RESUMO
Presenilins are components of the gamma-secretase protein complex that mediates intramembranous cleavage of betaAPP and Notch proteins. A C. elegans genetic screen revealed two genes, aph-1 and pen-2, encoding multipass transmembrane proteins, that interact strongly with sel-12/presenilin and aph-2/nicastrin. Human aph-1 and pen-2 partially rescue the C. elegans mutant phenotypes, demonstrating conserved functions. The human genes must be provided together to rescue the mutant phenotypes, and the inclusion of presenilin-1 improves rescue, suggesting that they interact closely with each other and with presenilin. RNAi-mediated inactivation of aph-1, pen-2, or nicastrin in cultured Drosophila cells reduces gamma-secretase cleavage of betaAPP and Notch substrates and reduces the levels of processed presenilin. aph-1 and pen-2, like nicastrin, are required for the activity and accumulation of gamma-secretase.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Proteínas de Caenorhabditis elegans/isolamento & purificação , Membrana Celular/metabolismo , Endopeptidases/metabolismo , Proteínas de Homeodomínio/isolamento & purificação , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide , Precursor de Proteína beta-Amiloide/genética , Animais , Ácido Aspártico Endopeptidases , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Membrana Celular/ultraestrutura , Células Cultivadas , Clonagem Molecular , Proteínas de Drosophila , Drosophila melanogaster , Elementos Facilitadores Genéticos/genética , Glucagon/metabolismo , Peptídeo 1 Semelhante ao Glucagon , Proteínas de Helminto/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Proteínas de Membrana/genética , Dados de Sequência Molecular , Mutação/genética , Fragmentos de Peptídeos/metabolismo , Presenilina-1 , Precursores de Proteínas/metabolismo , Receptores Notch , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Transdução de Sinais/genéticaRESUMO
Presenilin is the enzymatic component of gamma-secretase, a multisubunit intramembrane protease that processes several transmembrane receptors, such as the amyloid precursor protein (APP). Mutations in human Presenilins lead to altered APP cleavage and early-onset Alzheimer's disease. Presenilins also play an essential role in Notch receptor cleavage and signaling. The Notch pathway is a highly conserved signaling pathway that functions during the development of multicellular organisms, including vertebrates, Drosophila, and C. elegans. Recent studies have shown that Notch signaling is sensitive to perturbations in subcellular trafficking, although the specific mechanisms are largely unknown. To identify genes that regulate Notch pathway function, we have performed two genetic screens in Drosophila for modifiers of Presenilin-dependent Notch phenotypes. We describe here the cloning and identification of 19 modifiers, including nicastrin and several genes with previously undescribed involvement in Notch biology. The predicted functions of these newly identified genes are consistent with extracellular matrix and vesicular trafficking mechanisms in Presenilin and Notch pathway regulation and suggest a novel role for gamma-tubulin in the pathway.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas de Membrana/genética , Receptores Notch/genética , Alelos , Precursor de Proteína beta-Amiloide/genética , Animais , Cruzamentos Genéticos , Elementos Facilitadores Genéticos , Matriz Extracelular , Feminino , Masculino , Mutação , Presenilina-1 , Receptores Notch/metabolismo , Transdução de Sinais , Tubulina (Proteína)/metabolismoRESUMO
Human studies support the use of beta-lactams and tetracyclines in the treatment of leptospirosis. Additional agents from these and other classes of antimicrobials also have in vitro activity against Leptospira species, though corroborating in vivo data are limited or lacking. We evaluated the therapeutic efficacy of azithromycin, clarithromycin, and telithromycin in a lethal hamster model of leptospirosis using Leptospira interrogans serogroup Canicola serovar Portlandvere. A range of dosages for each antimicrobial was given to the infected animals on days 2 through 7 (5 days) of the 21-day survival model. All untreated control animals survived less than 10 days from infection. Ninety to 100% of doxycycline controls, treated for 5 days with 5 mg/kg of body weight of drug, survived to 21 days. Treatment with azithromycin (daily dose: 6.25, 12.5, 25, 50, 100, or 200 mg/kg) resulted in 100% survival at all evaluated doses. Animals receiving 20 mg/kg or more of clarithromycin (daily dose: 1, 5, 10, 15, 20, 40, 60, or 100 mg/kg) had improved survival. Ninety-eight percent of animals treated with telithromycin (daily dose: 1, 5, 10, 15, 20, or 40 mg/kg) survived. We conclude that all agents tested have demonstrated in vivo efficacy in treating acute leptospirosis. These results provide support for further evaluation of macrolide and ketolide antimicrobial agents in human trials.