RESUMO
The NLRP3 inflammasome plays a key role in responding to pathogens, and endogenous damage and mitochondria are intensively involved in inflammasome activation. The NLRP3 inflammasome forms multiprotein complexes and its sequential assembly is important for its activation. Here, we show that NLRP3 is ubiquitinated by the mitochondria-associated E3 ligase, MARCH5. Myeloid cell-specific March5 conditional knockout (March5 cKO) mice failed to secrete IL-1ß and IL-18 and exhibited an attenuated mortality rate upon LPS or Pseudomonas aeruginosa challenge. Macrophages derived from March5 cKO mice also did not produce IL-1ß and IL-18 after microbial infection. Mechanistically, MARCH5 interacts with the NACHT domain of NLRP3 and promotes K27-linked polyubiquitination on K324 and K430 residues of NLRP3. Ubiquitination-defective NLRP3 mutants on K324 and K430 residues are not able to bind to NEK7, nor form NLRP3 oligomers leading to abortive ASC speck formation and diminished IL-1ß production. Thus, MARCH5-dependent NLRP3 ubiquitination on the mitochondria is required for NLRP3-NEK7 complex formation and NLRP3 oligomerization. We propose that the E3 ligase MARCH5 is a regulator of NLRP3 inflammasome activation on the mitochondria.
Assuntos
Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Animais , Camundongos , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Interleucina-18/metabolismo , Ubiquitinação , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Caspase 1/metabolismoRESUMO
Human IgE is useful for immunological assays, such as sensitization of FcεRI-positive cells and IgE measurement. In this study, we report the development of a recombinant Ig fragment, designated IgCw-γεκ, as an alternative reagent to human IgE. IgCw-γεκ (â¼130 kDa) comprises two hybrid constant H chain regions (Cγ1-Cε2-4, each â¼53 kDa) and two constant κ L chains (Cκ, each â¼12 kDa) and lacks a V domain. The presence of Cγ1 instead of Cε1 within the H chain increased the production yield and facilitated assembly of the H and L chains. IgCw-γεκ was produced in cultured human embryonic kidney 293F cells, with a yield of â¼27 mg/l. IgCw-γεκ bound to human FcεRIαRs expressed on the surface of rat basophilic leukemia-2H3 cells. A ß-hexosaminidase release assay revealed that the biological activity of IgCw-γεκ was comparable with that of IgE. The IgE concentration measured using IgCw-γεκ as a standard was similar to that measured using IgE as a standard. These results suggest that the IgCw-γεκ molecule retains the basic characteristics of IgE, but does not cross-react with Ags, making it an alternative to the IgE isotype references used in a variety of immunological assays.
Assuntos
Imunoglobulina E/imunologia , Fragmentos de Imunoglobulinas/genética , Fragmentos de Imunoglobulinas/imunologia , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias kappa de Imunoglobulina/imunologia , Animais , Linhagem Celular , Células HEK293 , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias kappa de Imunoglobulina/genética , Indicadores e Reagentes , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologiaRESUMO
We report the production and application of a recombinant IgCw molecule, which is composed of only the constant domains of the heavy (CH) and light (CL) chains, lacking a variable (V) domain. We produced IgCw, especially human IgCw-γκ (98â¯kDa), composed of two human Cγ chains (37â¯kDa each) and two Cκ chains (12â¯kDa each), using HEK293F cell culture. We found that the yield of IgCw-γκ protein was â¼20â¯mg/L, which was comparable to that of full-size IgG; it bound to Fcγ receptor-positive cells with a low background noise on Fcγ receptor-negative cells; and IgCw-γκ can be used as a reference for measurement of Ig concentration. Moreover, Cγ and Cκ chains were easily isolated from IgCw-γκ by a single step of affinity chromatography in the presence of a reducing agent. These results demonstrate that the IgCw molecule has the potential to be used for certain in vitro and in vivo applications as an alternative to an irrelevant isotype control IgG, and to be used a favorable antigen for acquiring isotype-specific antibodies by immunizing animals.
Assuntos
Regiões Constantes de Imunoglobulina/química , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Leves de Imunoglobulina/química , Ensaio de Imunoadsorção Enzimática , Células HEK293 , Células HeLa , Humanos , Regiões Constantes de Imunoglobulina/genética , Imunoglobulina G , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Receptores de IgG/análise , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
The antigen-binding properties of single chain Fv antibodies (scFvs) can vary depending on the position and type of fusion tag used, as well as the host cells used for expression. The issue is even more complicated with a catalytic scFv antibody that binds and hydrolyses a specific antigen. Herein, we investigated the antigen-binding and -hydrolysing activities of the catalytic anti-nucleic acid antibody 3D8 scFv expressed in Escherichia coli or HEK293f cells with or without additional amino acid residues at the N- and C-termini. DNA-binding activity was retained in all recombinant forms. However, the DNA-hydrolysing activity varied drastically between forms. The DNA-hydrolysing activity of E. coli-derived 3D8 scFvs was not affected by the presence of a C-terminal human influenza haemagglutinin (HA) or His tag. By contrast, the activity of HEK293f-derived 3D8 scFvs was completely lost when additional residues were included at the N-terminus and/or when a His tag was incorporated at the C-terminus, whereas a HA tag at the C-terminus did not diminish activity. Thus, we demonstrate that the antigen-binding and catalytic activities of a catalytic antibody can be separately affected by the presence of additional residues at the N- and C-termini, and by the host cell type.
Assuntos
Anticorpos Catalíticos/metabolismo , DNA/metabolismo , Hemaglutininas/metabolismo , Histidina/metabolismo , Oligopeptídeos/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Anticorpos de Cadeia Única/metabolismo , Anticorpos Catalíticos/genética , Clonagem Molecular/métodos , DNA/química , Clivagem do DNA , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Células HEK293 , Hemaglutininas/genética , Histidina/genética , Humanos , Cinética , Oligopeptídeos/genética , Plasmídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Análise de Sequência de Proteína , Anticorpos de Cadeia Única/genéticaRESUMO
Viral protein neutralizing antibodies have been developed but they are limited only to the targeted virus and are often susceptible to antigenic drift. Here, we present an alternative strategy for creating virus-resistant cells and animals by ectopic expression of a nucleic acid hydrolyzing catalytic 3D8 single chain variable fragment (scFv), which has both DNase and RNase activities. HeLa cells (SCH07072) [corrected] expressing 3D8 scFv acquired significant resistance to DNA viruses. Virus challenging with Herpes simplex virus (HSV) in 3D8 scFv transgenic cells and fluorescence resonance energy transfer (FRET) assay based on direct DNA cleavage analysis revealed that the induced resistance in HeLa cells was acquired by the nucleic acid hydrolyzing catalytic activity of 3D8 scFv. In addition, pseudorabies virus (PRV) infection in WT C57BL/6 mice was lethal, whereas transgenic mice (STG90) that expressed high levels of 3D8 scFv mRNA in liver, muscle, and brain showed a 56% survival rate 5 days after PRV intramuscular infection. The antiviral effects against DNA viruses conferred by 3D8 scFv expression in HeLa cells as well as an in vivo mouse system can be attributed to the nuclease activity that inhibits viral genome DNA replication in the nucleus and/or viral mRNA translation in the cytoplasm. Our results demonstrate that the nucleic-acid hydrolyzing activity of 3D8 scFv confers viral resistance to DNA viruses in vitro in HeLa cells and in an in vivo mouse system.
Assuntos
DNA Viral/metabolismo , Desoxirribonucleases/imunologia , RNA Viral/metabolismo , Ribonucleases/imunologia , Anticorpos de Cadeia Única/genética , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Antivirais/metabolismo , Linhagem Celular Tumoral , Infecções por Vírus de DNA/imunologia , Vírus de DNA/genética , Vírus de DNA/imunologia , Desoxirribonucleases/genética , Transferência Ressonante de Energia de Fluorescência , Células HeLa , Herpesvirus Suídeo 1/genética , Herpesvirus Suídeo 1/imunologia , Humanos , Hidrólise , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pseudorraiva/genética , Pseudorraiva/imunologia , Ribonucleases/genética , Simplexvirus/genética , Simplexvirus/imunologia , Anticorpos de Cadeia Única/imunologiaRESUMO
Many murine monoclonal anti-DNA antibodies (Abs) derived from mice models for systemic lupus erythematosus have additional cell-penetration and/or nucleic acid-hydrolysis properties. Here, we examined the influence of deactivating each complementarity-determining region (CDR) within a multifunctional anti-nucleic acid antibody (Ab) that possesses these activities, the catalytic 3D8 single chain variable fragment (scFv). CDR-deactivated 3D8 scFv variants were generated by replacing all of the amino acids within each CDR with Gly/Ser residues. The structure of 3D8 scFv accommodated single complete CDR deactivations. Different functional activities of 3D8 scFv were affected differently depending on which CDR was deactivated. The only exception was CDR1, located within the light chain (LCDR1); deactivation of LCDR1 abolished all of the functional activities of 3D8 scFv. A hybrid Ab, HW6/3D8L1, in which the LCDR1 from an unrelated Ab (HW6) was replaced with the LCDR1 from 3D8, acquired all activities associated with the 3D8 scFv. These results suggest that the activity of a multifunctional 3D8 scFv Ab can be modulated by single complete CDR deactivation and that the LCDR1 plays a crucial role in maintaining Ab properties. This study presents a new approach for determining the role of individual CDRs in multifunctional Abs with important implications for the future of Ab engineering.
Assuntos
Regiões Determinantes de Complementaridade/imunologia , DNA/imunologia , RNA/imunologia , Anticorpos de Cadeia Única/imunologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/metabolismo , Células HeLa , Heparina/metabolismo , Humanos , Hidrólise , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/imunologia , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/metabolismoRESUMO
A minor fraction of cohesin complexes at chromosome arms is not removed by the prophase pathway, and maintained until metaphase and enriched at centromeres. Sgo1 localizes to chromosome arms from prophase to metaphase, and is indispensable for removing cohesin complexes from chromosome arms. However, it has not been established how the chromosome arm localization of Sgo1 leads to the establishment of cohesion on chromosomes. Here, we report that Aurora B kinase interacts with and phosphorylates Sgo1 in vitro and in vivo. Furthermore, the phosphorylation of Sgol by Aurora B kinase regulated the distribution of Sgo1 between centromeres and chromosome arms, and the expression of Aurora B kinase-dead mutants of Sgo1 caused mislocalization from centromeres to chromosome arms. These results suggest Aurora B kinase directly regulates the subcellular distribution of Sgo1 to facilitate the accurate separation of mitotic chromosomes.
Assuntos
Aurora Quinase B/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centrômero/metabolismo , Mitose , Aurora Quinase B/análise , Proteínas de Ciclo Celular/análise , Centrômero/ultraestrutura , Segregação de Cromossomos , Células HeLa , Humanos , FosforilaçãoRESUMO
Cross-presentation is important for initiating CTL responses against tumors. Delivery of exogenous Ags to the cross-presentation pathway in dendritic cells (DCs), using a number of different carriers, has been attempted to further understand the mechanisms underlying cross-presentation and to develop therapeutic tumor vaccines. The present study reports a new antigenic carrier molecule: a single-chain V region fragment (scFv) of a nucleic acid-hydrolyzing Ab, 3D8. A fusion protein comprising 3D8 scFv and the CTL epitope OVA(250-264) (chicken OVA aa 250-264) was internalized by DC2.4 DCs and processed via a proteasome-dependent, brefeldin- and cycloheximide-sensitive, chloroquine- and primaquine-insensitive pathway, resulting in loading of the CTL epitope onto H-2K(b). In vivo cross-presentation and cross-priming were efficient, even without adjuvant; injection of mice with 3D8 scFv-OVA(250-264) induced cross-presentation of the CTL epitope by draining lymph node CD11c(+) B7.1(+) MHC class II(high) DCs, elicited a CTL response, and suppressed the growth of tumors expressing the OVA epitope. This report shows that an anti-nucleic acid Ab is used to deliver exogenous Ag to the cross-presentation pathway and inhibit in vivo tumor growth.
Assuntos
Anticorpos Antinucleares/metabolismo , Antígenos de Neoplasias/metabolismo , Apresentação Cruzada/imunologia , Células Dendríticas/imunologia , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Anticorpos de Cadeia Única/metabolismo , Evasão Tumoral/imunologia , Animais , Anticorpos Antinucleares/genética , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Linhagem Celular Tumoral , Galinhas , Técnicas de Cocultura , Apresentação Cruzada/genética , Células Dendríticas/metabolismo , Células Dendríticas/patologia , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/metabolismo , Melanoma Experimental/genética , Camundongos , Camundongos Endogâmicos C57BL , Transporte Proteico/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Anticorpos de Cadeia Única/uso terapêutico , Evasão Tumoral/genéticaRESUMO
Introduction: The CH1 domain of IgG antibodies controls assembly and secretion, mediated by the molecular chaperone BiP via the endoplasmic reticulum protein quality control (ERQC) mechanism. However, it is not clear whether the variable domains are necessary for this process. Methods: Here, we generated IgG1 antibodies in which the V domain (VH and/or VL) was either removed or replaced, and then assessed expression, assembly, and secretion in HEK293 cells. Results: All Ig variants formed a covalent linkage between the Cγ1 and Cκ, were successfully secreted in an assembled form. Replacement of the cognate Vκ with a non-secretory pseudo Vκ (ψVκ) hindered secretion of individual or assembled secretion of neither heavy chains (HCs) nor light chains (LCs). The ψLC (ψVκ-Cκ) exhibited a less folded structure compared to the wild type (wt) LC, as evidenced by enhanced stable binding to the molecular chaperone BiP and susceptibility to proteolytic degradation. Molecular dynamics simulation demonstrated dramatic alterations in overall structure of ψFab (Fd-ψLC) from wt Fab. Discussion: These findings suggest that V domains do not initiate HC:LC assembly and secretion; instead, the critical factor governing IgG assembly and secretion is the CH-CL pairing. Additionally, the structural integrity of the VL domain is crucial for IgG secretion. These data offer valuable insight into the design of bioactive molecules based on an IgG backbone.
RESUMO
Here, we report the development of target-specific binding proteins based on the kringle domain (KD) ( approximately 80 residues), a ubiquitous modular structural unit occurring across eukaryotic species. By exploiting the highly conserved backbone folding by core residues, but using extensive sequence variations in the seven loop regions of naturally occurring human KDs, we generated a synthetic KD library on the yeast cell surface by randomizing 45 residues in the loops of a human KD template. We isolated KD variants that specifically bind to anticancer target proteins, such as human death receptor 4 (DR4) and/or DR5, and that function as agonists to induce apoptotic cell death in several cancer cell lines in vitro and inhibit tumor progression in mouse models. Combined treatments with KD variants possessing different recognition sites on the same target protein exerted synergisitic tumoricidal activities, compared to treatment with individual variants. In addition to the agonists, we isolated an antagonistic KD variant that binds human tumor necrosis factor-alpha (TNFalpha) and efficiently neutralizes TNFalpha-induced cytotoxicity in vitro and in vivo. The KD scaffold with seven flexible loops protruding from the central core was strongly sequence-tolerant to mutations in the loop regions, offering a potential advantage of distinct binding sites for target recognition on the single domain. Our results suggest that the KD scaffold can be used to develop target-specific binding proteins that function as agonists or antagonists toward given target molecules, indicative of their potential use as biotherapeutics.
Assuntos
Kringles , Domínios e Motivos de Interação entre Proteínas , Proteínas/química , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Modelos Moleculares , Dados de Sequência Molecular , Biblioteca de Peptídeos , Ligação Proteica , Engenharia de Proteínas , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteínas/genética , Proteínas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
IMPORTANCE: In this study, we confirmed the binding of M13KO7 to Potato virus Y (PVY) using enzyme-linked immunosorbent assay. M13KO7 is a "bald" bacteriophage in which no recombinant antibody is displayed. M13KO7 is easy to propagate by using Escherichia coli, making this method more reasonable in economic perspective. Based on this study, we suggest that M13KO7 detection system has applicability as a novel biological tool for the detection of PVY.
Assuntos
Bacteriófagos , Potyvirus , Bacteriófagos/genética , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Doenças das PlantasRESUMO
PURPOSE: Cytoplasmic delivery of a monoclonal antibody (mAb) with nucleic acid-hydrolyzing activity (3D8 scFv) using poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs) was investigated for persistent anti-viral effect. METHODS: 3D8 scFv-loaded PLGA (3D8-PLGA) NPs were prepared via a double emulsion method that was previously optimized. Flow cytometry and confocal microscopy was carried out to confirm the cellular uptake and cytoplasmic localization. immunochemical and fluorescence resonance energy transfer (FRET) assays tested the cytoplasmic release and hydrolyzing effect of 3D8 scFv, respectively. Anti-viral activity test was performed using MTT assay with vesicular stomatitis virus (VSV)-infected HeLa cells. RESULTS: 3D8-PLGA NPs were much more effectively taken into cells in dose- and time-dependent manner and localized in the cytosolic region, compared to free 3D8 scFv. 3D8 scFv was released and hydrolyzed RNAs in the cytoplasm, exhibiting the maxima at a period of time (12-24 h). Anti-viral activity test revealed that 3D8-PLGA NP has dose- and time-dependent anti-viral effect and the maximum effect at the dose of 2 mg/ml and the incubation of 3 days. CONCLUSIONS: Cytoplasmic delivery of 3D8 scFv via PLGA NPs could enhance the viability of infected cells in sustained manner due to preserved activity, much improved cellular uptake and sustained release.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Antivirais/administração & dosagem , Citosol/metabolismo , Ácido Láctico/administração & dosagem , Nanopartículas/administração & dosagem , Ácidos Nucleicos/metabolismo , Ácido Poliglicólico/administração & dosagem , Animais , Anticorpos Monoclonais/química , Antivirais/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Preparações de Ação Retardada , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos/métodos , Emulsões/administração & dosagem , Emulsões/química , Células HCT116 , Células HeLa , Humanos , Hidrólise , Ácido Láctico/química , Camundongos , Nanopartículas/química , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , RNA/metabolismoRESUMO
Targeting particular mRNAs for degradation is a fascinating approach to achieve gene silencing. Here we describe a new gene silencing tool exploiting a cell-penetrating, nucleic-acid hydrolyzing, single-domain antibody of the light-chain variable domain, 3D8 VL. We generated a synthetic library of 3D8 VL on the yeast surface by randomizing residues located in one of two beta-sheets. Using 18-bp single-stranded nucleic acids as target substrates, including the human Her2/neu-targeting sequence, we selected 3D8 VL variants that had approximately 100-1000-fold higher affinity and approximately 2-5-fold greater selective hydrolyzing activity for target substrates than for off targets. 3D8 VL variants efficiently penetrated into living cells to be accumulated in the cytosol and selectively decreased the amount of target sequence-carrying mRNAs as well as the proteins encoded by these mRNAs with minimal effects on off-target genes. In particular, one 3D8 VL variant targeting the Her2 sequence showed more efficient downregulation of Her2 expression than a small-interfering RNA targeting the same Her2 sequence, resulting in apoptotic cell death of Her2-overexpressing breast cancer cells. Our results demonstrate that cell-penetrating 3D8 VL variants with sequence-selective, nucleic-acid-hydrolyzing activity can selectively degrade target mRNAs in the cytosol, providing a new gene silencing tool mediated by antibody.
Assuntos
Anticorpos Catalíticos/metabolismo , Interferência de RNA , RNA Mensageiro/metabolismo , Anticorpos Catalíticos/química , Apoptose , Sequência de Bases , Linhagem Celular Tumoral , Citosol/metabolismo , DNA de Cadeia Simples/metabolismo , Técnicas de Silenciamento de Genes , Biblioteca Gênica , Humanos , Hidrólise , Cadeias Leves de Imunoglobulina/química , Região Variável de Imunoglobulina/química , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismoRESUMO
Preconditioning nerve injury enhances axonal regeneration of dorsal root ganglia (DRG) neurons in part by driving pro-regenerative perineuronal macrophage activation. How these macrophages influence the neuronal capacity of axon regeneration remains elusive. We report that oncomodulin (ONCM) is produced from the regeneration-associated macrophages and strongly influences regeneration of DRG sensory axons. We also attempted to promote sensory axon regeneration by nanogel-mediated delivery of ONCM to DRGs. Methods:In vitro neuron-macrophage interaction model and preconditioning sciatic nerve injury were used to verify the necessity of ONCM in preconditioning injury-induced neurite outgrowth. We developed a nanogel-mediated delivery system in which electrostatic encapsulation of ONCM by a reducible epsilon-poly(L-lysine)-nanogel (REPL-NG) enabled a controlled release of ONCM. Results: Sciatic nerve injury upregulated ONCM in DRG macrophages. ONCM in macrophages was necessary to produce pro-regenerative macrophages in the in vitro model of neuron-macrophage interaction and played an essential role in preconditioning-induced neurite outgrowth. ONCM increased neurite outgrowth in cultured DRG neurons by activating a distinct gene set, particularly neuropeptide-related genes. Increasing extracellularly secreted ONCM in DRGs sufficiently enhanced the capacity of neurite outgrowth. Intraganglionic injection of REPL-NG/ONCM complex allowed sustained ONCM activity in DRG tissue and achieved a remarkable long-range regeneration of dorsal column sensory axons beyond spinal cord lesion. Conclusion: NG-mediated ONCM delivery could be exploited as a therapeutic strategy for promoting sensory axon regeneration following spinal cord injury.
Assuntos
Axônios , Traumatismos dos Nervos Periféricos , Axônios/fisiologia , Proteínas de Ligação ao Cálcio , Humanos , Macrófagos/fisiologia , Nanogéis , Regeneração Nervosa/fisiologia , Traumatismos dos Nervos Periféricos/patologia , Medula EspinalRESUMO
The 3D8 single chain variable fragment (3D8 scFv) is an anti-nucleic acid antibody that can hydrolyze nucleic acids and enter the cytosol of cells without reaching the nucleus. The Tat peptide, derived from the basic region of the HIV-1 Tat protein, translocates to cell nuclei and has TAR RNA binding activity. In this study, we generated a Tat-grafted antibody ((H3)Tat-3D8) by replacing complementarity-determining region 3 (CDR3) within the VH domain of the 3D8 scFv with a Tat48â60 peptide (GRKKRRQRRRPPQ). (H3)Tat-3D8 retained the DNA-binding and DNA-hydrolyzing activity of the scFv, and translocated to the nuclei of HeLa cells and preferentially recognized TAR RNA. Thus, the properties associated with the Tat peptide were transferred to the antibody via Tat-grafting without loss of the intrinsic DNA-binding and hydrolyzing activities of the 3D8 scFv antibody.
Assuntos
Anticorpos Antinucleares/imunologia , Núcleo Celular/imunologia , HIV-1 , RNA Viral/imunologia , Proteínas de Ligação a RNA/imunologia , Anticorpos Antinucleares/metabolismo , Sequência de Bases , Núcleo Celular/metabolismo , DNA/imunologia , Células HeLa , Humanos , Hidrólise , Dados de Sequência Molecular , Estrutura Terciária de Proteína , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/metabolismoRESUMO
Cell-death signaling through the pro-apoptotic tumor necrosis factor-related apoptosis inducing ligand (TRAIL) receptors, death receptor 4 (DR4) and DR5, has shown tumor-selective apoptotic activity. Here, we examine susceptibility of various leukemia cell lines (HL-60, U937, K562, CCRF-CEM, CEM-CM3, and THP-1) to an anti-DR4 agonistic monoclonal antibody (mAb), AY4, in comparison with TRAIL. While most of the leukemia cell lines were intrinsically resistant to AY4 or TRAIL alone, the two T-cell acute lymphoblastic leukemia (T-ALL) lines, CEM-CM3 and CCRF-CEM cells, underwent synergistic caspase-dependent apoptotic cell death by combination of AY4 or TRAIL with a histone deacetylase inhibitor (HDACI), either suberoylanilide hydroxamic acid (SAHA) or valproic acid (VPA). All of the combined treatments synergistically downregulated several anti-apoptotic proteins (c-FLIP, Bcl-2, Bcl-X(L), XIAP, and survivin) without significant changing the expression levels of pro-apoptotic proteins (Bax and Bak) or the receptors (DR4 and DR5). Downregulation of c-FLIP to activate caspase-8 was a critical step for the synergistic apoptosis through both extrinsic and intrinsic apoptotic pathways. Our results demonstrate that the HDACIs have synergistic effects on DR4-specific mAb AY4-mediated cell death in the T-ALL cells with comparable competence to those exerted by TRAIL, providing a new strategy for the targeted treatment of human T-ALL cells.
Assuntos
Apoptose/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Receptores do Fator de Necrose Tumoral/imunologia , Receptores do Fator de Necrose Tumoral/metabolismo , Ácido Valproico/farmacologia , Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Western Blotting , Caspases/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo , Sinergismo Farmacológico , Citometria de Fluxo , Histona Desacetilases/metabolismo , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , RNA Interferente Pequeno , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Transdução de Sinais/efeitos dos fármacos , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , VorinostatRESUMO
T-cell immunoglobulin and mucin domain 3 (Tim3) plays an important role in the Th1-mediated immune response; however, its effect on the efficacy of tumor vaccines has not been fully evaluated. Here, we demonstrate the effect of Tim3 pathway inhibition on tumor growth in mice. Lewis lung carcinoma (3LL) cells expressing a Tim3 pathway inhibitor, when injected into mice, showed suppressed tumor growth and a reduced frequency of CD4(+)CD25(+)Foxp3(+) T-cells. Furthermore, Tim3 pathway inhibition significantly enhanced the efficacy of a prophylactic tumor vaccine and marginally enhanced the efficacy of a therapeutic tumor vaccine. However, when given in combination with the chemotherapeutic agent, 5-fluorouracil, the therapeutic tumor vaccine capable of Tim3 pathway inhibition had no additional anti-tumor effect. Our results show that Tim3 pathway inhibition can enhance tumor vaccine efficacy.
Assuntos
Vacinas Anticâncer/imunologia , Vacinas Anticâncer/uso terapêutico , Carcinoma Pulmonar de Lewis/prevenção & controle , Proteínas de Membrana/antagonistas & inibidores , Proteínas Supressoras de Tumor/antagonistas & inibidores , Animais , Antígenos CD4/metabolismo , Carcinoma Pulmonar de Lewis/imunologia , Fatores de Transcrição Forkhead/metabolismo , Receptor Celular 2 do Vírus da Hepatite A , Humanos , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T Reguladores/imunologiaRESUMO
Some proteins with ribonuclease (RNase) activity have been shown to suppress viral replication. A well-characterized recombinant antibody, 3D8 single-chain variable fragment (3D8 scFv), has RNA-hydrolyzing and cell-penetrating activities. Here, we investigated antiviral activity of 3D8 scFv against classical swine fever virus (CSFV). In a cell line expressing 3D8 scFv (C26), intracellular RNA-hydrolysis activity was higher compared to control PK-15 cells and viral replication was strongly suppressed at the viral RNA level, with the evidence of independency of IFN-alpha/beta induction. Exogenous treatment of 3D8 scFv, prior to or post-CSFV infection, was also shown to suppress CSFV replication at the viral RNA level. These observations suggest that antiviral activity of 3D8 scFv may be due to the intrinsic RNase activity of 3D8 scFv, which is capable of targeting viral RNA genomes or transcripts.
Assuntos
Anticorpos Antinucleares/farmacologia , Anticorpos Monoclonais/farmacologia , Antivirais/farmacologia , Vírus da Febre Suína Clássica/efeitos dos fármacos , Peste Suína Clássica/virologia , Proteínas Recombinantes/farmacologia , Anticorpos de Cadeia Única/farmacologia , Animais , Anticorpos Antinucleares/metabolismo , Anticorpos Monoclonais/metabolismo , Antivirais/metabolismo , Linhagem Celular , Peste Suína Clássica/imunologia , Regulação para Baixo , Hidrólise , Interferon Tipo I/imunologia , RNA Viral/imunologia , RNA Viral/metabolismo , Proteínas Recombinantes/metabolismo , Ribonucleases/metabolismo , Ribonucleases/farmacologia , Anticorpos de Cadeia Única/imunologia , Suínos , Replicação Viral/efeitos dos fármacosRESUMO
Intrabodies, antibodies expressed within cells, offer an interesting way to target intracellular molecules, making them potentially useful for biotechnology and medicine. However, it remains controversial whether full-size IgG intrabodies expressed in the reducing environment of the cytosol of mammalian cells are workable and structurally sound. Herein, we settle this issue with a systematic investigation of the structure and functionality of four chimeric IgG1s with distinct variable (V) domains but identical constant (C) domains. Full-size IgGs expressed in the cytosol of HEK293 cells were either assembly-competent or -incompetent, depending on the intrinsic properties of the V regions. Structural integrity of the C region is required for H:L association and the formation of a functional antigen-binding site. Partial intrachain disulfide bond formation occurs in both H and L chains of cytosolic IgG intrabodies, whereas interchain disulfide bond formation was absent and dispensable for functional assembly. IgG1s expressed in the cytosol and via the ER were shown to assemble differently. Our findings provide insight into the features and possible utilization of full-size IgGs as cytosolic antibodies in biotechnological and medical applications.
Assuntos
Citosol/metabolismo , Imunoglobulina G/química , Dobramento de Proteína , Multimerização Proteica , Animais , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Imunoglobulina G/metabolismo , CamundongosRESUMO
Though the activation of c-Jun NH2-terminal kinase (JNK) has been reported to be essential for autophagic cell death in response to various stressors, the molecular links between JNK activation and autophagic cell death signaling remain elusive. Here we report that, in the JNK-dependent autophagic cell death of HCT116 cells induced by an agonistic single chain variable fragment antibody, HW1, against human death receptor 5 (DR5), JNK activation upregulated Beclin-1 expression and induced Bcl-2 and p53 phosphorylation. Further, the p53-deficient HCT116 cells showed less susceptibility to the HW1-mediated autophagic cell death than the wild type cells, suggesting that JNK-mediated p53 phosphorylation promotes the autophagic cell death. Our results suggest that DR5-stimulated JNK activation and its consequent fluxes into the pro-autophagic signaling pathways contribute to the autophagic cell death in cancer cells.