RESUMO
NADPH oxidases (NOX) are transmembrane proteins, widely spread in eukaryotes and prokaryotes, that produce reactive oxygen species (ROS). Eukaryotes use the ROS products for innate immune defense and signaling in critical (patho)physiological processes. Despite the recent structures of human NOX isoforms, the activation of electron transfer remains incompletely understood. SpNOX, a homolog from Streptococcus pneumoniae, can serves as a robust model for exploring electron transfers in the NOX family thanks to its constitutive activity. Crystal structures of SpNOX full-length and dehydrogenase (DH) domain constructs are revealed here. The isolated DH domain acts as a flavin reductase, and both constructs use either NADPH or NADH as substrate. Our findings suggest that hydride transfer from NAD(P)H to FAD is the rate-limiting step in electron transfer. We identify significance of F397 in nicotinamide access to flavin isoalloxazine and confirm flavin binding contributions from both DH and Transmembrane (TM) domains. Comparison with related enzymes suggests that distal access to heme may influence the final electron acceptor, while the relative position of DH and TM does not necessarily correlate with activity, contrary to previous suggestions. It rather suggests requirement of an internal rearrangement, within the DH domain, to switch from a resting to an active state. Thus, SpNOX appears to be a good model of active NOX2, which allows us to propose an explanation for NOX2's requirement for activation.
Assuntos
NADPH Oxidases , Oxirredutases , Humanos , NADPH Oxidases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Raios X , Transporte de Elétrons , Oxirredutases/metabolismo , Flavinas/química , Flavinas/metabolismoRESUMO
Candida albicans and C. glabrata express exporters of the ATP-binding cassette (ABC) superfamily and address them to their plasma membrane to expel azole antifungals, which cancels out their action and allows the yeast to become multidrug resistant (MDR). In a way to understand this mechanism of defense, we describe the purification and characterization of Cdr1, the membrane ABC exporter mainly responsible for such phenotype in both species. Cdr1 proteins were functionally expressed in the baker yeast, tagged at their C-terminal end with either a His-tag for the glabrata version, cgCdr1-His, or a green fluorescent protein (GFP) preceded by a proteolytic cleavage site for the albicans version, caCdr1-P-GFP. A membrane Cdr1-enriched fraction was then prepared to assay several detergents and stabilizers, probing their level of extraction and the ATPase activity of the proteins as a functional marker. Immobilized metal-affinity and size-exclusion chromatographies (IMAC, SEC) were then carried out to isolate homogenous samples. Overall, our data show that although topologically and phylogenetically close, both proteins display quite distinct behaviors during the extraction and purification steps, and qualify cgCdr1 as a good candidate to characterize this type of proteins for developing future inhibitors of their azole antifungal efflux activity.
Assuntos
Antifúngicos , Azóis , Candida albicans , Farmacorresistência Fúngica , Proteínas Fúngicas , Proteínas de Membrana Transportadoras , Azóis/farmacologia , Azóis/química , Azóis/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/isolamento & purificação , Antifúngicos/farmacologia , Antifúngicos/química , Antifúngicos/isolamento & purificação , Candida albicans/efeitos dos fármacos , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Candida glabrata/efeitos dos fármacos , Candida glabrata/genética , Candida glabrata/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/químicaRESUMO
The quest for targeted therapies is critical in the battle against cancer. The RAS/MAP kinase pathway is frequently implicated in neoplasia, with ERK playing a crucial role as the most distal kinase in the RAS signaling cascade. Our previous research demonstrated that the interaction between ERK and MYD88, an adaptor protein in innate immunity, is crucial for RAS-dependent transformation and cancer cell survival. In this study, we examine the biological consequences of disrupting the ERK-MYD88 interaction through the ERK D-recruitment site (DRS), while preserving ERK's kinase activity. Our results indicate that EI-52, a small-molecule benzimidazole targeting ERK-MYD88 interaction induces an HRI-mediated integrated stress response (ISR), resulting in immunogenic apoptosis specific to cancer cells. Additionally, EI-52 exhibits anti-tumor efficacy in patient-derived tumors and induces an anti-tumor T cell response in mice in vivo. These findings suggest that inhibiting the ERK-MYD88 interaction may be a promising therapeutic approach in cancer treatment.