RESUMEN
Chlamydia trachomatis is an obligate intracellular bacterium that replicates within a vacuole termed an inclusion. At the end of their intracellular developmental cycle, chlamydiae are released either by lysis of the host cell or extrusion of the intact inclusion. The inclusion membrane is extensively modified by the insertion of type III secreted inclusion membrane proteins, Incs, which contribute to inclusion membrane structure and facilitate host-pathogen interactions. An interaction was identified between the inclusion membrane protein, MrcA, and the Ca2+ channel inositol-1,4,5-trisphosphate receptor, type 3 (ITPR3). ITPR3 was recruited and localized to active Src-family-kinase rich microdomains on the inclusion membrane as was the Ca2+ sensor, STIM1. Disruption of MrcA by directed mutagenesis resulted in loss of ITPR3 recruitment and simultaneous reduction of chlamydial release by extrusion. Complementation of MrcA restored ITPR3 recruitment and extrusion. Inhibition of extrusion was also observed following siRNA depletion of host ITPR3 or STIM1. Chlamydial extrusion was also inhibited by the calcium chelator BAPTA-AM. Each of these treatments resulted in a concomitant reduction in phosphorylation of the myosin regulatory light chain (MLC2) and a loss of myosin motor activity at the end of the developmental cycle which is consistent with the reduced extrusion formation. These studies suggest that Ca2+ signaling pathways play an important role in regulation of release mechanisms by C. trachomatis.
Asunto(s)
Infecciones por Chlamydia/metabolismo , Chlamydia trachomatis/metabolismo , Interacciones Huésped-Patógeno , Cuerpos de Inclusión/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Proteínas de la Membrana/metabolismo , Infecciones por Chlamydia/genética , Infecciones por Chlamydia/microbiología , Chlamydia trachomatis/genética , Células HeLa , Humanos , Cuerpos de Inclusión/microbiología , Receptores de Inositol 1,4,5-Trifosfato/genética , Proteínas de la Membrana/genética , FosforilaciónRESUMEN
Prion diseases are caused by the propagation of PrPSc, the pathological conformation of the PrPC prion protein. The molecular mechanisms underlying PrPSc propagation are still unsolved and no therapeutic solution is currently available. We thus sought to identify new anti-prion molecules and found that flunarizine inhibited PrPSc propagation in cell culture and significantly prolonged survival of prion-infected mice. Using an in silico therapeutic repositioning approach based on similarities with flunarizine chemical structure, we tested azelastine, duloxetine, ebastine, loperamide and metixene and showed that they all have an anti-prion activity. Like flunarizine, these marketed drugs reduced PrPSc propagation in cell culture and in mouse cerebellum organotypic slice culture, and inhibited the protein folding activity of the ribosome (PFAR). Strikingly, some of these drugs were also able to alleviate phenotypes due to PABPN1 nuclear aggregation in cell and Drosophila models of oculopharyngeal muscular dystrophy (OPMD). These data emphasize the therapeutic potential of anti-PFAR drugs for neurodegenerative and neuromuscular proteinopathies.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Flunarizina/administración & dosificación , Proteína I de Unión a Poli(A)/metabolismo , Enfermedades por Prión/metabolismo , Agregado de Proteínas/efectos de los fármacos , Pliegue de Proteína/efectos de los fármacos , Animales , Bloqueadores de los Canales de Calcio/administración & dosificación , Línea Celular , Bases de Datos Factuales , Drosophila , Femenino , Ratones , Ratones Transgénicos , Técnicas de Cultivo de Órganos , Proteína I de Unión a Poli(A)/antagonistas & inhibidores , Proteína I de Unión a Poli(A)/genética , Enfermedades por Prión/tratamiento farmacológico , Enfermedades por Prión/genética , Proteínas Priónicas/antagonistas & inhibidores , Proteínas Priónicas/genética , Proteínas Priónicas/metabolismo , Agregado de Proteínas/fisiología , OvinosRESUMEN
6AP and GA are potent inhibitors of yeast and mammalian prions and also specific inhibitors of PFAR, the protein-folding activity borne by domain V of the large rRNA of the large subunit of the ribosome. We therefore explored the link between PFAR and yeast prion [PSI(+)] using both PFAR-enriched mutants and site-directed methylation. We demonstrate that PFAR is involved in propagation and de novo formation of [PSI(+)]. PFAR and the yeast heat-shock protein Hsp104 partially compensate each other for [PSI(+)] propagation. Our data also provide insight into new functions for the ribosome in basal thermotolerance and heat-shocked protein refolding. PFAR is thus an evolutionarily conserved cell component implicated in the prion life cycle, and we propose that it could be a potential therapeutic target for human protein misfolding diseases.
Asunto(s)
Proteínas de Choque Térmico/metabolismo , Factores de Terminación de Péptidos/metabolismo , Priones/metabolismo , Pliegue de Proteína , Ribosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Guanabenzo/farmacología , Proteínas de Choque Térmico/genética , Mutación , Factores de Terminación de Péptidos/genética , Fenantridinas/farmacología , Priones/genética , Pliegue de Proteína/efectos de los fármacos , ARN Ribosómico/metabolismo , Ribosomas/genética , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Guanabenz (GA) is an orally active α2-adrenergic agonist that has been used for many years for the treatment of hypertension. We recently described that GA is also active against both yeast and mammalian prions in an α2-adrenergic receptor-independent manner. These data suggest that this side-activity of GA could be explored for the treatment of prion-based diseases and other amyloid-based disorders. In this perspective, the potent antihypertensive activity of GA happens to be an annoying side-effect that could limit its use. In order to get rid of GA agonist activity at α2-adrenergic receptors, we performed a structure-activity relationship study around GA based on changes of the chlorine positions on the benzene moiety and then on the modifications of the guanidine group. Hence, we identified the two derivatives 6 and 7 that still possess a potent antiprion activity but were totally devoid of any agonist activity at α2-adrenergic receptors. Similarly to GA, 6 and 7 were also able to inhibit the protein folding activity of the ribosome (PFAR) which has been suggested to be involved in prion appearance/maintenance. Therefore, these two GA derivatives are worth being considered as drug candidates.
Asunto(s)
Agonistas de Receptores Adrenérgicos alfa 2/farmacología , Guanabenzo/análogos & derivados , Guanabenzo/farmacología , Fármacos Neuroprotectores/farmacología , Priones/efectos de los fármacos , Agonistas de Receptores Adrenérgicos alfa 2/química , Animales , Células CHO , Bovinos , Cerebelo/efectos de los fármacos , Cerebelo/fisiopatología , Cricetulus , Escherichia coli , Guanabenzo/química , Humanos , Ratones Endogámicos C57BL , Ratones Transgénicos , Estructura Molecular , Fármacos Neuroprotectores/química , Proteínas PrPSc/metabolismo , Enfermedades por Prión/tratamiento farmacológico , Enfermedades por Prión/fisiopatología , Pliegue de Proteína/efectos de los fármacos , Receptores Adrenérgicos alfa 2/metabolismo , Ribosomas/efectos de los fármacos , Ribosomas/metabolismo , Relación Estructura-Actividad , Técnicas de Cultivo de Tejidos , LevadurasRESUMEN
Using a yeast-based assay, a previously unsuspected antiprion activity was found for imiquimod (IQ), a potent Toll-like receptor 7 (TLR7) agonist already used for clinical applications. The antiprion activity of IQ was first detected against yeast prions [PSI (+) ] and [URE3], and then against mammalian prion both ex vivo in a cell-based assay and in vivo in a transgenic mouse model for prion diseases. In order to facilitate structure-activity relationship studies, we conducted a new synthetic pathway which provides a more efficient means of producing new IQ chemical derivatives, the activity of which was tested against both yeast and mammalian prions. The comparable antiprion activity of IQ and its chemical derivatives in the above life forms further emphasizes the conservation of prion controlling mechanisms throughout evolution. Interestingly, this study also demonstrated that the antiprion activity of IQ and IQ-derived compounds is independent from their ability to stimulate TLRs. Furthermore, we found that IQ and its active chemical derivatives inhibit the protein folding activity of the ribosome (PFAR) in vitro.