4) pyrimidine-pyrimidone photoproducts. S. pombe DNA endonuclease (SPDE) catalyzes a single ATP-independent incision immediately 5' to the UV photoproduct and generates termini containing 3' hydroxyl and 5' phosphoryl groups. Based on these properties, we propose that SPDE may function in a DNA repair capacity, representing the initial recognition/cleavage step of a DNA excision repair pathway.
