Assuntos
Metabolismo dos Lipídeos , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas/metabolismo , Animais , Transporte Biológico , Citoesqueleto/metabolismo , Exocitose/fisiologia , Complexo de Golgi/metabolismo , Humanos , Fusão de Membrana/fisiologia , Ligação Proteica , Saccharomyces cerevisiaeRESUMO
Using recombinant vaccinia virus, we have expressed in mammalian cells the cDNA coding for the precursor of dermorphin, a D-alanine containing opioid peptide from the skin of the South American frog Phyllomedusa sauvagei. HeLa cells and AtT-20 cells produced prodermorphin where proline-6 of dermorphin was partly hydroxylated. This was demonstrated by digesting the partially purified precursors with trypsin and carboxypeptidase B. After immunoprecipitation and separation by HPLC, two decapeptides were detected which differed by the presence of proline or hydroxy-proline at position 6. This demonstrates that HeLa cells as well as AtT-20 cells can perform the post-translational conversion of certain proline residues to hydroxyproline in a foreign hormone precursor expressed in these cells.
Assuntos
Hidroxiprolina/metabolismo , Oligopeptídeos/biossíntese , Oligopeptídeos/metabolismo , Neoplasias Hipofisárias/patologia , Precursores de Proteínas/metabolismo , Ranidae/genética , Proteínas Recombinantes de Fusão/biossíntese , Sequência de Aminoácidos , Animais , Sequência Consenso , DNA Complementar/genética , Células HeLa , Humanos , Hidroxilação , Camundongos , Dados de Sequência Molecular , Proteínas de Neoplasias/metabolismo , Oligopeptídeos/genética , Peptídeos Opioides , Neoplasias Hipofisárias/metabolismo , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Prolina/metabolismo , Precursores de Proteínas/genética , Proteínas Recombinantes de Fusão/genética , Células Tumorais CultivadasRESUMO
We have investigated the sorting and processing of the amphibian precursor prepro-dermorphin in mammalian cells. Dermorphin, a D-alanine-containing peptide with potent opioid activity, has been isolated from the skin of the frog Phyllomedusa sauvagei. The maturation of this peptide from the precursor involves several posttranslational steps. Recombinant vaccinia viruses were used to infect AtT-20, PC12, and HeLa cells to study the sorting and processing of prepro-dermorphin. While this precursor was not processed in any of the examined cell lines, AtT-20 cells were able to process approximately 40% of a chimeric precursor consisting of the first 241 amino acids of prepro-enkephalin fused to a carboxy-terminal part of pro-dermorphin. By immunogold-EM, we could show that the chimeric protein, but not pro-dermorphin, was sorted to dense-core secretion granules. The processing products could be released upon stimulation by 8-Br-cAMP. We conclude that the pro-enkephalin part of the fusion protein contains the information for targeting to the regulated pathway of secretion, while this sorting information is missing in pro-dermorphin. This indicates that sorting mechanisms may differ between amphibian and mammalian cells.
