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1.
Proc Natl Acad Sci U S A ; 119(23): e2201562119, 2022 06 07.
Artículo en Inglés | MEDLINE | ID: mdl-35653561

RESUMEN

The utilization of avidity to drive and tune functional responses is fundamental to antibody biology and often underlies the mechanisms of action of monoclonal antibody drugs. There is increasing evidence that antibodies leverage homotypic interactions to enhance avidity, often through weak transient interfaces whereby self-association is coupled with target binding. Here, we comprehensively map the Fab­Fab interfaces of antibodies targeting DR5 and 4-1BB that utilize homotypic interaction to promote receptor activation and demonstrate that both antibodies have similar self-association determinants primarily encoded within a germline light chain complementarity determining region 2 (CDRL2). We further show that these determinants can be grafted onto antibodies of distinct target specificity to substantially enhance their activity. An expanded characterization of all unique germline CDRL2 sequences reveals additional self-association sequence determinants encoded in the human germline repertoire. Our results suggest that this phenomenon is unique to CDRL2, and is correlated with the less frequent antigen interaction and lower somatic hypermutation associated with this loop. This work reveals a previously unknown avidity mechanism in antibody native biology that can be exploited for the engineering of biotherapeutics.


Asunto(s)
Afinidad de Anticuerpos , Regiones Determinantes de Complementariedad , Células Germinativas , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/genética , Quimioterapia , Fragmentos Fab de Inmunoglobulinas
2.
J Transl Med ; 19(1): 517, 2021 12 20.
Artículo en Inglés | MEDLINE | ID: mdl-34930320

RESUMEN

BACKGROUND: Over the past decade, human Interleukin 33 (hIL-33) has emerged as a key contributor to the pathogenesis of numerous inflammatory diseases. Despite the existence of several commercial hIL-33 assays spanning multiple platform technologies, their ability to provide accurate hIL-33 concentration measurements and to differentiate between active (reduced) and inactive (oxidized) hIL-33 in various matrices remains uncertain. This is especially true for lower sample volumes, matrices with low hIL-33 concentrations, and matrices with elevated levels of soluble Interleukin 1 Receptor-Like 1 (sST2), an inactive form of ST2 that competes with membrane bound ST2 for hIL-33 binding. RESULTS: We tested the performance of several commercially available hIL-33 detection assays in various human matrices and found that most of these assays lacked the sensitivity to accurately detect reduced hIL-33 at biologically relevant levels (sub-to-low pg/mL), especially in the presence of human sST2 (hsST2), and/or lacked sufficient target specificity. To address this, we developed and validated a sensitive and specific enzyme-linked immunosorbent assay (ELISA) capable of detecting reduced and total hIL-33 levels even in the presence of high concentrations of sST2. By incorporating the immuno-polymerase chain reaction (iPCR) platform, we further increased the sensitivity of this assay for the reduced form of hIL-33 by ~ 52-fold. Using this hIL-33 iPCR assay, we detected hIL-33 in postmortem human vitreous humor (VH) samples from donors with age-related macular degeneration (AMD) and found significantly increased hIL-33 levels when compared to control individuals. No statistically significant difference was observed in aqueous humor (AH) from AMD donors nor in plasma and nasosorption fluid (NF) from asthma patients compared to control individuals. CONCLUSIONS: Unlike existing commercial hIL-33 assays, our hIL-33 bioassays are highly sensitive and specific and can accurately quantify hIL-33 in various human clinical matrices, including those with high levels of hsST2. Our results provide a proof of concept of the utility of these assays in clinical trials targeting the hIL-33/hST2 pathway.


Asunto(s)
Asma , Degeneración Macular , Bioensayo , Biomarcadores , Desarrollo de Medicamentos , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Interleucina-33 , Sensibilidad y Especificidad
3.
J Biol Chem ; 293(3): 906-919, 2018 01 19.
Artículo en Inglés | MEDLINE | ID: mdl-29191832

RESUMEN

Anti-hinge antibodies (AHAs) are an autoantibody subclass that, following proteolytic cleavage, recognize cryptic epitopes exposed in the hinge regions of immunoglobulins (Igs) and do not bind to the intact Ig counterpart. AHAs have been postulated to exacerbate chronic inflammatory disorders such as inflammatory bowel disease and rheumatoid arthritis. On the other hand, AHAs may protect against invasive microbial pathogens and cancer. However, despite more than 50 years of study, the origin and specific B cell compartments that express AHAs remain elusive. Recent research on serum AHAs suggests that they arise during an active immune response, in contrast to previous proposals that they derive from the preexisting immune repertoire in the absence of antigenic stimuli. We report here the isolation and characterization of AHAs from memory B cells, although anti-hinge-reactive B cells were also detected in the naive B cell compartment. IgG AHAs cloned from a single human donor exhibited restricted specificity for protease-cleaved F(ab')2 fragments and did not bind the intact IgG counterpart. The cloned IgG-specific AHA-variable regions were mutated from germ line-derived sequences and displayed a high sequence variability, confirming that these AHAs underwent class-switch recombination and somatic hypermutation. Consistent with previous studies of serum AHAs, several of these clones recognized a linear, peptide-like epitope, but one clone was unique in recognizing a conformational epitope. All cloned AHAs could restore immune effector functions to proteolytically generated F(ab')2 fragments. Our results confirm that a diverse set of epitope-specific AHAs can be isolated from a single human donor.


Asunto(s)
Autoanticuerpos/metabolismo , Linfocitos B/metabolismo , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Autoanticuerpos/inmunología , Linfocitos B/inmunología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Inmunoglobulina G/inmunología , Inmunoglobulina G/metabolismo , Metaloproteinasa 3 de la Matriz/metabolismo , Metaloproteinasa 7 de la Matriz/metabolismo
4.
J Immunol ; 193(1): 111-9, 2014 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-24879793

RESUMEN

IL-13 can bind to two distinct receptors: a heterodimer of IL-13Rα1/IL-4Rα and IL-13Rα2. Whereas IL-13Rα1/IL-4Rα engagement by IL-13 leads to the activation of STAT6, the molecular events triggered by IL-13 binding to IL-13Rα2 remain incompletely understood. IL-4 can bind to and signal through the IL-13Rα1/IL-4Rα complex but does not interact with IL-13Rα2. Idiopathic pulmonary fibrosis is a progressive and generally fatal parenchymal lung disease of unknown etiology with no current pharmacologic treatment options that substantially prolong survival. Preclinical models of fibrotic diseases have implicated IL-13 activity on multiple cell types, including macrophages and fibroblasts, in initiating and perpetuating pathological fibrosis. In this study, we show that IL-13, IL-4, IL-13Rα2, and IL-13-inducible target genes are expressed at significantly elevated levels in lung tissue from patients with idiopathic pulmonary fibrosis compared with control lung tissue. IL-4 and IL-13 induce virtually identical transcriptional responses in human monocytes, macrophages, and lung fibroblasts. IL-13Rα2 expression can be induced in lung fibroblasts by IL-4 or IL-13 via a STAT6-dependent mechanism, or by TNF-α via a STAT6-independent mechanism. Endogenously expressed IL-13Rα2 decreases, but does not abolish, sensitivity of lung fibroblasts to IL-13 and does not affect sensitivity to IL-4. Genome-wide transcriptional analyses of lung fibroblasts stimulated with IL-13 in the presence of Abs that selectively block interactions of IL-13 with IL-13Rα1/IL-4Rα or IL-13Rα2 show that endogenously expressed IL-13Rα2 does not activate any unique IL-13-mediated gene expression patterns, confirming its role as a decoy receptor for IL-13 signaling.


Asunto(s)
Fibroblastos/inmunología , Regulación de la Expresión Génica/inmunología , Fibrosis Pulmonar Idiopática/inmunología , Subunidad alfa2 del Receptor de Interleucina-13/inmunología , Interleucina-13/inmunología , Pulmón/inmunología , Transducción de Señal/inmunología , Femenino , Fibroblastos/patología , Estudio de Asociación del Genoma Completo , Humanos , Fibrosis Pulmonar Idiopática/patología , Subunidad alfa1 del Receptor de Interleucina-13/inmunología , Interleucina-4/inmunología , Subunidad alfa del Receptor de Interleucina-4/inmunología , Pulmón/patología , Macrófagos/inmunología , Macrófagos/patología , Masculino , Monocitos/inmunología , Monocitos/patología , Factor de Transcripción STAT6/inmunología , Factor de Necrosis Tumoral alfa/inmunología
5.
J Biol Chem ; 288(37): 26583-93, 2013 Sep 13.
Artículo en Inglés | MEDLINE | ID: mdl-23880771

RESUMEN

Human bispecific antibodies have great potential for the treatment of human diseases. Although human IgG1 bispecific antibodies have been generated, few attempts have been reported in the scientific literature that extend bispecific antibodies to other human antibody isotypes. In this paper, we report our work expanding the knobs-into-holes bispecific antibody technology to the human IgG4 isotype. We apply this approach to generate a bispecific antibody that targets IL-4 and IL-13, two cytokines that play roles in type 2 inflammation. We show that IgG4 bispecific antibodies can be generated in large quantities with equivalent efficiency and quality and have comparable pharmacokinetic properties and lung partitioning, compared with the IgG1 isotype. This work broadens the range of published therapeutic bispecific antibodies with natural surface architecture and provides additional options for the generation of bispecific antibodies with differing effector functions through the use of different antibody isotypes.


Asunto(s)
Anticuerpos Biespecíficos/inmunología , Regulación de la Expresión Génica , Inmunoglobulina G/inmunología , Interleucina-13/metabolismo , Interleucina-4/metabolismo , Animales , Anticuerpos Biespecíficos/biosíntesis , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Línea Celular Tumoral , Proliferación Celular , Femenino , Humanos , Inmunoglobulina G/biosíntesis , Pulmón/inmunología , Pulmón/metabolismo , Macaca fascicularis , Masculino , Ratones , Ratones Endogámicos BALB C , Plásmidos/metabolismo , Ingeniería de Proteínas/métodos , Resonancia por Plasmón de Superficie
6.
PLoS One ; 19(4): e0297122, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38662671

RESUMEN

Site specific biotinylation of AviTagged recombinant proteins using BirA enzyme is a widely used protein labeling technology. However, due to the incomplete biotinylation reactions and the lack of a purification method specific for the biotinylated proteins, it is challenging to purify the biotinylated sample when mixed with the non-biotinylated byproduct. Here, we have developed a monoclonal antibody that specifically recognizes the non-biotinylated AviTag but not the biotinylated sequence. After a ten-minute incubation with the resin that is conjugated with the antibody, the non-biotinylated AviTagged protein is trapped on the resin while the fully biotinylated material freely passes through. Therefore, our AviTrap (anti-AviTag antibody conjugated resin) provides an efficient solution for enriching biotinylated AviTagged proteins via a simple one-step purification.


Asunto(s)
Anticuerpos Monoclonales , Biotinilación , Anticuerpos Monoclonales/química , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Humanos , Biotina/química , Animales , Ligasas de Carbono-Nitrógeno/química , Ligasas de Carbono-Nitrógeno/metabolismo
7.
Cell Rep Med ; 4(8): 101130, 2023 08 15.
Artículo en Inglés | MEDLINE | ID: mdl-37490914

RESUMEN

Signal regulatory protein (SIRPα) is an immune inhibitory receptor expressed by myeloid cells to inhibit immune cell phagocytosis, migration, and activation. Despite the progress of SIRPα and CD47 antagonist antibodies to promote anti-cancer immunity, it is not yet known whether SIRPα receptor agonism could restrain excessive autoimmune tissue inflammation. Here, we report that neutrophil- and monocyte-associated genes including SIRPA are increased in inflamed tissue biopsies from patients with rheumatoid arthritis and inflammatory bowel diseases, and elevated SIRPA is associated with treatment-refractory ulcerative colitis. We next identify an agonistic anti-SIRPα antibody that exhibits potent anti-inflammatory effects in reducing neutrophil and monocyte chemotaxis and tissue infiltration. In preclinical models of arthritis and colitis, anti-SIRPα agonistic antibody ameliorates autoimmune joint inflammation and inflammatory colitis by reducing neutrophils and monocytes in tissues. Our work provides a proof of concept for SIRPα receptor agonism for suppressing excessive innate immune activation and chronic inflammatory disease treatment.


Asunto(s)
Colitis , Neoplasias , Humanos , Fagocitosis , Neoplasias/tratamiento farmacológico , Neutrófilos/metabolismo , Inflamación/patología , Colitis/metabolismo
8.
Sci Transl Med ; 14(627): eabf8188, 2022 01 12.
Artículo en Inglés | MEDLINE | ID: mdl-35020406

RESUMEN

Exacerbations of symptoms represent an unmet need for people with asthma. Bacterial dysbiosis and opportunistic bacterial infections have been observed in, and may contribute to, more severe asthma. However, the molecular mechanisms driving these exacerbations remain unclear. We show here that bacterial lipopolysaccharide (LPS) induces oncostatin M (OSM) and that airway biopsies from patients with severe asthma present with an OSM-driven transcriptional profile. This profile correlates with activation of inflammatory and mucus-producing pathways. Using primary human lung tissue or human epithelial and mesenchymal cells, we demonstrate that OSM is necessary and sufficient to drive pathophysiological features observed in severe asthma after exposure to LPS or Klebsiella pneumoniae. These findings were further supported through blockade of OSM with an OSM-specific antibody. Single-cell RNA sequencing from human lung biopsies identified macrophages as a source of OSM. Additional studies using Osm-deficient murine macrophages demonstrated that macrophage-derived OSM translates LPS signals into asthma-associated pathologies. Together, these data provide rationale for inhibiting OSM to prevent bacterial-associated progression and exacerbation of severe asthma.


Asunto(s)
Asma , Oncostatina M/metabolismo , Animales , Asma/patología , Humanos , Pulmón/patología , Macrófagos/metabolismo , Ratones , Moco , Oncostatina M/genética
9.
J Immunother Cancer ; 7(1): 207, 2019 08 06.
Artículo en Inglés | MEDLINE | ID: mdl-31387641

RESUMEN

BACKGROUND: One of the mechanisms by which tumors evade immune surveillance is through shedding of the major histocompatibility complex (MHC) class I chain-related protein A and B (MICA/B) from their cell surface. MICA/B are ligands for the activating receptor NKG2D on NK and CD8 T cells. This shedding reduces cell surface levels of MICA/B and impairs NKG2D recognition. Shed MICA/B can also mask NKG2D receptor and is thought to induce NKG2D internalization, further compromising immune surveillance by NK cells. METHODS: We isolated human primary NK cells from normal donors and tested the suppressive activity of soluble recombinant MICA in vitro. Utilizing a panel of novel anti-MICA antibodies, we further examined the stimulatory activities of anti-MICA antibodies that reversed the suppressive effects of soluble MICA. RESULTS: We show that suppressive effects of soluble MICA (sMICA) on NK cell cytolytic activity was not due to the down-regulation of cell surface NKG2D. In the presence of an α3 domain-specific MICA antibody, which did not obstruct NKG2D binding, sMICA-mediated NK cell suppression was completely reversed. Reversal of NK cell inhibition by sMICA was mediated by immune complex formation that agonized NKG2D signaling. Furthermore, this restorative activity was dependent on antibody Fc effector function as the introduction of Fc mutations to abrogate Fc receptor binding failed to reverse sMICA-mediated NK cell suppression. Furthermore, MICA immune complexes preformed with an α3 domain-specific antibody (containing a wild-type Fc) induced IFN-γ and TNF-α secretion by NK cells in the absence of cancer cells, whereas MICA immune complexes preformed with the Fc effectorless antibody failed to induce IFN-γ and TNF-α secretion. Finally, we demonstrated that MICA immune complexes formed with the α3 domain-specific antibody activates NKG2D on NK cells leading to the release of IFN-γ. CONCLUSIONS: Our results demonstrate that an α3 domain-specific MICA antibody can circumvent sMICA-mediated suppression of NK cell cytolytic activity. Moreover, our data suggest that MICA immune complexes formed with α3-specific antibodies can activate NKG2D receptor and restore NK cell function in a Fc-dependent manner. The clinical utility of α3 domain-specific MICA/B antibodies may hold great promise as a new strategy for cancer immunotherapy.


Asunto(s)
Complejo Antígeno-Anticuerpo/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Inmunoterapia/métodos , Células Asesinas Naturales/inmunología , Línea Celular , Humanos , Transfección
10.
Elife ; 82019 06 25.
Artículo en Inglés | MEDLINE | ID: mdl-31237236

RESUMEN

Outer membrane proteins (OMPs) in Gram-negative bacteria dictate permeability of metabolites, antibiotics, and toxins. Elucidating the structure-function relationships governing OMPs within native membrane environments remains challenging. We constructed a diverse library of >3000 monoclonal antibodies to assess the roles of extracellular loops (ECLs) in LptD, an essential OMP that inserts lipopolysaccharide into the outer membrane of Escherichia coli. Epitope binning and mapping experiments with LptD-loop-deletion mutants demonstrated that 7 of the 13 ECLs are targeted by antibodies. Only ECLs inaccessible to antibodies were required for the structure or function of LptD. Our results suggest that antibody-accessible loops evolved to protect key extracellular regions of LptD, but are themselves dispensable. Supporting this hypothesis, no α-LptD antibody interfered with essential functions of LptD. Our experimental workflow enables structure-function studies of OMPs in native cellular environments, provides unexpected insight into LptD, and presents a method to assess the therapeutic potential of antibody targeting.


Asunto(s)
Anticuerpos Monoclonales/metabolismo , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Animales , Antibacterianos/farmacología , Sitios de Unión , Mapeo Epitopo , Epítopos/metabolismo , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Ratones Endogámicos BALB C , Estructura Secundaria de Proteína , Ratas Sprague-Dawley , Relación Estructura-Actividad
11.
Sci Rep ; 8(1): 7136, 2018 05 08.
Artículo en Inglés | MEDLINE | ID: mdl-29740124

RESUMEN

Outer membrane proteins (OMPs) in Gram-negative bacteria are essential for a number of cellular functions including nutrient transport and drug efflux. Escherichia coli BamA is an essential component of the OMP ß-barrel assembly machinery and a potential novel antibacterial target that has been proposed to undergo large (~15 Å) conformational changes. Here, we explored methods to isolate anti-BamA monoclonal antibodies (mAbs) that might alter the function of this OMP and ultimately lead to bacterial growth inhibition. We first optimized traditional immunization approaches but failed to identify mAbs that altered cell growth after screening >3000 hybridomas. We then developed a "targeted boost-and-sort" strategy that combines bacterial cell immunizations, purified BamA protein boosts, and single hybridoma cell sorting using amphipol-reconstituted BamA antigen. This unique workflow improves the discovery efficiency of FACS + mAbs by >600-fold and enabled the identification of rare anti-BamA mAbs with bacterial growth inhibitory activity in the presence of a truncated lipopolysaccharide layer. These mAbs represent novel tools for dissecting the BamA-mediated mechanism of ß-barrel folding and our workflow establishes a new template for the efficient discovery of novel mAbs against other highly dynamic membrane proteins.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/aislamiento & purificación , Proteínas de la Membrana Bacteriana Externa/inmunología , Escherichia coli/inmunología , Proteínas de Escherichia coli/inmunología , Inmunización , Conformación Proteica , Pliegue de Proteína , Transporte de Proteínas/genética , Transporte de Proteínas/inmunología , Vacunación
12.
Sci Rep ; 5: 17488, 2015 Dec 03.
Artículo en Inglés | MEDLINE | ID: mdl-26631978

RESUMEN

Rapid identification of residues that influence antibody expression and thermostability is often needed to move promising therapeutics into the clinic. To establish a method that can assess small expression differences, we developed a Bacterial Antibody Display (BAD) system that overcomes previous limitations, enabling the use of full-length formats for antibody and antigen in a live cell setting. We designed a unique library of individual framework variants using natural diversity introduced by somatic hypermutation, and screened half-antibodies for increased expression using BAD. We successfully identify variants that dramatically improve expression yields and in vitro thermostability of two therapeutically relevant antibodies in E. coli and mammalian cells. While we study antibody expression, bacterial display can now be expanded to examine the processes of protein folding and translocation. Additionally, our natural library design strategy could be applied during antibody humanization and library design for in vitro display methods to maintain expression and formulation stability.


Asunto(s)
Anticuerpos/genética , Escherichia coli/genética , Ingeniería de Proteínas/métodos , Anticuerpos/metabolismo , Regulación de la Expresión Génica , Variación Genética , Interleucina-13/inmunología , Biblioteca de Péptidos , Estabilidad Proteica , Receptor EphB3 , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factor A de Crecimiento Endotelial Vascular/inmunología
13.
J Mol Biol ; 425(8): 1330-9, 2013 Apr 26.
Artículo en Inglés | MEDLINE | ID: mdl-23357170

RESUMEN

The cytokine interleukin 13 (IL-13) is a major effector molecule for T-helper type 2 inflammation and is pathogenic in allergic diseases such as asthma. The effects of IL-13 are mediated via a pathway that is initiated by binding to a heterodimeric receptor consisting of IL-13Rα1 and IL-4Rα. Antibodies raised against IL-13 can block its inflammatory effects by interfering with binding to either of the two receptor polypeptides. Lebrikizumab is a monoclonal anti-IL-13 antibody that has shown clinical benefit in a phase II study for the treatment of moderate-to-severe uncontrolled asthma. Here we report the molecular structure of IL-13 in complex with the Fab from lebrikizumab by X-ray crystallography at 1.9Å resolution. We show that lebrikizumab inhibits IL-13 signaling by binding to IL-13 with very high affinity and blocking IL-13 binding to IL-4Rα. In addition, we use site-directed mutations to identify the most important antibody contributors to binding. Our studies define key features of lebrikizumab binding and its mechanism of action that may contribute to its clinical effects.


Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Factores Inmunológicos/química , Factores Inmunológicos/metabolismo , Interleucina-13/antagonistas & inhibidores , Cristalografía por Rayos X , Análisis Mutacional de ADN , Modelos Moleculares , Unión Proteica , Conformación Proteica , Transducción de Señal
14.
Mol Cell Biochem ; 288(1-2): 179-89, 2006 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-16718380

RESUMEN

The conserved N-domain of the STAT proteins has been implicated in several activities crucial to cytokine signaling including receptor recruitment and STAT activation, cooperative DNA binding and STAT-dependent gene expression. We evaluated the role of the STAT3 N-domain in the IL-6 signal transduction pathway leading to Socs3 gene expression, an essential mechanism that controls the quality and magnitude of IL-6-dependent transcriptional responses. Based on the model for STAT N-domain function in cooperative gene expression and the presence of tandem STAT binding motifs in the murine Socs3 promoter, we anticipated that stabilizing interactions between adjacent STAT3 dimers via N-domain sequences might be essential for Socs3 gene expression. This was underscored by the tight conservation in the location and sequence of the tandem STAT binding sites between the murine and human Socs3 promoters. Using reconstitution into Stat3-/- mouse embryonic fibroblasts (Stat3-/- MEFs), we find that a STAT3 N-domain deletion mutant (Delta 133STAT3) is activated by tyrosine phosphorylation in response to IL-6 and then undergoes dephosphorylation with kinetics similar to full-length STAT3. These results highlight important differences compared to other STATs where the N-domain has been shown to mediate activation (STAT4) or dephosphorylation (STAT1). STAT3 binds predominantly to a single STAT consensus site in the Socs3 promoter, despite the presence of an adjacent STAT motif. Significantly, Delta 133STAT3 stimulates expression of the endogenous Socs3 gene in Stat3-/- MEFs upon IL-6 treatment with an activity similar to reconstituted STAT3, demonstrating that the N-domain is dispensable for Socs3 gene expression. We propose that the Socs3 gene in its chromosomal context is activated by the IL-6/STAT3 pathway independent of STAT3 N-domain sequences.


Asunto(s)
Interleucina-6/farmacología , Proteínas Recombinantes de Fusión/farmacología , Factor de Transcripción STAT3/química , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Proteínas Supresoras de la Señalización de Citocinas/genética , Animales , Secuencia de Bases , Secuencia Conservada , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Interleucina-6/genética , Ratones , Datos de Secuencia Molecular , Células 3T3 NIH , Regiones Promotoras Genéticas , Estructura Terciaria de Proteína/genética , Receptores de Interleucina/genética , Receptores de Interleucina-6 , Proteínas Recombinantes de Fusión/genética , Factor de Transcripción STAT3/genética , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Transfección
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