RESUMO
Activation of the complement system represents an important effector mechanism of endogenous and therapeutic Abs. However, efficient complement activation is restricted to a subset of Abs due to the requirement of multivalent interactions between the Ab Fc regions and the C1 complex. In the present study, we demonstrate that Fc-independent recruitment of C1 by modular bispecific single-domain Abs that simultaneously bind C1q and a surface Ag can potently activate the complement system. Using Ags from hematological and solid tumors, we show that these bispecific Abs are cytotoxic to human tumor cell lines that express the Ag and that the modular design allows a functional exchange of the targeting moiety. Direct comparison with clinically approved Abs demonstrates a superior ability of the bispecific Abs to induce complement-dependent cytotoxicity. The efficacy of the bispecific Abs to activate complement strongly depends on the epitope of the C1q binding Ab, demonstrating that the spatial orientation of the C1 complex upon Ag engagement is a critical factor for efficient complement activation. Collectively, our data provide insight into the mechanism of complement activation and provide a new platform for the development of immunotherapies.
Assuntos
Antineoplásicos , Complemento C1q , Humanos , Complemento C1q/metabolismo , Proteínas do Sistema Complemento , Ativação do Complemento , Linhagem Celular TumoralRESUMO
BACKGROUND INFORMATION: Interferons are a family of cytokines with growth inhibitory and antiviral functions, which exert their biological actions through the expression of interferon-stimulated genes (ISGs). The human ISG12 family of proteins comprises ISG12A, ISG12B, ISG12C and ISG6-16. Due to differential splicing and a gene variation, the human ISG12A protein exists as a full-length ISG12A form and three ISG12A variants. ISG12 genes have been found transcriptionally dysregulated in many disorders. High levels of ISG12A mRNA have been found in breast and ovarian cancers. Loss of heterozygosity at the position of the ISG12 genes often occurs in ovarian carcinomas and lymphoblastic leukemias. Both ISG12A and ISG6-16 are up-regulated in psoriasis. RESULTS: We demonstrate here that expression of the human full-length ISG12A protein sensitises cells for TNFα and the BH3 mimetic gossypol induced apoptosis, and the other ISG12A variants as well as ISG12B and ISG12C can induce apoptosis directly in HEK293 cells. Also ISG6-16 sensitises HEK293 cells for gossypol-induced apoptosis. In the ISG12 motif, two putative Bcl-2 homology (BH)3 like motifs were found, which may be decisive for the apoptotic properties of the ISG12 proteins. A series of BH3 mutants was made in ISG12AΔ-S, the smallest apoptosis-inducing ISG12A variant and our results indicate that ISG12AΔ-S indeed possesses features resembling those of BH3-only proteins. Supporting this notion are our findings that the full-length ISG12A co-immunoprecipitates with the Bcl-2 protein, and the apoptotic properties of the ISG12A variants are reduced in Bcl-2 expressing HEK293 cells. In addition, full-length ISG12A is able to form homodimers, which suggests a possible involvement in pore formation during apoptosis. The full-length ISG12A, the three ISG12A variants and the ISG12B proteins were found to be localised in the mitochondria. CONCLUSIONS: Our results suggest that the ISG12 family of proteins has an important role for the apoptotic properties induced by type 1 interferon. SIGNIFICANCE: The ISG12 family constitute small hydrophic proteins involved in apoptosis. This is the first comparison of the apoptotic potentials of the full-length ISG12A protein and the three ISG12A variants. The differential apoptotic potentials of these proteins might have an impact on the strategies to monitor and interpret their dysregulation associated with many disorders.
Assuntos
Apoptose , Proteínas de Membrana/fisiologia , Sequência de Aminoácidos , Sequência Conservada , Gossipol/farmacologia , Células HEK293 , Células HeLa , Humanos , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Mutação de Sentido Incorreto , Ligação Proteica , Isoformas de Proteínas/fisiologia , Transporte Proteico , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Upon infection of Escherichia coli by bacteriophage Qß, the virus-encoded ß-subunit recruits host translation elongation factors EF-Tu and EF-Ts and ribosomal protein S1 to form the Qß replicase holoenzyme complex, which is responsible for amplifying the Qß (+)-RNA genome. Here, we use X-ray crystallography, NMR spectroscopy, as well as sequence conservation, surface electrostatic potential and mutational analyses to decipher the roles of the ß-subunit and the first two oligonucleotide-oligosaccharide-binding domains of S1 (OB1-2) in the recognition of Qß (+)-RNA by the Qß replicase complex. We show how three basic residues of the ß subunit form a patch located adjacent to the OB2 domain, and use NMR spectroscopy to demonstrate for the first time that OB2 is able to interact with RNA. Neutralization of the basic residues by mutagenesis results in a loss of both the phage infectivity in vivo and the ability of Qß replicase to amplify the genomic RNA in vitro. In contrast, replication of smaller replicable RNAs is not affected. Taken together, our data suggest that the ß-subunit and protein S1 cooperatively bind the (+)-stranded Qß genome during replication initiation and provide a foundation for understanding template discrimination during replication initiation.
Assuntos
Allolevivirus/fisiologia , Genoma Viral , Q beta Replicase/química , Replicação Viral , Allolevivirus/genética , Dimerização , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Q beta Replicase/genética , Q beta Replicase/metabolismo , RNA Viral/biossíntese , RNA Viral/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismoRESUMO
The classical pathway of complement is important for protection against pathogens and in maintaining tissue homeostasis, but excessive or aberrant activation is directly linked to numerous pathologies. We describe the development and in vitro characterization of C1qNb75, a single domain antibody (nanobody) specific for C1q, the pattern recognition molecule of the classical pathway. C1qNb75 binds to the globular head modules of human C1q with sub-nanomolar affinity and impedes classical pathway mediated hemolysis by IgG and IgM. Crystal structure analysis revealed that C1qNb75 recognizes an epitope primarily located in the C1q B-chain that overlaps with the binding sites of IgG and IgM. Thus, C1qNb75 competitively prevents C1q from binding to IgG and IgM causing blockade of complement activation by the classical pathway. Overall, C1qNb75 represents a high-affinity nanobody-based inhibitor of IgG- and IgM-mediated activation of the classical pathway and may serve as a valuable reagent in mechanistic and functional studies of complement, and as an efficient inhibitor of complement under conditions of excessive CP activation.
Assuntos
Complemento C1q/imunologia , Receptores de Reconhecimento de Padrão/metabolismo , Anticorpos de Domínio Único/metabolismo , Afinidade de Anticorpos , Células Cultivadas , Ativação do Complemento , Complemento C1q/antagonistas & inibidores , Via Clássica do Complemento , Cristalografia por Raios X , Humanos , Imunoglobulina G/metabolismo , Imunoglobulina M/metabolismo , Ligação Proteica , Conformação Proteica , Receptores de Reconhecimento de Padrão/genética , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/genética , Relação Estrutura-AtividadeRESUMO
ß-Mannosidase is a lysosomal enzyme from the glycosyl hydrolase family 2 that cleaves the single ß(1-4)-linked mannose at the nonreducing end of N-glycosylated proteins, and plays an important role in the polysaccharide degradation pathway. Mutations in the MANBA gene, which encodes the ß-mannosidase, can lead to the lysosomal storage disease ß-mannosidosis, as well as nystagmus, an eye condition characterized by involuntary eye movements. Here, we present the first structures of a mammalian ß-mannosidase in both the apo- and mannose-bound forms. The structure is similar to previously determined ß-mannosidase structures with regard to domain organization and fold, however, there are important differences that underlie substrate specificity between species. Additionally, in contrast to most other ligand-bound ß-mannosidases from bacterial and fungal sources where bound sugars were in a boat-like conformation, we find the mannose in the chair conformation. Evaluation of known disease mutations in the MANBA gene provides insight into their impact on disease phenotypes. Together, these results will be important for the design of therapeutics for treating diseases caused by ß-mannosidase deficiency. DATABASE: Structural data are available in the Protein Data Bank under the accession numbers 6DDT and 6DDU.
Assuntos
Manose/metabolismo , Mutação , Nistagmo Patológico/enzimologia , beta-Manosidase/química , beta-Manosidose/enzimologia , Sequência de Aminoácidos , Animais , Domínio Catalítico , Glicosilação , Humanos , Camundongos , Nistagmo Patológico/genética , Nistagmo Patológico/patologia , Fenótipo , Conformação Proteica , Homologia de Sequência , Especificidade por Substrato , beta-Manosidase/genética , beta-Manosidase/metabolismo , beta-Manosidose/genética , beta-Manosidose/patologiaRESUMO
Homology-directed repair (HDR) induced by site specific DNA double-strand breaks with CRISPR-Cas9 is a precision gene editing approach that occurs at low frequency in comparison to indel forming non-homologous end joining (NHEJ). In order to obtain high HDR percentages in mammalian cells, we engineered a Cas9 protein fused to a monoavidin domain to bind biotinylated donor DNA. In addition, we used the cationic polymer, polyethylenimine, to deliver Cas9-donor DNA complexes into cells. Improved HDR percentages of up to 90% in three loci tested (CXCR4, EMX1, and TLR) in standard HEK293T cells were observed. Our results suggest that donor DNA biotinylation and Cas9-donor conjugation in addition to delivery influence HDR efficiency.