RESUMEN
The G protein-coupled receptors of the Frizzled (FZD) family, in particular FZD1,2,7, are receptors that are exploited by Clostridioides difficile toxin B (TcdB), the major virulence factor responsible for pathogenesis associated with Clostridioides difficile infection. We employ a live-cell assay examining the affinity between full-length FZDs and TcdB. Moreover, we present cryoelectron microscopy structures of TcdB alone and in complex with full-length FZD7, which reveal that large structural rearrangements of the combined repetitive polypeptide domain are required for interaction with FZDs and other TcdB receptors, constituting a first step for receptor recognition. Furthermore, we show that bezlotoxumab, an FDA-approved monoclonal antibody to treat Clostridioides difficile infection, favors the apo-TcdB structure and thus disrupts binding with FZD7. The dynamic transition between the two conformations of TcdB also governs the stability of the pore-forming region. Thus, our work provides structural and functional insight into how conformational dynamics of TcdB determine receptor binding.
Asunto(s)
Toxinas Bacterianas , Compuestos de Boro , Clostridioides difficile , Infecciones por Clostridium , Humanos , Microscopía por CrioelectrónRESUMEN
Heterotrimeric G proteins constitute the primary transducers of G proteincoupled receptor (GPCR) signaling. In addition to mediating ligand-induced GPCR activation, G proteins transduce basal signaling in various physiological and pathophysiological settings evoked by constitutively active, native GPCRs or disease-related receptor mutants. Optical biosensors have been developed and optimized to monitor GPCR ligandinduced activation of G proteins, but these biosensors cannot be used to detect constitutively active GPCRs. Here, we designed and validated eight bioluminescence resonance energy transfer (BRET)based G protein sensors that can measure the activity of all four major families of G proteins. We also established a protocol to identify constitutive GPCR or G protein signaling in live cells. These G proteinbased, tricistronic activity sensors (G-CASE) rely on the encoding of all three G protein subunits by a single plasmid, enabling their expression at the desired relative amounts and resulting in reduced signal variability in mammalian cells. We also present an experimental protocol to use the G-CASE sensor toolbox to quantify constitutive signaling of native and mutated GPCRs through these heterotrimeric transducers. This approach will help to characterize constitutively active GPCRs and their role in health and disease.
Asunto(s)
Técnicas Biosensibles , Proteínas de Unión al GTP , Proteínas de Unión al GTP/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transducción de SeñalRESUMEN
Cardiovascular disease remains the leading cause of morbidity and mortality globally. Extracellular vesicles (EVs), a group of heterogeneous nanosized cell-derived vesicles, have attracted great interest as liquid biopsy material for biomarker discovery in a variety of diseases including cardiovascular disease. Because EVs inherit bioactive components from parent cells and are able to transfer their contents to recipient cells, EVs hold great promise as potential cell-free therapeutics and drug delivery systems. However, the development of EV-based diagnostics, therapeutics or drug delivery systems has been challenging due to the heterogenicity of EVs in biogenesis, size and cellular origin, the lack of standardized isolation and purification methods as well as the low production yield. In this review, we will provide an overview of the recent advances in EV-based biomarker discovery, highlight the potential usefulness of EVs and EV mimetics for therapeutic treatment and drug delivery in cardiovascular disease. In view of the fast development in this field, we will also discuss the challenges of current methodologies for isolation, purification and fabrication of EVs and potential alternatives.
Asunto(s)
Enfermedades Cardiovasculares/metabolismo , Vesículas Extracelulares/metabolismo , Biomarcadores/metabolismo , Sistemas de Liberación de Medicamentos , Vesículas Extracelulares/química , HumanosRESUMEN
The WNT signaling system governs critical processes during embryonic development and tissue homeostasis, and its dysfunction can lead to cancer. Details concerning selectivity and differences in relative binding affinities of 19 mammalian WNTs to the cysteine-rich domain (CRD) of their receptors-the ten mammalian Frizzleds (FZDs)-remain unclear. Here, we used eGFP-tagged mouse WNT-3A for a systematic analysis of WNT interaction with every human FZD paralogue in HEK293A cells. Employing HiBiT-tagged full-length FZDs, we studied eGFP-WNT-3A binding kinetics, saturation binding, and competition binding with commercially available WNTs in live HEK293A cells using a NanoBiT/BRET-based assay. Further, we generated receptor chimeras to dissect the contribution of the transmembrane core to WNT-CRD binding. Our data pinpoint distinct WNT-FZD selectivity and shed light on the complex WNT-FZD binding mechanism. The methodological development described herein reveals yet unappreciated details of the complexity of WNT signaling and WNT-FZD interactions, providing further details with respect to WNT-FZD selectivity.
RESUMEN
Pharmacogenomic drug labels in the Summary of Product Characteristics (SmPC) provide an instrument for clinical implementation of pharmacogenomics. We compared pharmacogenomic guidance by Clinical Pharmacogenetics Implementation Consortium (CPIC), Dutch Pharmacogenetics Working Group (DPWG), the US Food and Drug Administration (FDA), and by the European agencies the European Medicines Agency (EMA), College ter Beoordeling van Geneesmiddelen Medicines Evaluation Board (CBG-MEB), and Federal Institute for Drugs and Medical Devices (FIDMD), collectively assigned as EMA/FIDMD+MEB shortened as EMA/FM. Of 54 drugs with an actionable gene-drug interaction in the CPIC and DPWG guidelines, only 50% had actionable pharmacogenomic information in the SmPCs and the agencies were in agreement in only 18% of the cases. We further compared 450 additional drugs, lacking CPIC or DPWG guidance, and found 126 actionable gene-drug labels by the FDA and/or the EMA/FM. Based on these 126 drugs in addition to the 54 above, the consensus of actionable pharmacogenomic labeling between the FDA and the EMA/FM was only 54%. In conclusion, guidelines provided by CPIC/DPWG are only partly implemented into the SmPCs and the implementation of pharmacogenomic drug labels into the clinics would strongly gain from a higher extent of consensus between agencies.