RESUMEN
Interleukin 33 (IL-33) is a member of the IL-1 family of cytokines with a growing number of target cells and a plethora of biological functions. Although it has commonalities with other IL-1 cytokines, IL-33 exhibits some unique features. Here we review the biology of IL-33 and its receptor and develop a working model that describes two 'lives' for IL-33-one intracellular and one extracellular. Under healthy conditions, constitutively produced, intracellular IL-33 participates in maintaining barrier function by regulating gene expression as a nuclear protein. In parallel, nuclear IL-33 functions as a stored alarmin that is released when barriers are breached. Extracellular IL-33 coordinates immune defense and repair mechanisms while also initiating differentiation of helper T cells as the adaptive immune response is triggered.
Asunto(s)
Alarminas/metabolismo , Interleucina-33/metabolismo , Animales , Regulación de la Expresión Génica , Humanos , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , Interleucina-33/química , Interleucina-33/genética , Unión Proteica , Transporte de Proteínas , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Transducción de SeñalRESUMEN
The extracellular forms of the IL-1 cytokines are active through binding to specific receptors on the surface of target cells. IL-1 ligands bind to the extracellular portion of their ligand-binding receptor chain. For signaling to take place, a non-binding accessory chain is recruited into a heterotrimeric complex. The intracellular approximation of the Toll-IL-1-receptor (TIR) domains of the 2 receptor chains is the event that initiates signaling. The family of IL-1 receptors (IL-1R) includes 10 structurally related members, and the distantly related soluble protein IL-18BP that acts as inhibitor of the cytokine IL-18. Over the years the receptors of the IL-1 family have been known with many different names, with significant confusion. Thus, we will use here a recently proposed unifying nomenclature. The family includes several ligand-binding chains (IL-1R1, IL-1R2, IL-1R4, IL-1R5, and IL-1R6), 2 types of accessory chains (IL-1R3, IL-1R7), molecules that act as inhibitors of signaling (IL-1R2, IL-1R8, IL-18BP), and 2 orphan receptors (IL-1R9, IL-1R10). In this review, we will examine how the receptors of the IL-1 family regulate the inflammatory and anti-inflammatory functions of the IL-1 cytokines and are, more at large, involved in modulating defensive and pathological innate immunity and inflammation. Regulation of the IL-1/IL-1R system in the brain will be also described, as an example of the peculiarities of organ-specific modulation of inflammation.
Asunto(s)
Inflamación/inmunología , Interleucina-1/metabolismo , Receptores de Interleucina-1/metabolismo , Animales , Humanos , Inmunidad Innata , Inmunomodulación , Interleucina-18/metabolismo , Transducción de SeñalRESUMEN
Interleukin-33 (IL-33) is an unconventional member of the IL-1 family: it is a dual function cytokine. Many different cell types, tissue cells and leukocytes, produce IL-33 either constitutively or after stimulation and release it by a poorly defined molecular mechanism. Free IL-33 acts as a classical cytokine by binding to target cells expressing receptors for IL-33 minimally consisting of ST2 and IL-1RAcP. Depending on the target cell type IL-33 will stimulate cell-type specific signal transduction mechanisms and thereby change the biosynthetic profile of the respective cell. In addition, it is stored in the nucleus of cells and may be released after cell stress, death by injury or necrosis, acting as an alarmin by orchestrating a sterile inflammation. Furthermore, IL-33 has intracrine functions in the cell producing it, which are independent of IL-33 receptors. Intracellular IL-33 is predominantly found in the nucleus associated to the chromatin and may exert gene regulatory function by yet poorly defined mechanisms. It is the aim of this review to address two basic biological aspects of the IL-33/IL-33 receptor system. First, to summarize the current understanding of the fate and function of intracellular IL-33, and second, to discuss recent advances in the knowledge of the molecular composition, function and regulation of the IL-33 receptor complex, including initial signaling mechanisms.
Asunto(s)
Proteína Accesoria del Receptor de Interleucina-1/metabolismo , Interleucinas/metabolismo , Sistema de Señalización de MAP Quinasas , Receptores de Superficie Celular/metabolismo , Puntos de Control del Ciclo Celular/genética , Núcleo Celular/metabolismo , Cromatina , Humanos , Inflamación/inmunología , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Proteína 1 Similar al Receptor de Interleucina-1 , Interleucina-33 , Interleucinas/biosíntesis , Interleucinas/genética , Estructura Terciaria de Proteína , Receptores de Superficie Celular/genética , Factor de Transcripción ReIA/biosíntesis , Factor de Transcripción ReIA/genética , Activación Transcripcional/genéticaRESUMEN
Background: As long-term, regular aesthetic botulinum neurotoxin A (BoNT-A) use becomes more commonplace, it is vital to understand real-world risk factors and impact of BoNT-A immunoresistance. The first Aesthetic Council on Ethical Use of Neurotoxin Delivery panel discussed issues relating to BoNT-A immunoresistance from the health care professionals' (HCPs') perspective. Understanding the implications of BoNT-A immunoresistance from the aesthetic patient's viewpoint allows HCPs to better support patients throughout their aesthetic treatment journey. Methods: A real-world consumer study surveyed 363 experienced aesthetic BoNT-A recipients across six Asia-Pacific territories. The survey mapped participants' BoNT-A aesthetic treatment journey and characterized awareness and attitudes relating to BoNT-A immunoresistance and treatment implications. At the second Aesthetic Council on Ethical use of Neurotoxin Delivery meeting, panelists discussed survey findings and developed consensus statements relating to the impact of BoNT-A immunoresistance on the aesthetic treatment journey. Results: Aesthetic BoNT-A patients' depth of knowledge about BoNT-A immunoresistance remains low, and risk/benefit communications need to be more lay-friendly. The initial consultation is the most important touchpoint for HCPs to raise awareness of BoNT-A immunoresistance as a potential side effect considering increased risk with repeated high-dose treatments. HCPs should be cognizant of differences across BoNT-A formulations due to the presence of certain excipients and pharmacologically unnecessary components that can increase immunogenicity. Standardized screening for clinical signs of secondary nonresponse and a framework for diagnosing and managing immunoresistance-related secondary nonresponse were proposed. Conclusion: These insights can help patients and HCPs make informed treatment decisions to achieve desired aesthetic outcomes while preserving future treatment options with BoNT-A.
RESUMEN
IL-1alpha, like IL-1beta, possesses multiple inflammatory and immune properties. However, unlike IL-1beta, the cytokine is present intracellularly in healthy tissues and is not actively secreted. Rather, IL-1alpha translocates to the nucleus and participates in transcription. Here we show that intracellular IL-1alpha is a chromatin-associated cytokine and highly dynamic in the nucleus of living cells. During apoptosis, IL-1alpha concentrates in dense nuclear foci, which markedly reduces its mobile nature. In apoptotic cells, IL-1alpha is retained within the chromatin fraction and is not released along with the cytoplasmic contents. To simulate the in vivo inflammatory response to cells undergoing different mechanisms of death, lysates of cells were embedded in Matrigel plugs and implanted into mice. Lysates from cells undergoing necrosis recruited cells of the myeloid lineage into the Matrigel, whereas lysates of necrotic cells lacking IL-1alpha failed to recruit an infiltrate. In contrast, lysates of cells undergoing apoptotic death were inactive. Cells infiltrating the Matrigel were due to low concentrations (20-50 pg) of the IL-1alpha precursor containing the receptor interacting C-terminal, whereas the N-terminal propiece containing the nuclear localization site failed to do so. When normal keratinocytes were subjected to hypoxia, the constitutive IL-1alpha precursor was released into the supernatant. Thus, after an ischemic event, the IL-1alpha precursor is released by hypoxic cells and incites an inflammatory response by recruiting myeloid cells into the area. Tissues surrounding the necrotic site also sustain damage from the myeloid cells. Nuclear trafficking and differential release during necrosis vs. apoptosis demonstrate that inflammation by IL-1alpha is tightly controlled.
Asunto(s)
Apoptosis , Cromatina/metabolismo , Interleucina-1alfa/metabolismo , Animales , Western Blotting , Hipoxia de la Célula , Línea Celular Tumoral , Núcleo Celular/metabolismo , Células Cultivadas , Medios de Cultivo Condicionados/química , Medios de Cultivo Condicionados/farmacología , Fibroblastos/citología , Fibroblastos/metabolismo , Recuperación de Fluorescencia tras Fotoblanqueo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Interleucina-1alfa/genética , Interleucina-1alfa/farmacología , Queratinocitos/citología , Queratinocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Células Mieloides/efectos de los fármacos , Células Mieloides/metabolismo , Células Mieloides/patología , Necrosis , Transporte de Proteínas , TransfecciónRESUMEN
With increasing off-label aesthetic indications using higher botulinum neurotoxin A (BoNT-A) doses and individuals starting treatment at a younger age, particularly in Asia, there is a greater risk of developing immunoresistance to BoNT-A. This warrants more in-depth discussions by aesthetic practitioners to inform patients and guide shared decision-making. A panel comprising international experts and experienced aesthetic practitioners in Hong Kong discussed the implications and impact of immunoresistance to BoNT-A in contemporary aesthetic practice, along with practical strategies for risk management. Following discussions on a clinical case example and the results of an Asia-Pacific consumer study, the panel concurred that it is a priority to raise awareness of the possibility and long-term implications of secondary non-response due to immunoresistance to BoNT-A. Where efficacy and safety are comparable, a formulation with the lowest immunogenicity is preferred. The panel also strongly favored a thorough initial consultation to establish the patient's treatment history, explain treatment side effects, including the causes and consequences of immunoresistance, and discuss treatment goals. Patients look to aesthetic practitioners for guidance, placing an important responsibility on practitioners to adopt risk-mitigating strategies and adequately communicate important risks to patients to support informed and prudent BoNT-A treatment decisions.
Asunto(s)
Toxinas Botulínicas Tipo A , Humanos , Toxinas Botulínicas Tipo A/toxicidad , Hong KongRESUMEN
Botulinum neurotoxin A (BoNT-A) injection is the most widely performed aesthetic procedure and a first-line therapeutic option for various medical conditions. The potential for BoNT-A immunoresistance and secondary nonresponse related to neutralizing antibody (NAb) formation warrants attention as the range of BoNT-A aesthetic applications continues to expand. Methods: An international multidisciplinary panel reviewed published evidence on BoNT-A immunoresistance in aesthetic and therapeutic applications and discussed best practices integrating clinical, ethical, and aesthetic considerations. Consensus statements relating to awareness, assessment, and management of the risk of NAb-related secondary nonresponse in aesthetic practice were developed. Results: There was a consensus that, as doses used in aesthetic practice become like those in therapeutics, rates of NAb formation may be expected to increase. However, the true extent of NAb formation in aesthetics is likely underestimated due to limitations of published evidence and variability in treatment patterns of aesthetic patients. Since BoNT-A therapy is often lifelong, practitioners need to recognize immunogenicity as a potential complication that might affect future therapeutic use and strive to minimize modifiable risk factors. The selection and use of a BoNT-A product with the least immunogenic potential from the beginning may thus be advantageous, especially when treatment with high doses is planned. Conclusions: In view of current trends in BoNT-A aesthetic use, it is essential for practitioners to conduct thorough clinical assessments, inform patients of treatment risks, and develop BoNT-A treatment plans to minimize immunogenicity. This can help preserve the option of continued or future BoNT-A treatment with satisfactory outcomes.
RESUMEN
Lack of the IL-1 receptor accessory protein (IL-1RAcP) abrogates responses to IL-33 and IL-1 in the mouse thymoma clone EL-4 D6/76 cells. Reconstitution with full-length IL-1RAcP is sufficient to restore responsiveness to IL-33 and IL-1. IL-33 activates IL-1 receptor-associated kinase-1, cJun-N-terminal kinase, and the NF-kappaB pathway in an IL-1RAcP-dependent manner and results in IL-2 release. IL-33 is able to induce the release of proinflammatory cytokines in bone marrow-derived (BMD) mast cells, indicating that IL-33 may have a proinflammatory potential like its relatives IL-1 and IL-18, in addition to its Th2-skewing properties in the adaptive response described previously. Blocking of murine IL-1RAcP with the neutralizing antibody 4C5 inhibits response of mouse thymoma cells and BMD mast cells to IL-33. The interaction of either membrane-bound or soluble forms of IL-1RAcP and IL-33Ralpha-chain depends on the presence of IL-33, as demonstrated by coimmunoprecipitation assays. These data demonstrate that IL-1RAcP is indispensable for IL-33 signaling. Furthermore, they suggest that IL-1RAcP is used by more than one alpha-chain of the IL-1 receptor family and thus may resemble a common beta-chain of that family.
Asunto(s)
Proteína Accesoria del Receptor de Interleucina-1/metabolismo , Interleucinas/farmacología , Activación de Linfocitos/inmunología , Mastocitos/efectos de los fármacos , Mastocitos/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Animales , Línea Celular , Humanos , Interleucina-33 , Activación de Linfocitos/efectos de los fármacos , Mastocitos/metabolismo , Ratones , Unión Proteica , Linfocitos T/metabolismoRESUMEN
Ligand binding in the TLR/IL-1R family results in the transient formation of an intracellular signaling complex, which contains, amongst others, the serine/threonine-specific kinase IL-1R-associated kinase 1 (IRAK-1). Concomitantly, the kinase function of IRAK-1 becomes activated, resulting in massive autophosphorylation and finally in the dissociation of the initially constituted signaling complex. The death domain (DD) of IRAK-1 mediates the interaction with other molecules of the signaling complex, e.g., the adaptor MyD88, the silencer Tollip, and the activator kinase IRAK-4. The conserved threonine at position 66 (T66), located within the DD, is a putative autophosphorylation target site. Here, we provide evidence that T66 critically impacts the secondary structure of the IRAK-1 DD. Thereby, it ensures the transient manner of interactions between IRAK-1 and the other signaling molecules. This essential role, however, is not regulated by phosphorylation of T66 itself.
Asunto(s)
Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Factor 88 de Diferenciación Mieloide/metabolismo , Treonina/química , Células Cultivadas , Transferencia Resonante de Energía de Fluorescencia , Vectores Genéticos , Humanos , Immunoblotting , Inmunoprecipitación , Quinasas Asociadas a Receptores de Interleucina-1/química , Quinasas Asociadas a Receptores de Interleucina-1/genética , Interleucina-2/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Riñón/metabolismo , Peso Molecular , Factor 88 de Diferenciación Mieloide/genética , FN-kappa B/genética , FN-kappa B/metabolismo , Fosforilación , Transducción de Señal , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Treonina/genética , Treonina/metabolismoRESUMEN
The Toll/interleukin-1 (IL-1) receptor (TIR) family comprises two groups of transmembrane proteins, which share functional and structural properties. The members of the IL-1 receptor (IL-1R) subfamily are characterized by three extracellular immunoglobulin (Ig)-like domains. They form heterodimeric signaling receptor complexes consisting of receptor and accessory proteins. The members of the Toll-like receptor (TLR) subfamily recognize alarm signals that can be derived either from pathogens or the host itself. TLRs possess leucine-rich repeats in their extracellular part. TLRs can form dimeric receptor complexes consisting of two different TLRs or homodimers in the case of TLR4. The TLR4 receptor complex requires supportive molecules for optimal response to its ligand lipopolysaccharide (LPS). A hallmark of the TIR family is the cytoplasmic TIR domain that is indispensable for signal transduction. The TIR domain serves as a scaffold for a series of protein-protein interactions which result in the activation of a unique signaling module consisting of MyD88, interleukin-1 receptor associated kinase (IRAK) family members and Tollip, which is used exclusively by TIR family members. Subsequently, several central signaling pathways are activated in parallel, the activation of NFkappaB being the most prominent event of the inflammatory response. Recent developments indicate that in addition to the common signaling module MyD88/IRAK/Tollip, other molecules can modulate signaling by TLRs, especially of TLR4, resulting in differential biological answers to distinct pathogenic structures. Subtle differences in TLR signaling pathways are now becoming apparent, which reveal how the innate immune system decides at a very early stage the direction in which the adaptive immune response will develop. The creation of pathogen-specific mediator environments by dendritic cells defines whether a cellular or humoral response will be activated in response to the pathogen.
Asunto(s)
Proteínas de Drosophila , Péptidos y Proteínas de Señalización Intracelular , Glicoproteínas de Membrana/fisiología , Receptores de Superficie Celular/fisiología , Receptores de Interleucina-1/fisiología , Proteínas Adaptadoras Transductoras de Señales , Animales , Antígenos de Diferenciación/fisiología , Proteínas Portadoras/fisiología , Humanos , Quinasas Asociadas a Receptores de Interleucina-1 , Interleucina-18/química , Subunidad alfa del Receptor de Interleucina-18 , Glicoproteínas de Membrana/química , Modelos Moleculares , Factor 88 de Diferenciación Mieloide , Proteínas Quinasas/fisiología , Estructura Terciaria de Proteína , Receptores de Superficie Celular/química , Receptores Inmunológicos/fisiología , Receptores de Interleucina/química , Receptores de Interleucina/fisiología , Receptores de Interleucina-1/química , Receptores de Interleucina-18 , Transducción de Señal , Receptor Toll-Like 4 , Receptores Toll-LikeRESUMEN
The dual function cytokines IL-1α, IL-33 and IL-37 are members of the IL-1 cytokine family. Besides of being able to bind to their cognate receptors on target cells, they can act intracellularly in the producing cell. All three are able to translocate to the nucleus and have been discussed to affect gene expression. In order to compare and quantitate nuclear translocation of these IL-1 family members we established a robust technique which enables to measure nuclear localization on a single cell level by flow cytometry. Vectors encoding fusion proteins of different IL-1 family members with enhanced green fluorescent protein were cloned and cell lines transiently transfected with these. Fluorescent fusion proteins in intact cells or in isolated nuclei were detected subsequently by fluorescence microscopy and flow cytometry, respectively. Depending on the cellular system, cells and nuclei were distinguishable by flow cytometry in forward scatter/sideward scatter. Fluorescent fusion proteins were detectable in isolated nuclei up to three days following preparation. Signal intensity of fusion proteins of IL-33 and IL-37 in isolated nuclei but not of IL-1α, was markedly increased by fixation with paraformaldehyde, directly following cell lysis, indicating that IL-1α binds stronger to nuclear structures than IL-33 and IL-37. Nuclear translocation of fluorescent IL-37 fusion proteins in a stably transfected RAW264.7 mouse macrophage cell line required stimulation with lipopolysaccharide. Applying this method we demonstrated that a prolonged lag phase of more than 15h before LPS-stimulated nuclear translocation was detected. In summary, we present a robust method to analyze and quantitate nuclear localization of IL-1 cytokine family members.
Asunto(s)
Núcleo Celular/metabolismo , Citometría de Flujo/métodos , Interleucina-1/metabolismo , Interleucina-1alfa/metabolismo , Interleucinas/metabolismo , Transporte Activo de Núcleo Celular , Animales , Línea Celular , Células HEK293 , Células HeLa , Humanos , Interleucina-1/genética , Interleucina-1alfa/genética , Interleucina-33 , Interleucinas/genética , Lipopolisacáridos/farmacología , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Microscopía Fluorescente , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Análisis de la Célula Individual/métodosRESUMEN
Alveolar IL-8 has been reported to early identify patients at-risk to develop ARDS. However, it remains unknown how alveolar IL-8 is related to pulmonary and systemic inflammation in patients predisposed for ARDS. We studied 24 patients 2-6h after multiple trauma. Patients with IL-8 >200 pg/ml in bronchoalveolar lavage (BAL) were assigned to the group at high risk for ARDS (H, n = 8) and patients with BAL IL-8 <200 pg/ml to the group at low risk for ARDS (L, n = 16). ARDS developed within 24h after trauma in 5 patients at high and at least after 1 week in 2 patients at low risk for ARDS (p = 0.003). High-risk patients had also increased BAL IL-6, TNF-α, IL-1ß, IL-10 and IL-1ra levels (p<0.05). BAL neutrophil counts did not differ between patient groups (H vs. L, 12% (3-73%) vs. 6% (2-32%), p = 0.1) but correlated significantly with BAL IL-8, IL-6 and IL-1ra. High-risk patients had increased plasma levels of pro- but not anti-inflammatory mediators. The enhanced alveolar and systemic inflammation associated with alveolar IL-8 release should be considered to identify high-risk patients for pulmonary complications after multiple trauma to adjust surgical and other treatment strategies to the individual risk profile.
Asunto(s)
Líquido del Lavado Bronquioalveolar/inmunología , Traumatismo Múltiple/inmunología , Síndrome de Dificultad Respiratoria/inmunología , Adulto , Anciano , Susceptibilidad a Enfermedades , Ensayo de Inmunoadsorción Enzimática , Femenino , Mortalidad Hospitalaria , Humanos , Proteína Antagonista del Receptor de Interleucina 1/inmunología , Interleucina-10/inmunología , Interleucina-8/inmunología , Recuento de Leucocitos , Masculino , Persona de Mediana Edad , Traumatismo Múltiple/mortalidad , Traumatismo Múltiple/fisiopatología , Valor Predictivo de las Pruebas , Pronóstico , Estudios Prospectivos , Síndrome de Dificultad Respiratoria/mortalidad , Síndrome de Dificultad Respiratoria/fisiopatología , Factor de Necrosis Tumoral alfa/inmunología , Adulto JovenRESUMEN
Ligand binding in the Toll-like/interleukin-1 receptor family results in the recruitment of an intracellular signaling complex. IRAK-1, which is centrally involved in this complex, is able to homo-oligomerize and to bind to Tollip and the adapters MyD88 and IRAK-4. The interactions of IRAK-1 with MyD88 or Tollip are mediated by the N-terminal part of IRAK-1, containing the death domain with the highly conserved threonine at position 66 (T66). Mutation of this amino acid into alanine or aspartic acid stabilized binding to MyD88, Tollip, and IRAK-4, allowing the definitive experimental proof, that all these interactions are mediated by the death domain of IRAK-1. Homo-oligomerization of IRAK-1, which is mediated by the death domain too, is not affected by mutation of T66. Finally, mutation of IRAK-1 at T66 not only allowed stable binding to the signaling adapters, but also enhanced its signaling capacity.
Asunto(s)
Quinasas Asociadas a Receptores de Interleucina-1/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Factor 88 de Diferenciación Mieloide/metabolismo , Receptores de Muerte Celular/fisiología , Secuencia de Aminoácidos , Línea Celular Tumoral , Humanos , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Estructura Terciaria de Proteína , Transducción de Señal , Treonina/fisiología , TimomaRESUMEN
Interleukin-1 receptor-associated-kinases (IRAKs) are signal transduction mediators of the Toll/IL-1 receptor family, which comprise several transmembrane proteins involved in host defense mechanisms. Today four different human IRAKs (hu-IRAK-1, hu-IRAK-2, hu-IRAK-M, hu-IRAK-4) and two murine IRAKs (mouse pelle like kinase (mPLK) and mu-IRAK-4) have been described. Here we report the identification and characterization of murine IRAK-M (mu-IRAK-M), a mouse homologue to human IRAK-M (hu-IRAK-M). These IRAK-M molecules show 71% sequence identity, a comparable cellular expression, and functional similarities with respect to signal transduction capacity and kinase activity, suggesting functional homology in signalling in human and mouse cells.
Asunto(s)
Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , Animales , Northern Blotting , Células Cultivadas , ADN Complementario/metabolismo , Genes Reporteros , Humanos , Immunoblotting , Interleucina-1/metabolismo , Quinasas Asociadas a Receptores de Interleucina-1 , Ratones , FN-kappa B/metabolismo , Fosforilación , Reacción en Cadena de la Polimerasa , Pruebas de Precipitina , Estructura Terciaria de Proteína , ARN/metabolismo , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Distribución TisularRESUMEN
Interleukin-1 receptor-associated kinases (IRAKs) are pivotal signaling elements of the Toll/IL-1 receptor (TIL) family, which play a role in innate immune responses by coordinating host defence mechanisms. Presently four different forms of human IRAK molecules are cloned (hu-IRAK-1, hu-IRAK-2, hu-IRAK-M, and hu-IRAK-4). In the murine system, only three genes have been identified so far, mouse Pelle-Like Kinase (mPLK), which corresponds to human IRAK-1, mu-IRAK-M, and mu-IRAK-4. Here we report the molecular cloning and characterization of murine IRAK-2 (mu-IRAK-2), a mouse homolog to human IRAK-2 (hu-IRAK-2). Murine and human IRAK-2 molecules show 67% sequence identity, they are ubiquitiously expressed, and both practically lack autophoshorylation kinase activity. The murine molecule reveals two remarkable differences to its human counterpart: it shows a C-terminal extension and it has no stimulatory effect on IL-1 induced NF-kappa B activation when compared to hu-IRAK-2, suggesting subtle functional differences in signaling by IRAK-2 in human and mouse cells.
Asunto(s)
Proteínas Quinasas/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Expresión Génica , Humanos , Quinasas Asociadas a Receptores de Interleucina-1 , Ratones , Datos de Secuencia Molecular , FN-kappa B/metabolismo , Fosforilación , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
IL-1, IL-18 and LPS are recognized by specific receptor complexes of the Toll/IL-1R family, characterized by a common intracellular domain indispensable for downstream signaling. Upon ligand binding, these receptors activate the central MyD88-IRAK-TRAF6 signaling module, resulting in the activation of NF-kappaB. Ligated receptors also induce activation of other signaling cascades, suchas the PI3-kinase (PI3-K) and the p38 mitogen-activated protein kinase (MAPK) pathways. Unlike the p38MAPK pathway, which couples to the central signaling module, the PI3-K pathway seems to directly interact with the receptor molecules. Thus, activation of the PI3-K pathway is thought to be independent of the IRAK-containing signaling module. Employing two cell lines, we show that the PI3-K pathways can be activated by IL-1, IL-18 or LPS with comparable, but cell type specific kinetics, which can be correlated to biological consequences. This indicates that activation of the PI3-K pathways may be regulated by an element common for all three receptor types, the MyD88-IRAK-TRAF6 module being a candidate for this function. Using an IRAK-1-deficient cell line, we demonstrate that the IRAK-1-containing signaling module is essential for the IL-1-induced activation of the PI3-K pathway. Possible models of the interaction between IRAK-1 and the PI3-K pathway are discussed.
Asunto(s)
Interleucina-1/farmacología , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/metabolismo , Células 3T3 , Androstadienos/farmacología , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Inhibidores Enzimáticos/farmacología , Quinasas Asociadas a Receptores de Interleucina-1 , Interleucina-18/farmacología , Interleucina-2/biosíntesis , Cinética , Lipopolisacáridos/farmacología , Ratones , Proteínas Quinasas Activadas por Mitógenos/biosíntesis , Modelos Inmunológicos , Fosfatidilinositol 3-Quinasas/biosíntesis , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Proteínas Quinasas/deficiencia , Proteínas Proto-Oncogénicas c-akt , Wortmanina , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
The Toll/interleukin 1 (IL-1) receptor family plays an important role in both innate and adaptive immunity. These receptors are characterized by a C-terminal homology motif called the Toll/IL-1 receptor (TIR) domain. A principal function of the TIR domain is mediating homotypic protein-protein interactions in the signal transduction pathway. To suggest interaction sites of TIR domains in the IL-1 receptor complex, we modeled the putative three-dimensional structure of the TIR domain within the co-receptor chain, IL-1 receptor accessory protein. The model was based on homology with the crystal structures of human TLR1 and TLR2. The final structure of the IL-1 receptor accessory protein TIR domain suggests the conserved regions box 1 and 2, including Pro-446, as well as box 3 within the C-terminal alpha-helix as possible protein-protein interaction sites due to their exposure and their electrostatic potential. Pro-446, corresponding to the Pro/His mutation in dominant negative TLR4, is located in the third loop at the outmost edge of the TIR domain and does not play any structural role. Inhibition of IL-1 responsiveness seen after substitution of Pro-446 by charged amino acids is due to the loss of an interaction site for other TIR domains. Amino acids 527-534 as part of the loop close to the conserved box 3 are critical for recruitment of myeloid differentiation factor 88 and to a lesser extent for IL-1 responsiveness. Modeling suggests that native folding of the TIR domain may be approached by the responsive deletion mutants delta528-534 and delta527-533, whereas the C-terminal beta-strand and/or alpha-helix is displaced in the nonresponsive mutant delta527-534.
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Receptores de Interleucina-1/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular Tumoral , Cartilla de ADN , Proteína Accesoria del Receptor de Interleucina-1 , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Receptores de Interleucina-1/química , Receptores de Interleucina-1/genética , Homología de Secuencia de AminoácidoRESUMEN
The interleukin-1 receptor-associated kinase 1 (IRAK-1) is an important adapter in the signaling complex of the Toll/interleukin-1 (IL-1) receptor family. Formation of the signaling IL-1 receptor complex results in the activation and hyperphosphorylation of IRAK-1, which leads to a pronounced shift of its apparent molecular mass in gel electrophoresis. Presently, the individual residues phosphorylated in IRAK-1 and the consequences for IRAK-1 function are unknown. We define sequential phosphorylation steps in IRAK-1, which are, in vitro, autophosphorylation. First, IRAK-1 is phosphorylated at Thr209. By fluorescence energy transfer experiments, we demonstrate that Thr209 phosphorylation results in a conformational change of the kinase domain, permitting further phosphorylations to take place. Substitution of Thr209 by alanine results in a kinase-inactive IRAK-1. Second, Thr387 in the activation loop is phosphorylated, leading to full enzymatic activity. Third, IRAK-1 autophosphorylates several times in the proline-, serine-, and threonine-rich ProST region between the N-terminal death domain and kinase domain. Hyperphosphorylation of this region leads to dissociation of IRAK-1 from the upstream adapters MyD88 and Tollip but leaves its interaction with the downstream adapter TRAF6 unaffected. This identifies IRAK-1 as a novel type of adapter protein, which employs its own kinase activity to introduce negative charges adjacent to the protein interaction domain, which anchors IRAK-1 at the active receptor complex. Thus, IRAK-1 regulates its own availability as an adapter molecule by sequential autophosphorylation.
Asunto(s)
Interleucina-1/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Proteínas Quinasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Antígenos de Diferenciación/química , Proteínas Portadoras/química , Línea Celular , Clonación Molecular , Dimerización , Transferencia Resonante de Energía de Fluorescencia , Vectores Genéticos , Humanos , Immunoblotting , Quinasas Asociadas a Receptores de Interleucina-1 , Espectrometría de Masas , Modelos Biológicos , Datos de Secuencia Molecular , Factor 88 de Diferenciación Mieloide , Fosforilación , Pruebas de Precipitina , Conformación Proteica , Estructura Terciaria de Proteína , Receptores Inmunológicos/química , Receptores de Interleucina-1/metabolismo , Transducción de Señal , Espectrometría de Masa por Ionización de Electrospray , Treonina/química , Factores de Tiempo , TransfecciónRESUMEN
In human monocytes and macrophages, interferon-gamma (IFNgamma) augmented mRNA and surface expression of toll-like receptor 4 (TLR4), a crucial component of the signaling receptor complex for bacterial lipopolysaccharide (LPS). Expression of the accessory component MD-2 and of the adapter protein MyD88 was also increased. LPS increased TLR4 mRNA levels, but concomitantly decreased its surface expression. IFNgamma counteracted the LPS-induced downregulation of TLR4. IFNgamma-primed monocytes showed increased responsiveness to LPS in terms of phosphorylation of the interleukin-1 receptor-associated kinase (IRAK; immediately downstream of the MyD88 adapter protein), NF-kB DNA binding activity, and, accordingly, of cytokine (tumor necrosis factor alpha [TNFalpha] and interleukin-12 [IL-12]) production. These results suggest that enhanced TLR4 expression underlies the long-known priming by IFNgamma of mononuclear phagocytes for pathogen recognition and killing as well as its synergism with LPS in macrophage activation.