RESUMO
Partial cDNAs of different isoforms of protein phosphatase 2Cbeta (PP2Cbeta or PPM1B) have been characterized in mammals. We disclose here the full cDNAs of two major PP2Cbeta isoforms from human, rat and mouse. These cDNAs (2.6 and 3.3 kb) are able to encode 53 kDa (PP2Cbetal) and 43 kDa (PP2Cbetas) polypeptides, respectively. The isoforms are co-expressed ubiquitously with the highest level in skeletal muscle, as assessed by Northern-blot analysis. Western and in situ analyses using monoclonal antibodies against PP2Cbeta confirmed the existence of two isoforms in the cytoplasm. Comparative sequence analysis revealed that both cDNAs consist of six exons with an alternate usage of the 3' exons that underlies the differences between them. The genomic structure of PP2Cbeta is similar to that of other PP2C paralogs and includes a non-coding first exon followed by a large intron and a large second exon that encoded most of the catalytic domain. Both variants of the ending exon include large non-coding regions. All non-translated regions (NTRs) are highly conserved between the orthologous genes, indicating their regulatory function. The 5'-NTR is long (379 bp), includes upstream start codons and is predicted to contain stable secondary structures. Such features inhibit translation initiation by the scanning mechanism. Introduction of this NTR element into a bi-luciferase expression-cassette enabled expression of the second cistron, suggesting that it might serve as an internal ribosome entry site, or it contains a cryptic promoter. Overexpression of PP2Cbeta under CMV-promoter in 293 cells led to cell-growth arrest or cell death.
Assuntos
Processamento Alternativo/genética , Sequência Conservada/genética , Fosfoproteínas Fosfatases/genética , Proteínas de Saccharomyces cerevisiae , Transcrição Gênica/genética , Animais , Sequência de Bases , Domínio Catalítico , Morte Celular , Divisão Celular , Linhagem Celular , Clonagem Molecular , Citoplasma/enzimologia , Éxons/genética , Etiquetas de Sequências Expressas , Perfilação da Expressão Gênica , Humanos , Íntrons/genética , Isoenzimas/análise , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Fosfoproteínas Fosfatases/análise , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/imunologia , Biossíntese de Proteínas/genética , Proteína Fosfatase 2 , Proteína Fosfatase 2C , Transporte Proteico , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RatosRESUMO
Prion protein (PrP)(Sc), the only known component of the prion, is present mostly in the brains of animals and humans affected with prion diseases. We now show that a protease-resistant PrP isoform can also be detected in the urine of hamsters, cattle, and humans suffering from transmissible spongiform encephalopathies. Most important, this PrP isoform (UPrP(Sc)) was also found in the urine of hamsters inoculated with prions long before the appearance of clinical signs. Interestingly, intracerebrally inoculation of hamsters with UPrP(Sc) did not cause clinical signs of prion disease even after 270 days, suggesting it differs in its pathogenic properties from brain PrP(Sc). We propose that the detection of UPrP(Sc) can be used to diagnose humans and animals incubating prion diseases, as well as to increase our understanding on the metabolism of PrP(Sc) in vivo.
