RESUMEN
The intrinsic tenase (Xase) complex, formed by factors (f) VIIIa and fIXa, forms on activated platelet surfaces and catalyzes the activation of factor X to Xa, stimulating thrombin production in the blood coagulation cascade. The structural organization of the membrane-bound Xase complex remains largely unknown, hindering our understanding of the structural underpinnings that guide Xase complex assembly. Here, we aimed to characterize the Xase complex bound to a lipid nanodisc with biolayer interferometry (BLI), Michaelis-Menten kinetics, and small-angle X-ray scattering (SAXS). Using immobilized lipid nanodiscs, we measured binding rates and nanomolar affinities for fVIIIa, fIXa, and the Xase complex. Enzyme kinetic measurements demonstrated the assembly of an active enzyme complex in the presence of lipid nanodiscs. An ab initio molecular envelope of the nanodisc-bound Xase complex allowed us to computationally model fVIIIa and fIXa docked onto a flexible lipid membrane and identify protein-protein interactions. Our results highlight multiple points of contact between fVIIIa and fIXa, including a novel interaction with fIXa at the fVIIIa A1-A3 domain interface. Lastly, we identified hemophilia A/B-related mutations with varying severities at the fVIIIa/fIXa interface that may regulate Xase complex assembly. Together, our results support the use of SAXS as an emergent tool to investigate the membrane-bound Xase complex and illustrate how mutations at the fVIIIa/fIXa dimer interface may disrupt or stabilize the activated enzyme complex.
Asunto(s)
Factor IXa , Factor VIIIa/metabolismo , Lípidos , Cisteína Endopeptidasas , Factor IXa/química , Factor IXa/genética , Factor IXa/metabolismo , Proteínas de Neoplasias , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
Chimeric antigen receptor (CAR) T-cell therapy has been widely successful in the treatment of B-cell malignancies, including B-cell lymphoma, mantle cell lymphoma, and multiple myeloma; and three generations of CAR designs have led to effective FDA approved therapeutics. Traditionally, CAR antigen specificity is derived from a monoclonal antibody where the variable heavy (VH) and variable light (VL) chains are connected by a peptide linker to form a single-chain variable fragment (scFv). While this provides a level of antigen specificity parallel to that of an antibody and has shown great success in the clinic, this design is not universally successful. For instance, issues of stability, immunogenicity, and antigen escape hinder the translational application of some CARs. As an alternative, natural receptor- or ligand-based designs may prove advantageous in some circumstances compared to scFv-based designs. Herein, the advantages and disadvantages of scFv-based and natural receptor- or ligand-based CAR designs are discussed. In addition, several translational aspects of natural receptor- and ligand-based CAR approaches that are being investigated in preclinical and clinical studies will be examined.
Asunto(s)
Citotoxicidad Inmunológica , Receptores Quiméricos de Antígenos/metabolismo , Animales , Ensayos Clínicos como Asunto , Humanos , Inmunoterapia Adoptiva , Ligandos , Anticuerpos de Cadena Única/inmunologíaRESUMEN
BACKGROUND: Coagulation factor VIII represents one of the oldest protein-based therapeutics, serving as an effective hemophilia A treatment for half a century. Optimal treatment consists of repeated intravenous infusions of blood coagulation factor VIII (FVIII) per week for life. Despite overall treatment success, significant limitations remain, including treatment invasiveness, duration, immunogenicity, and cost. These issues have inspired research into the development of bioengineered FVIII products and gene therapies. OBJECTIVES: To structurally characterize a bioengineered construct of FVIII, termed ET3i, which is a human/porcine chimeric B domain-deleted heterodimer with improved expression and slower A2 domain dissociation following proteolytic activation by thrombin. METHODS: The structure of ET3i was characterized with X-ray crystallography and tandem mass spectrometry-based glycoproteomics. RESULTS: Here, we report the 3.2 Å crystal structure of ET3i and characterize the distribution of N-linked glycans with LC-MS/MS glycoproteomics. This structure shows remarkable conservation with the human FVIII protein and provides a detailed view of the interface between the A2 domain and the remaining FVIII structure. With two FVIII molecules in the crystal, we observe two conformations of the C2 domain relative to the remaining FVIII structure. The improved model and stereochemistry of ET3i served as a scaffold to generate an improved, refined structure of human FVIII. With the original datasets at 3.7 Å and 4.0 Å resolution, this new structure resulted in improved refinement statistics. CONCLUSIONS: These improved structures yield a more confident model for next-generation engineering efforts to develop FVIII therapeutics with longer half-lives, higher expression levels, and lower immunogenicity.