RESUMEN
Dysregulated activation of the complement system is implicated in the onset or progression of several diseases. Most clinical-stage complement inhibitors target the inactive complement proteins present at high concentrations in plasma, which increases target-mediated drug disposition and necessitates high drug levels to sustain therapeutic inhibition. Furthermore, many efforts are aimed at inhibiting only terminal pathway activity, which leaves opsonin-mediated effector functions intact. We describe the discovery of SAR443809, a specific inhibitor of the alternative pathway C3/C5 convertase (C3bBb). SAR443809 selectively binds to the activated form of factor B (factor Bb) and inhibits alternative pathway activity by blocking the cleavage of C3, leaving the initiation of classical and lectin complement pathways unaffected. Ex vivo experiments with patient-derived paroxysmal nocturnal hemoglobinuria erythrocytes show that, although terminal pathway inhibition via C5 blockade can effectively inhibit hemolysis, proximal complement inhibition with SAR443809 inhibits both hemolysis and C3b deposition, abrogating the propensity for extravascular hemolysis. Finally, intravenous and subcutaneous administration of the antibody in nonhuman primates demonstrated sustained inhibition of complement activity for several weeks after injection. Overall, SAR443809 shows strong potential for treatment of alternative pathway-mediated disorders.
Asunto(s)
Factor B del Complemento , Vía Alternativa del Complemento , Animales , Factor B del Complemento/antagonistas & inhibidores , Eritrocitos/efectos de los fármacos , Hemólisis/efectos de los fármacos , Convertasas de Complemento C3-C5/antagonistas & inhibidores , Vía Alternativa del Complemento/efectos de los fármacos , Enfermedades del Sistema Inmune/tratamiento farmacológico , Enfermedades del Sistema Inmune/enzimología , Humanos , Macaca fascicularis , Anticuerpos/administración & dosificación , Proteolisis/efectos de los fármacosRESUMEN
Interaction of factor VIII (FVIII) with von Willebrand factor (VWF) is mediated by the VWF D'D3 domains and thrombin-mediated release is essential for hemostasis after vascular injury. VWF-D'D3 mutations resulting in loss of FVIII binding are the underlying cause of von Willebrand disease (VWD) type 2N. Furthermore, the FVIII-VWF interaction has significant implications for the development of therapeutics for bleeding disorders, particularly hemophilia A, in which endogenous VWF clearance imposes a half-life ceiling on replacement FVIII therapy. To understand the structural basis of FVIII engagement by VWF, we solved the structure of BIVV001 by cryo-electron microscopy to 2.9 Å resolution. BIVV001 is a bioengineered clinical-stage FVIII molecule for the treatment of hemophilia A. In BIVV001, VWF-D'D3 is covalently linked to an Fc domain of a B domain-deleted recombinant FVIII (rFVIII) Fc fusion protein, resulting in a stabilized rFVIII/VWF-D'D3 complex. Our rFVIII/VWF structure resolves BIVV001 architecture and provides a detailed spatial understanding of previous biochemical and clinical observations related to FVIII-VWF engagement. Notably, the FVIII acidic a3 peptide region (FVIII-a3), established as a critical determinant of FVIII/VWF complex formation, inserts into a basic groove formed at the VWF-D'/rFVIII interface. Our structure shows direct interaction of sulfated Y1680 in FVIII-a3 and VWF-R816 that, when mutated, leads to severe hemophilia A or VWD type 2N, respectively. These results provide insight on this key coagulation complex, explain the structural basis of many hemophilia A and VWD type 2N mutations, and inform studies to further elucidate how VWF dissociates rapidly from FVIII upon activation.
Asunto(s)
Microscopía por Crioelectrón/métodos , Factor VIII/química , Proteínas Recombinantes de Fusión/química , Factor de von Willebrand/química , Combinación de Medicamentos , Humanos , Modelos Moleculares , Conformación Proteica , Dominios Proteicos , Mapeo de Interacción de Proteínas , Proteínas Recombinantes de Fusión/ultraestructuraRESUMEN
Essentials Non-factor VIII (FVIII) therapies for hemophilia A, such as bispecific antibodies (bsAbs), are in development. Bispecific antibodies are intrinsically different from FVIII and lack many of the same regulatory mechanisms. These differences complicate assignment and interpretation of FVIII-equivalent activity. Inability to assign FVIII equivalence compromises our capacity to assess hemostatic potential of bsAb therapies. BACKGROUND: Activated factor VIII (FVIIIa) mimetic bsAbs aim to enable prophylactic treatment of hemophilia A patients with and without inhibitors. With different mechanisms of action, benchmarking their activity against FVIII to determine efficacious yet safe dosage is difficult. OBJECTIVE: To compare the activities of sequence identical emicizumab (SI-Emi) and another bsAb, BS-027125, to recombinant FVIII (rFVIII) using clinical and nonclinical assays and to evaluate our ability to assign a FVIII-equivalent value to bsAbs and implications thereof. METHODS: Activities of SI-Emi, BS-027125, and rFVIII were measured by one-stage clotting assay, chromogenic factor Xa generation assay, and thrombin generation assay. We also assessed the activity of anti-FIXa and anti-FX bivalent homodimers of each bsAb and probed the effect of different reagents in thrombin generation assay (TGA). RESULTS: The FVIII-like activity of SI-Emi and BS-027125 ranged greatly across each assay, varying both by parameter measured within an assay and by reagents used. Notably, SI-Emi anti-FIXa bivalent homodimer had meaningful activity in several assays, whereas BS-027125 anti-FIXa bivalent homodimer only had activity in the chromogenic assay. Surprisingly, SI-Emi displayed activity in the absence of phospholipids, while BS-027125 had minimal phospholipid-independent activity. CONCLUSIONS: Bispecific antibodies demonstrate little consistency between assays tested here owing to intrinsic differences between FVIII and bsAbs. While some trends are shared, the bsAbs also differ in mechanism. These inconsistencies complicate assignment of FVIII-equivalent values to bsAbs. Ultimately, a deeper mechanistic understanding of bsAbs as well as bsAb-tailored assays are needed to monitor and predict their hemostatic potential and long-term efficacy and safety confidently.
Asunto(s)
Anticuerpos Biespecíficos/farmacología , Anticuerpos Monoclonales Humanizados/farmacología , Mimetismo Biológico , Factor VIII/farmacología , Hematínicos/farmacología , Hemofilia A/tratamiento farmacológico , Hemostasis/efectos de los fármacos , Equivalencia Terapéutica , Pruebas de Coagulación Sanguínea , Factor VIII/inmunología , Factor Xa/metabolismo , Hemofilia A/sangre , Hemofilia A/diagnóstico , Hemofilia A/inmunología , Humanos , Resonancia por Plasmón de Superficie , Trombina/metabolismoRESUMEN
D assemblies make up half of the von Willebrand factor (VWF), yet are of unknown structure. D1 and D2 in the prodomain and D'D3 in mature VWF at Golgi pH form helical VWF tubules in Weibel Palade bodies and template dimerization of D3 through disulfides to form ultralong VWF concatemers. D'D3 forms the binding site for factor VIII. The crystal structure of monomeric D'D3 with cysteine residues required for dimerization mutated to alanine was determined at an endoplasmic reticulum (ER)-like pH. The smaller C8-3, TIL3 (trypsin inhibitor-like 3), and E3 modules pack through specific interfaces as they wind around the larger, N-terminal, Ca2+-binding von Willebrand D domain (VWD) 3 module to form a wedge shape. D' with its TIL' and E' modules projects away from D3. The 2 mutated cysteines implicated in D3 dimerization are buried, providing a mechanism for protecting them against premature disulfide linkage in the ER, where intrachain disulfide linkages are formed. D3 dimerization requires co-association with D1 and D2, Ca2+, and Golgi-like acidic pH. Associated structural rearrangements in the C8-3 and TIL3 modules are required to expose cysteine residues for disulfide linkage. Our structure provides insight into many von Willebrand disease mutations, including those that diminish factor VIII binding, which suggest that factor VIII binds not only to the N-terminal TIL' domain of D' distal from D3 but also extends across 1 side of D3. The organizing principle for the D3 assembly has implications for other D assemblies and the construction of higher-order, disulfide-linked assemblies in the Golgi in both VWF and mucins.
Asunto(s)
Factor VIII/metabolismo , Multimerización de Proteína , Factor de von Willebrand/química , Sitios de Unión , Cristalografía por Rayos X , Disulfuros , Retículo Endoplásmico/química , Aparato de Golgi/química , Humanos , Concentración de Iones de Hidrógeno , Espectroscopía de Resonancia Magnética , Biogénesis de Organelos , Unión Proteica , Dominios Proteicos , Factor de von Willebrand/metabolismoAsunto(s)
Pruebas de Coagulación Sanguínea/métodos , Hemofilia A/sangre , Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/uso terapéutico , Anticuerpos Monoclonales Humanizados/inmunología , Anticuerpos Monoclonales Humanizados/uso terapéutico , Pruebas de Coagulación Sanguínea/normas , Factor VIII/inmunología , Hemofilia A/tratamiento farmacológico , HumanosRESUMEN
The nuclear pore complex (NPC) enables transport across the nuclear envelope. It is one of the largest multiprotein assemblies in the cell, built from about 30 proteins called nucleoporins (Nups), organized into distinct subcomplexes. Structure determination of the NPC is a major research goal. The assembled â¼40-112 MDa NPC can be visualized by cryoelectron tomography (cryo-ET), while Nup subcomplexes are studied crystallographically. Docking the crystal structures into the cryo-ET maps is difficult because of limited resolution. Further, intersubcomplex contacts are not well characterized. Here, we systematically investigated direct interactions between Nups. In a comprehensive, structure-based, yeast two-hybrid interaction matrix screen, we mapped protein-protein interactions in yeast and human. Benchmarking against crystallographic and coaffinity purification data from the literature demonstrated the high coverage and accuracy of the data set. Novel intersubcomplex interactions were validated biophysically in microscale thermophoresis experiments and in intact cells through protein fragment complementation. These intersubcomplex interaction data provide direct experimental evidence toward possible structural arrangements of architectural elements within the assembled NPC, or they may point to assembly intermediates. Our data favors an assembly model in which major architectural elements of the NPC, notably the Y-complex, exist in different structural contexts within the scaffold.
Asunto(s)
Proteínas de Complejo Poro Nuclear/metabolismo , Mapeo de Interacción de Proteínas/métodos , Proteoma/metabolismo , Saccharomyces cerevisiae/metabolismo , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Simulación del Acoplamiento Molecular , Proteínas de Complejo Poro Nuclear/química , Conformación Proteica , Multimerización de Proteína , Proteoma/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
His-tag affinity purification is one of the most commonly used methods to purify recombinant proteins expressed in E. coli. One drawback of using the His-tag is the co-purification of contaminating histidine-rich E. coli proteins. We engineered a new E. coli expression strain, LOBSTR (low background strain), which eliminates the most abundant contaminants. LOBSTR is derived from the E. coli BL21(DE3) strain and carries genomically modified copies of arnA and slyD, whose protein products exhibit reduced affinities to Ni and Co resins, resulting in a much higher purity of the target protein. The use of LOBSTR enables the pursuit of challenging low-expressing protein targets by reducing background contamination with no additional purification steps, materials, or costs, and thus pushes the limits of standard His-tag purifications.
Asunto(s)
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Histidina/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Cromatografía de Afinidad , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas Recombinantes/genéticaRESUMEN
The nuclear pore complex (NPC) regulates all traffic between the cytoplasm and the nucleus. It is a large protein assembly composed of multiple copies of â¼30 nucleoporins (nups). Structural studies of the NPC have been limited by its considerable size and complexity. Progress toward understanding the structure of this nanomachine has benefited from its modular nature, which allows for this 40-60 MDa assembly to be broken down into subcomplexes that can be studied individually. While recent work by both crystallographers and electron microscopists has greatly enhanced our model of the NPC, the resolution gap between crystal and EM structures remains too large to confidently place individual proteins within the context of the fully assembled NPC. In an effort to arrive at a veritable model of the NPC, we solved the structure of several scaffold nups and defined the ancestral coatomer element (ACE1) common to a set of nucleoporins and COPII vesicle coat proteins. Subsequently, we proposed a lattice-like model of the NPC, analogous to the COPII lattice, in which ACE1 proteins form the edge elements and ß-propellers form the vertex elements. Here, we review our recent studies, speculate on how interactions between subcomplexes of the NPC are mediated, and outline the steps and challenges that lay ahead on the path to understanding this enormous assembly in molecular detail.
Asunto(s)
Vesículas Cubiertas/metabolismo , Poro Nuclear/metabolismo , Vesículas Cubiertas/química , Cristalografía por Rayos X , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Modelos Moleculares , Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/metabolismo , Estructura Cuaternaria de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
Nucleocytoplasmic transport is mediated by nuclear pore complexes (NPCs), enormous protein assemblies residing in circular openings in the nuclear envelope. The NPC is modular, with transient and stable components. The stable core is essentially built from two multiprotein complexes, the Y-shaped heptameric Nup84 complex and the Nic96 complex, arranged around an eightfold axis. We present the crystal structure of Nup120(1-757), one of the two short arms of the Y-shaped Nup84 complex. The protein adopts a compact oval shape built around a novel bipartite alpha-helical domain intimately integrated with a beta-propeller domain. The domain arrangement is substantially different from the Nup85*Seh1 complex, which forms the other short arm of the Y. With the data presented here, we establish that all three branches of the Y-shaped Nup84 complex are tightly connected by helical interactions and that the beta-propellers likely form interaction site(s) to neighboring complexes.
Asunto(s)
Proteínas de Complejo Poro Nuclear/química , Estructura Terciaria de Proteína , Proteínas de Saccharomyces cerevisiae/química , Cromatografía en Gel , Cristalización , Cristalografía por Rayos X , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Microscopía Fluorescente , Modelos Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Mutación , Poro Nuclear/química , Poro Nuclear/metabolismo , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Unión Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Electricidad EstáticaRESUMEN
Nuclear pore complexes (NPCs) facilitate nucleocytoplasmic transport. These massive assemblies comprise an eightfold symmetric scaffold of architectural proteins and central-channel phenylalanine-glycine-repeat proteins forming the transport barrier. We determined the nucleoporin 85 (Nup85)*Seh1 structure, a module in the heptameric Nup84 complex, at 3.5 angstroms resolution. Structural, biochemical, and genetic analyses position the Nup84 complex in two peripheral NPC rings. We establish a conserved tripartite element, the ancestral coatomer element ACE1, that reoccurs in several nucleoporins and vesicle coat proteins, providing structural evidence of coevolution from a common ancestor. We identified interactions that define the organization of the Nup84 complex on the basis of comparison with vesicle coats and confirmed the sites by mutagenesis. We propose that the NPC scaffold, like vesicle coats, is composed of polygons with vertices and edges forming a membrane-proximal lattice that provides docking sites for additional nucleoporins.