RESUMEN
HIV-1 Rev mediates the nuclear export of intron-containing viral RNA transcripts and is essential for viral replication. Rev is imported into the nucleus by the host protein importin ß (Impß), but how Rev associates with Impß is poorly understood. Here, we report biochemical, mutational, and biophysical studies of the Impß/Rev complex. We show that Impß binds two Rev monomers through independent binding sites, in contrast to the 1:1 binding stoichiometry observed for most Impß cargos. Peptide scanning data and charge-reversal mutations identify the N-terminal tip of Rev helix α2 within Rev's arginine-rich motif (ARM) as a primary Impß-binding epitope. Cross-linking mass spectrometry and compensatory mutagenesis data combined with molecular docking simulations suggest a structural model in which one Rev monomer binds to the C-terminal half of Impß with Rev helix α2 roughly parallel to the HEAT-repeat superhelical axis, whereas the other monomer binds to the N-terminal half. These findings shed light on the molecular basis of Rev recognition by Impß and highlight an atypical binding behavior that distinguishes Rev from canonical cellular Impß cargos.
Asunto(s)
VIH-1 , beta Carioferinas , VIH-1/metabolismo , Modelos Estructurales , Simulación del Acoplamiento Molecular , ARN Viral/metabolismo , beta Carioferinas/genética , beta Carioferinas/metabolismoRESUMEN
Invasive fungal infections cause significant morbidity and mortality among immunocompromised individuals, posing an urgent need for new antifungal therapeutic strategies. Here we investigate a chromatin-interacting module, the bromodomain (BD) from the BET family of proteins, as a potential antifungal target in Candida albicans, a major human fungal pathogen. We show that the BET protein Bdf1 is essential in C. albicans and that mutations inactivating its two BDs result in a loss of viability in vitro and decreased virulence in mice. We report small-molecule compounds that inhibit C. albicans Bdf1 with high selectivity over human BDs. Crystal structures of the Bdf1 BDs reveal binding modes for these inhibitors that are sterically incompatible with the human BET-binding pockets. Furthermore, we report a dibenzothiazepinone compound that phenocopies the effects of a Bdf1 BD-inactivating mutation on C. albicans viability. These findings establish BET inhibition as a promising antifungal therapeutic strategy and identify Bdf1 as an antifungal drug target that can be selectively inhibited without antagonizing human BET function.
Asunto(s)
Antifúngicos/farmacología , Candida albicans/efectos de los fármacos , Candidiasis/tratamiento farmacológico , Proteínas Fúngicas/antagonistas & inhibidores , Terapia Molecular Dirigida , Factores de Transcripción/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Antifúngicos/síntesis química , Compuestos de Azabiciclo/síntesis química , Compuestos de Azabiciclo/farmacología , Azepinas/farmacología , Benzodiazepinas/farmacología , Sitios de Unión , Candida albicans/crecimiento & desarrollo , Candida albicans/metabolismo , Candida albicans/patogenicidad , Candidiasis/microbiología , Cristalografía por Rayos X , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Expresión Génica , Humanos , Ratones , Modelos Moleculares , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Piridinas/síntesis química , Piridinas/farmacología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Factores de Transcripción/química , Factores de Transcripción/genética , Triazoles/farmacologíaRESUMEN
Two challenges were identified to improve the place of PEP in combined prevention: (1) improvement of healthcare professionals’ knowledge, practices and attitudes; and (2) revision of the guidelines concerning first-line prescription, the conditions for access to PEP, and sexual health support..
Asunto(s)
Infecciones por VIH/prevención & control , Profilaxis Posexposición , Adulto , Femenino , Accesibilidad a los Servicios de Salud , Humanos , MasculinoRESUMEN
The Wnt pathway inhibitors DKK1 and sclerostin (SOST) are important therapeutic targets in diseases involving bone loss or damage. It has been appreciated that Wnt coreceptors LRP5/6 are also important, as human missense mutations that result in bone overgrowth (bone mineral density, or BMD, mutations) cluster to the E1 propeller domain of LRP5. Here, we report a crystal structure of LRP6 E1 bound to an antibody, revealing that the E1 domain is a peptide recognition module. Remarkably, the consensus E1 binding sequence is a close match to a conserved tripeptide motif present in all Wnt inhibitors that bind LRP5/6. We show that this motif is important for DKK1 and SOST binding to LRP6 and for inhibitory function, providing a detailed structural explanation for the effect of the BMD mutations.