RESUMEN
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.
Asunto(s)
Inflamasomas , Proteína con Dominio Pirina 3 de la Familia NLR , Animales , Ratones , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Interleucina-18/metabolismo , Ubiquitinación , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Caspasa 1/metabolismoRESUMEN
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.
Asunto(s)
Inmunoglobulina E/inmunología , Fragmentos de Inmunoglobulinas/genética , Fragmentos de Inmunoglobulinas/inmunología , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas kappa de Inmunoglobulina/inmunología , Animales , Línea Celular , Células HEK293 , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/genética , Indicadores y Reactivos , Ratas , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunologíaRESUMEN
Organochlorine pesticides (OCPs) are widely used in certain countries. We determined atmospheric concentrations, distribution patterns, and seasonal variations of OCPs at four sites in South Korea for 1 year. Samples of 22 OCPs were collected using a high-volume air sampler, and measured via the isotope dilution method with HRGC/HRMS. In South Korea, pentachlorobenzene (PeCB), hexachlorocyclohexane (HCB), and endosulfan (EnSF) were dominant, accounting for > 87% of total OCPs. Spatial distributions showed significant differences and the highest levels were observed in Seosan (295.2 pg·m-3), indicating the compounding potential of diverse sources as Seosan has concentrated large-scale industrial complexes and agricultural activity (Seoul: 243.6 pg·m-3 > Jeju: 193.5 pg·m-3 > Baengnyeong: 178.2 pg·m-3). The isomeric ratios of OCPs in the South Korean atmosphere indicated that the dominant sources of HCB and dichlorodiphenyltrichloroethane were primarily used in the past; meanwhile, chlordane (CHL) and EnSFs were derived from recent material inputs. Seasonally, OCP concentrations largely peaked in summer with minimum values in winter. This apparent temperature dependence suggests the re-volatilization of accumulated chemicals into the atmosphere. Additionally, an air mass back trajectory indicated the influence of pollutants released from a reservoir through long-range atmospheric transport in the summer. In particular, restricted OCPs are primarily released into the atmosphere by inadvertent sources, such as industrial activities and volatilization from contaminated areas. Thus, severe OCP pollution in Korea is due to the mobile nature of the particles. These data can be useful for the continuous monitoring of long-range transported air pollutants that are transferred between countries.
Asunto(s)
Contaminantes Atmosféricos , Hidrocarburos Clorados , Plaguicidas , Contaminantes Atmosféricos/análisis , Atmósfera/química , Monitoreo del Ambiente , Hidrocarburos Clorados/análisis , Plaguicidas/análisis , Estaciones del AñoRESUMEN
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.
Asunto(s)
Regiones Constantes de Inmunoglobulina/química , Cadenas Pesadas de Inmunoglobulina/química , Cadenas Ligeras de Inmunoglobulina/química , Ensayo de Inmunoadsorción Enzimática , Células HEK293 , Células HeLa , Humanos , Regiones Constantes de Inmunoglobulina/genética , Inmunoglobulina G , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/genética , Receptores de IgG/análisis , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMEN
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.
Asunto(s)
Anticuerpos Catalíticos/metabolismo , ADN/metabolismo , Hemaglutininas/metabolismo , Histidina/metabolismo , Oligopéptidos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Anticuerpos de Cadena Única/metabolismo , Anticuerpos Catalíticos/genética , Clonación Molecular/métodos , ADN/química , División del ADN , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Células HEK293 , Hemaglutininas/genética , Histidina/genética , Humanos , Cinética , Oligopéptidos/genética , Plásmidos/química , Plásmidos/metabolismo , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Análisis de Secuencia de Proteína , Anticuerpos de Cadena Única/genéticaRESUMEN
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.
Asunto(s)
ADN Viral/metabolismo , Desoxirribonucleasas/inmunología , ARN Viral/metabolismo , Ribonucleasas/inmunología , Anticuerpos de Cadena Única/genética , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Antivirales/metabolismo , Línea Celular Tumoral , Infecciones por Virus ADN/inmunología , Virus ADN/genética , Virus ADN/inmunología , Desoxirribonucleasas/genética , Transferencia Resonante de Energía de Fluorescencia , Células HeLa , Herpesvirus Suido 1/genética , Herpesvirus Suido 1/inmunología , Humanos , Hidrólisis , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Seudorrabia/genética , Seudorrabia/inmunología , Ribonucleasas/genética , Simplexvirus/genética , Simplexvirus/inmunología , Anticuerpos de Cadena Única/inmunologíaRESUMEN
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.
Asunto(s)
Regiones Determinantes de Complementariedad/inmunología , ADN/inmunología , ARN/inmunología , Anticuerpos de Cadena Única/inmunología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/genética , Regiones Determinantes de Complementariedad/metabolismo , Células HeLa , Heparina/metabolismo , Humanos , Hidrólisis , Cadenas Ligeras de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/inmunología , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/metabolismoRESUMEN
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.
Asunto(s)
Aurora Quinasa B/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centrómero/metabolismo , Mitosis , Aurora Quinasa B/análisis , Proteínas de Ciclo Celular/análisis , Centrómero/ultraestructura , Segregación Cromosómica , Células HeLa , Humanos , FosforilaciónRESUMEN
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.
Asunto(s)
Anticuerpos Antinucleares/metabolismo , Antígenos de Neoplasias/metabolismo , Reactividad Cruzada/inmunología , Células Dendríticas/inmunología , Melanoma Experimental/inmunología , Melanoma Experimental/terapia , Anticuerpos de Cadena Única/metabolismo , Escape del Tumor/inmunología , Animales , Anticuerpos Antinucleares/genética , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Línea Celular Tumoral , Pollos , Técnicas de Cocultivo , Reactividad Cruzada/genética , Células Dendríticas/metabolismo , Células Dendríticas/patología , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/metabolismo , Melanoma Experimental/genética , Ratones , Ratones Endogámicos C57BL , Transporte de Proteínas/inmunología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Anticuerpos de Cadena Única/uso terapéutico , Escape del Tumor/genéticaRESUMEN
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.
RESUMEN
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.
Asunto(s)
Kringles , Dominios y Motivos de Interacción de Proteínas , Proteínas/química , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Modelos Moleculares , Datos de Secuencia Molecular , Biblioteca de Péptidos , Unión Proteica , Ingeniería de Proteínas , Pliegue de Proteína , Estructura Terciaria de Proteína , Proteínas/genética , Proteínas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
Asunto(s)
Bacteriófagos , Potyvirus , Bacteriófagos/genética , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/genética , Enfermedades de las PlantasRESUMEN
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.
Asunto(s)
Anticuerpos Catalíticos/metabolismo , Interferencia de ARN , ARN Mensajero/metabolismo , Anticuerpos Catalíticos/química , Apoptosis , Secuencia de Bases , Línea Celular Tumoral , Citosol/metabolismo , ADN de Cadena Simple/metabolismo , Técnicas de Silenciamiento del Gen , Biblioteca de Genes , Humanos , Hidrólisis , Cadenas Ligeras de Inmunoglobulina/química , Región Variable de Inmunoglobulina/química , Receptor ErbB-2/antagonistas & inhibidores , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismoRESUMEN
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.
Asunto(s)
Axones , Traumatismos de los Nervios Periféricos , Axones/fisiología , Proteínas de Unión al Calcio , Humanos , Macrófagos/fisiología , Nanogeles , Regeneración Nerviosa/fisiología , Traumatismos de los Nervios Periféricos/patología , Médula EspinalRESUMEN
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.
Asunto(s)
Anticuerpos Antinucleares/inmunología , Núcleo Celular/inmunología , VIH-1 , ARN Viral/inmunología , Proteínas de Unión al ARN/inmunología , Anticuerpos Antinucleares/metabolismo , Secuencia de Bases , Núcleo Celular/metabolismo , ADN/inmunología , Células HeLa , Humanos , Hidrólisis , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , ARN Viral/metabolismo , Proteínas de Unión al ARN/metabolismo , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/metabolismoRESUMEN
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.
Asunto(s)
Apoptosis/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Ácidos Hidroxámicos/farmacología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Receptores del Factor de Necrosis Tumoral/inmunología , Receptores del Factor de Necrosis Tumoral/metabolismo , Ácido Valproico/farmacología , Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Western Blotting , Caspasas/metabolismo , Línea Celular Tumoral , Regulación hacia Abajo , Sinergismo Farmacológico , Citometría de Flujo , Histona Desacetilasas/metabolismo , Humanos , Proteínas Inhibidoras de la Apoptosis/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , ARN Interferente Pequeño , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF , Transducción de Señal/efectos de los fármacos , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , VorinostatRESUMEN
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.
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Vacunas contra el Cáncer/inmunología , Vacunas contra el Cáncer/uso terapéutico , Carcinoma Pulmonar de Lewis/prevención & control , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Animales , Antígenos CD4/metabolismo , Carcinoma Pulmonar de Lewis/inmunología , Factores de Transcripción Forkhead/metabolismo , Receptor 2 Celular del Virus de la Hepatitis A , Humanos , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Ratones , Ratones Endogámicos C57BL , Linfocitos T Reguladores/inmunologíaRESUMEN
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.
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Anticuerpos Antinucleares/farmacología , Anticuerpos Monoclonales/farmacología , Antivirales/farmacología , Virus de la Fiebre Porcina Clásica/efectos de los fármacos , Peste Porcina Clásica/virología , Proteínas Recombinantes/farmacología , Anticuerpos de Cadena Única/farmacología , Animales , Anticuerpos Antinucleares/metabolismo , Anticuerpos Monoclonales/metabolismo , Antivirales/metabolismo , Línea Celular , Peste Porcina Clásica/inmunología , Regulación hacia Abajo , Hidrólisis , Interferón Tipo I/inmunología , ARN Viral/inmunología , ARN Viral/metabolismo , Proteínas Recombinantes/metabolismo , Ribonucleasas/metabolismo , Ribonucleasas/farmacología , Anticuerpos de Cadena Única/inmunología , Porcinos , Replicación Viral/efectos de los fármacosRESUMEN
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.
Asunto(s)
Citosol/metabolismo , Inmunoglobulina G/química , Pliegue de Proteína , Multimerización de Proteína , Animales , Retículo Endoplásmico/metabolismo , Células HEK293 , Humanos , Inmunoglobulina G/metabolismo , RatonesRESUMEN
Humanization of nonhuman antibodies (Abs) has been carried out mainly for Abs which bind to antigen without catalytic activity. Here we report humanization of mouse-originated 3D8 (m3D8) mAbs (scFv, VH, and VL) with DNA hydrolyzing catalytic activity by grafting the complementarity determining regions (CDRs) into the corresponding regions of a fixed human framework scaffold, generating humanized 3D8 (h3D8) Abs in the respective format of scFv, VH, and VL. h3D8 Abs retained comparable DNA binding and hydrolyzing activities to those of the corresponding m3D8 Abs. Our results suggest that CDRs of anti-DNA hydrolyzing Abs might possess the intrinsic properties of DNA binding and hydrolyzing activities.