RESUMEN
In the present study, we characterized tumor necrosis factor receptor-associated factor 2/7 (lcTRAF2/7) and TNFR1-associated death domain protein (lcTRADD) in Larimichthys crocea (L. crocea) and examined their expression profiles in tissues of Vibrio-challenged and unchallenged fish. The coding sequences of lcTRAF2, lcTRAF7, and lcTRADD were 1488, 2454, and 744 nucleotides, and they encoded proteins of 495, 344, and 248 amino acids, respectively. The results of phylogenetic analysis revealed that lcTRAF2, lcTRAF7, and lcTRADD were closest to Oplegnathus fasciatus (85%), Xiphophorus maculatus (97%), and Acanthochromis polyacanthus (65%), respectively. Multiple sequence alignment showed that lcTRAF2 and lcTRAF7 were highly conserved with other vertebrate TRAFs in their functional domains; however, lcTRADD was poorly conserved. The results of quantitative real-time polymerase chain reaction analysis indicated that lcTRAF2, lcTRAF7, and lcTRADD were constitutively expressed in the spleen, liver, kidney, heart, brain, gill, bladder, skin, fin, eye, and muscle. After challenging fish with Vibrio parahaemolyticus, the mRNA expression levels of lcTRAF2, lcTRAF7, and lcTRADD were upregulated in liver, spleen, and kidney. Immunofluorescence staining revealed that lcTRAF2 and lcTRADD were cytoplasmic in localization, whereas lcTRAF7 targeted both the cytoplasm and nucleus. In addition, the NF-κB protein level was upregulated after lipopolysaccharide stimulation in lcTRAF2, lcTRAF7, or lcTRADD overexpressing cells. Taken collectively, these results have improved our understanding of the functions of TRAF2, TRAF7, and TRADD in pathogenic infections in teleosts.
Asunto(s)
Enfermedades de los Peces/inmunología , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Regulación de la Expresión Génica/inmunología , Inmunidad Innata/genética , Perciformes/genética , Perciformes/inmunología , Secuencia de Aminoácidos , Animales , Proteínas de Peces/química , Perfilación de la Expresión Génica/veterinaria , Filogenia , Alineación de Secuencia/veterinaria , Proteína de Dominio de Muerte Asociada a Receptor de TNF/química , Proteína de Dominio de Muerte Asociada a Receptor de TNF/genética , Proteína de Dominio de Muerte Asociada a Receptor de TNF/inmunología , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/química , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/genética , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/inmunología , Vibriosis/inmunología , Vibriosis/veterinaria , Vibrio parahaemolyticus/fisiologíaRESUMEN
The protozoan parasite Toxoplasma gondii secretes proteins from specialized organelles, the rhoptries, and dense granules, which are involved in the modulation of host cell processes. Dense granule protein GRA15 activates the nuclear factor kappa B (NF-κB) pathway, which plays an important role in cell death, innate immunity, and inflammation. Exactly how GRA15 activates the NF-κB pathway is unknown. Here we show that GRA15 interacts with tumor necrosis factor receptor-associated factors (TRAFs), which are adaptor proteins functioning upstream of the NF-κB transcription factor. We identified several TRAF binding sites in the GRA15 amino acid sequence and showed that these are involved in NF-κB activation. Furthermore, a TRAF2 knockout cell line has impaired GRA15-mediated NF-κB activation. Thus, we determined the mechanism for GRA15-dependent NF-κB activation.IMPORTANCE The parasite Toxoplasma can cause birth defects and severe disease in immunosuppressed patients. Strain differences in pathogenicity exist, and these differences are due to polymorphic effector proteins that Toxoplasma secretes into the host cell to coopt host cell functions. The effector protein GRA15 of some Toxoplasma strains activates the nuclear factor kappa B (NF-κB) pathway, which plays an important role in cell death, innate immunity, and inflammation. We show that GRA15 interacts with TNF receptor-associated factors (TRAFs), which are adaptor proteins functioning upstream of the NF-κB transcription factor. Deletion of TRAF-binding sites in GRA15 greatly reduces its ability to activate the NF-κB pathway, and TRAF2 knockout cells have impaired GRA15-mediated NF-κB activation. Thus, we determined the mechanism for GRA15-dependent NF-κB activation.
Asunto(s)
FN-kappa B/metabolismo , Proteínas Protozoarias/metabolismo , Transducción de Señal , Toxoplasma/fisiología , Toxoplasmosis/metabolismo , Toxoplasmosis/parasitología , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/metabolismo , Sitios de Unión , Expresión Génica , Interacciones Huésped-Patógeno , Humanos , Unión Proteica , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/químicaRESUMEN
Determining the relationship between protein folding pathways on and off the ribosome remains an important area of investigation in biology. Studies on isolated domains have shown that alteration of the separation of residues in a polypeptide chain, while maintaining their spatial contacts, may affect protein stability and folding pathway. Due to the vectorial emergence of the polypeptide chain from the ribosome, chain connectivity may have an important influence upon cotranslational folding. Using MATH, an all ß-sandwich domain, we investigate whether the connectivity of residues and secondary structure elements is a key determinant of when cotranslational folding can occur on the ribosome. From Φ-value analysis, we show that the most structured region of the transition state for folding in MATH includes the N and C terminal strands, which are located adjacent to each other in the structure. However, arrest peptide force-profile assays show that wild-type MATH is able to fold cotranslationally, while some C-terminal residues remain sequestered in the ribosome, even when destabilized by 2-3â¯kcalâ¯mol-1. We show that, while this pattern of Φ-values is retained in two circular permutants in our studies of the isolated domains, one of these permutants can fold only when fully emerged from the ribosome. We propose that in the case of MATH, onset of cotranslational folding is determined by the ability to form a sufficiently stable folding nucleus involving both ß-sheets, rather than by the location of the terminal strands in the ribosome tunnel.
Asunto(s)
Ribosomas/metabolismo , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/química , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/metabolismo , Cinética , Modelos Moleculares , Biosíntesis de Proteínas , Conformación Proteica en Lámina beta , Pliegue de Proteína , Estabilidad Proteica , Estructura Secundaria de ProteínaRESUMEN
TRAF-interacting protein (TRAIP), a negative regulator of TNF-induced-nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation, inhibits adaptor protein TRAF2 by direct interaction and is critical in apoptosis, cell proliferation, antiviral response, and embryonic development. Although the critical function of TRAIP in NF-κB signaling is well-known, the molecular inhibitory mechanism of TRAIP remains unclear. We found that the TRAIP coiled-coil domain altered its stoichiometry between dimer and trimer in a concentration-dependent manner. Additionally, the TRAIP RING domain induced even higher-ordered assembly, which was necessary for interacting with the TRAF-N domain of TRAF2 but not TRAF1. Characterization of the TRAF-N domains of TRAF1 and TRAF2, the tentative TRAIP-binding region of TRAFs, suggested the molecular basis of the inhibitory effect of TRAIP on TRAF2 in NF-κB signaling.
Asunto(s)
Factor 2 Asociado a Receptor de TNF/metabolismo , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/metabolismo , Humanos , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Mapas de Interacción de Proteínas , Multimerización de Proteína , Estabilidad Proteica , Factor 2 Asociado a Receptor de TNF/química , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/químicaRESUMEN
The seven members of the tumor necrosis factor receptor (TNF-R)-associated factor (TRAF) family of intracellular proteins were originally discovered and characterized as signaling adaptor molecules coupled to the cytoplasmic regions of receptors of the TNF-R superfamily. Functionally, TRAFs act both as a scaffold and/or enzymatic proteins to regulate activation of mitogen-activated protein kinases (MAPKs) and transcription factors of nuclear factor-κB family (NF-κB). Given the wide variety of stimuli intracellularly conveyed by TRAF proteins, they are physiologically involved in multiple biological processes, including embryonic development, tissue homeostasis, and regulation of innate and adaptive immune responses. In the last few years, it has become increasingly evident the involvement of TRAF7, the last member of the TRAF family to be discovered, in the genesis and progression of several human cancers, placing TRAF7 in the spotlight as a novel tumor suppressor protein. In this paper, we review and discuss the literature recently produced on this subject. J. Cell. Physiol. 232: 1233-1238, 2017. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.
Asunto(s)
Carcinogénesis/metabolismo , Carcinogénesis/patología , Neoplasias/metabolismo , Neoplasias/patología , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/metabolismo , Animales , Humanos , Modelos Biológicos , Mutación/genética , Dominios Proteicos , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/química , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/genéticaRESUMEN
TRIP (Tumor Necrosis Factor (TNF) Receptor-Associated Factor (TRAF)-Interacting Protein), a member of the TNF superfamily, plays a crucial role in the modulation of inflammation in vertebrates. However, no information about TRIP is available in teleosts. In this study, the full-length cDNA of TRIP, containing a 5'UTR of 112 bp, an ORF of 1359 bp, and a 3'UTR of 29 bp before the poly (A) tail, was cloned from grass carp, Ctenopharyngodon idella. The TRIP gene encoded a protein of 452 amino acids with an estimated molecular mass of 51.06 KD and a predicted theoretical isoelectric point (pI) of 9.11. Quantitative real-time PCR analysis revealed that TRIP mRNA was expressed in all the tissues examined in grass carp, with the highest expression in the kidney, followed by the intestine and thymus. However, lower levels of expression were also detected in fat, spleen, liver, gonad and heart. Subcellular localization and two-hybrid analysis revealed that TRIP was located in the nucleus and that it interacted with TRAF1 and TRAF2 in HEK293T cells. Furthermore, similar to TNF-α, IL-10 and TRIP mRNA expression was upregulated in the spleen of fish fed high-fat or high-carbohydrate diets, suggesting that TRIP might be associated with the response to excessive energy intake. The mRNA relative expression of TRIP was significantly reduced (P < 0.05) after hepatocyte of C. idella was treated with 2 µg/mL lipopolysaccharide (LPS) for 4 h, while the expression levels of inflammatory cytokines TNF-α and IL-10 were significantly increased (P < 0.05). Taken together, these results indicate that TRIP might play important roles in immune defense and has the potential to be used as a anti-inflammation target in grass carp.
Asunto(s)
Carpas/genética , Carbohidratos de la Dieta/administración & dosificación , Proteínas de Peces/genética , Regulación de la Expresión Génica , Lípidos/administración & dosificación , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/genética , Alimentación Animal/análisis , Animales , Carpas/inmunología , Carpas/metabolismo , Clonación Molecular , ADN Complementario/genética , ADN Complementario/metabolismo , Dieta/veterinaria , Proteínas de Peces/química , Proteínas de Peces/metabolismo , Células HEK293 , Humanos , Filogenia , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de Secuencia de Proteína/veterinaria , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/química , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/metabolismoRESUMEN
Mammalian Tyrosyl-DNA phosphodiesterase 2 (Tdp2) reverses Topoisomerase 2 (Top2) DNA-protein crosslinks triggered by Top2 engagement of DNA damage or poisoning by anticancer drugs. Tdp2 deficiencies are linked to neurological disease and cellular sensitivity to Top2 poisons. Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions. Modeling of a proposed Tdp2 reaction coordinate, combined with mutagenesis and biochemical studies support a single Mg(2+)-ion mechanism assisted by a phosphotyrosyl-arginine cation-π interface. We further identify a Tdp2 active site SNP that ablates Tdp2 Mg(2+) binding and catalytic activity, impairs Tdp2 mediated NHEJ of tyrosine blocked termini, and renders cells sensitive to the anticancer agent etoposide. Collectively, our results provide a structural mechanism for Tdp2 engagement of heterogeneous DNA damage that causes Top2 poisoning, and indicate that evaluation of Tdp2 status may be an important personalized medicine biomarker informing on individual sensitivities to chemotherapeutic Top2 poisons.
Asunto(s)
Daño del ADN , ADN-Topoisomerasas de Tipo II/metabolismo , Hidrolasas Diéster Fosfóricas/química , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/química , Animales , Dominio Catalítico , ADN/química , ADN/metabolismo , Aductos de ADN/química , Aductos de ADN/metabolismo , Reparación del ADN por Unión de Extremidades , ADN-Topoisomerasas de Tipo II/química , Proteínas de Unión al ADN , Humanos , Magnesio/química , Ratones , Ratones Noqueados , Modelos Moleculares , Mutación , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Fosfotirosina/metabolismo , Polimorfismo de Nucleótido Simple , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/genética , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/metabolismoRESUMEN
Tumor necrosis factor receptor-associated factors (TRAFs) constitute a family of adapter proteins that act in numerous signaling pathways important in human biology and disease. The MATH domain of TRAF proteins binds peptides found in the cytoplasmic domains of signaling receptors, thereby connecting extracellular signals to downstream effectors. Beyond several very general motifs, the peptide binding preferences of TRAFs have not been extensively characterized, and differences between the binding preferences of TRAF paralogs are poorly understood. Here we report a screening system that we established to explore TRAF peptide-binding specificity using deep mutational scanning of TRAF-peptide ligands. We displayed single- and double-mutant peptide libraries based on the TRAF-binding sites of CD40 or TANK on the surface of Escherichia coli and screened them for binding to TRAF2, TRAF3, and TRAF5. Enrichment analysis of the library sequencing results showed differences in the permitted substitution patterns in the TANK versus CD40 backgrounds. The three TRAF proteins also demonstrated different preferences for binding to members of the CD40 library, and three peptides from that library that were analyzed individually showed striking differences in affinity for the three TRAFs. These results illustrate a previously unappreciated level of binding specificity between these close paralogs and demonstrate that established motifs are overly simplistic. The results from this work begin to outline differences between TRAF family members, and the experimental approach established herein will enable future efforts to investigate and redesign TRAF peptide-binding specificity.
Asunto(s)
Análisis Mutacional de ADN/métodos , Péptidos/metabolismo , Factor 2 Asociado a Receptor de TNF/metabolismo , Factor 3 Asociado a Receptor de TNF/metabolismo , Factor 5 Asociado a Receptor de TNF/metabolismo , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Sitios de Unión , Antígenos CD40/metabolismo , Humanos , Modelos Moleculares , Unión Proteica , Estructura Secundaria de Proteína , Factor 2 Asociado a Receptor de TNF/química , Factor 2 Asociado a Receptor de TNF/genética , Factor 3 Asociado a Receptor de TNF/química , Factor 3 Asociado a Receptor de TNF/genética , Factor 5 Asociado a Receptor de TNF/química , Factor 5 Asociado a Receptor de TNF/genética , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/química , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/genéticaRESUMEN
The tumor necrosis factor (TNF) receptor associated factors (TRAFs) are the major signal transducers for the TNF receptor superfamily and the interleukin-1 receptor/Toll-like receptor (IL-1R/TLR) superfamily, which regulate a variety of cellular activities and innate immune responses. TRAF genes have been extensively studied in various species, including vertebrates and invertebrates. However, as one of the key component of NF-κB pathway, TRAF genes have not been systematically characterized in marine invertebrates. In this study, we identified and characterized five TRAF genes, PyTRAF2, PyTRAF3, PyTRAF4, PyTRAF6 and PyTRAF7, in the Yesso scallop (Patinopecten yessoensis). Phylogenetic and protein structural analyses were conducted to determine their identities and evolutionary relationships. In comparison with the TRAF genes from vertebrate species, the structural features were all relatively conserved in the PyTRAF genes. To gain insights into the roles of TRAF genes during scallop innate immune responses, quantitative real-time PCR was used to investigate the expression profiles in the different stages of scallop development, in the healthy adult tissues, and in the hemocytes after bacterial infection with Micrococcus luteus and Vibrio anguillarum. Based on the qRT-PCR analysis, the expression of most of the PyTRAFs was significantly induced in the acute phases (3-6 h) after infection with Gram-positive (M. luteus) and Gram-negative (V. anguillarum) bacteria, and many more dramatic changes in PyTRAFs expression were observed after V. anguillarum challenge. Notably, the strong response in the up-regulation of PyTRAF6 post-bacterial challenge was distinct from that previously reported in scallops and crabs but was similar to that of other shellfish, Echinodermata and even teleost fish. The high level expressions of PyTRAFs in the hemocytes and the gill, and their specific expression patterns after challenges provide insights into the versatile roles and responses of TRAFs in the innate immune system against Gram-negative bacterial pathogens in bivalves.
Asunto(s)
Regulación de la Expresión Génica , Micrococcus luteus/fisiología , Pectinidae/genética , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/genética , Vibrio/fisiología , Animales , Hemocitos/inmunología , Inmunidad Innata , Datos de Secuencia Molecular , Especificidad de Órganos , Pectinidae/inmunología , Pectinidae/metabolismo , Pectinidae/microbiología , Filogenia , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ADN , Análisis de Secuencia de Proteína , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/química , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/metabolismoRESUMEN
TTRAP (TRAF and TNF receptor-associated protein) is latest identified cytosolic protein that serves as a negative regulator for TNF signaling pathway. In this study, a member of TNF superfamily, TTRAP gene (designed as EcTTRAP) was cloned from grouper, Epinephelus coioides. There was an Exo_endo_phos type domain in EcTTRAP, and it was well conserved when compared with other TTRAPs, especially the endonuclease activity related motifs. EcTTRAP exhibited prominent endonuclease activity against the genome DNA from Escherichia coli, Vibrio vulnificus and E. coli JM109. Intracellular localization revealed that EcTTRAP expression distributed in both cytoplasm and nucleus. Real-time PCR analysis indicates that EcTTRAP is expressed in all selective grouper tissues, with the higher expression level in muscle, skin and gills. EcTTRAP was identified as a remarkably (P < 0.01) up-regulated protein responding to Singapore grouper iridovirus (SGIV) infection. Overexpression of EcTTRAP inhibited NF-κB activation, meanwhile the C terminal portion of the protein was found to be responsive domain for the inhibition. Stable transfection of FHM cells with EcTTRAP inhibited apoptosis induced by SGIV. Overexpression of EcTTRAP in grouper spleen (GS) cells inhibited the replication of SGIV. The present results provided new evidences for the potential roles of such molecule in E. coioides, and further confirmed the existence of TTRAP modulated TNF signaling pathway in grouper.
Asunto(s)
Lubina/genética , Lubina/inmunología , Infecciones por Virus ADN/veterinaria , Enfermedades de los Peces/inmunología , Proteínas de Peces/genética , Regulación de la Expresión Génica , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/genética , Secuencia de Aminoácidos , Animales , Fenómenos Fisiológicos Bacterianos , Lubina/metabolismo , Citocinas/genética , Citocinas/metabolismo , Infecciones por Virus ADN/inmunología , Infecciones por Virus ADN/virología , Enfermedades de los Peces/virología , Proteínas de Peces/química , Proteínas de Peces/metabolismo , Especificidad de Órganos , Filogenia , Ranavirus/fisiología , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Alineación de Secuencia/veterinaria , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/química , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/metabolismoRESUMEN
Immune signaling cascades have an indispensable role in the host defense of almost all the organisms. Tumor necrosis factor (TNF) signaling is considered as a prominent signaling pathway in vertebrate as well as invertebrate species. Within the signaling cascade, TNF receptor-associated factor (TRAF) and TNF receptor-associated protein (TTRAP) has been shown to have a crucial role in the modulation of immune signaling in animals. Here, we attempted to characterize a novel molluskan ortholog of TTRAP (AbTTRAP) from disk abalone (Haliotis discus discus) and analyzed its expression levels under pathogenic stress. The complete coding sequence of AbTTRAP consisted of 1071 nucleotides, coding for a 357 amino acid peptide, with a predicted molecular mass of 40 kDa. According to our in-silico analysis, AbTTRAP resembled the typical TTRAP domain architecture, including a 5'-tyrosyl DNA phosphodiesterase domain. Moreover, phylogenetic analysis revealed its common ancestral invertebrate origin, where AbTTRAP was clustered with molluskan counterparts. Quantitative real time PCR showed universally distributed expression of AbTTRAP in selected tissues of abalone, from which more prominent expression was detected in hemocytes. Upon stimulation with two pathogen-derived mitogens, lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid (poly I:C), transcript levels of AbTTRAP in hemocytes and gill tissues were differentially modulated with time. In addition, the recombinant protein of AbTTRAP exhibited prominent endonuclease activity against abalone genomic DNA, which was enhanced by the presence of Mg(2+) in the medium. Collectively, these results reinforce the existence of the TNF signaling cascade in mollusks like disk abalone, further implicating the putative regulatory behavior of TTRAP in invertebrate host pathology.
Asunto(s)
Gastrópodos/genética , Regulación de la Expresión Génica , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Endonucleasas/metabolismo , Gastrópodos/metabolismo , Lipopolisacáridos , Datos de Secuencia Molecular , Especificidad de Órganos , Filogenia , Poli I-C , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/química , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/metabolismoRESUMEN
Tumor necrosis factor receptor-associated factor 6 (TRAF6) is one of the key adapter molecules in Toll-like receptor signal transduction that triggers downstream cascades involved in innate immunity. In the present study, a TRAF6 (named as Et-TRAF6) was identified from the marine fish grouper, Epinephelus tauvina by RACE PCR. The full-length cDNA of Et-TRAF6 comprised 1949 bp with a 1713 bp open reading frame (ORF) that encodes a putative protein of 570 amino acids. Similar to most TRAF6s, Et-TRAF6 includes one N-terminal RING domain (78aa-116aa), two zinc fingers of TRAF-type (159aa-210aa and 212aa-269aa), one coiled-coil region (370aa-394aa), and one conserved C-terminal meprin and TRAF homology (MATH) domain (401aa-526aa). Quantitative real-time PCR analysis revealed that Et-TRAF6 mRNA is expressed in all tested tissues, with the predominant expression in the stomach and intestine. The expression of Et-TRAF6 was up-regulated in the liver after challenge with Lipoteichoic acid (LTA), Peptidoglycan (PGN), Zymosan, polyinosine-polycytidylic acid [Poly(I:C)] and Polydeoxyadenylic acid · Polythymidylic acid sodium salt [Poly(dA:dT)]. The expression of Et-TRAF6 was also up-regulated in the liver after infection with Vibrio alginolyticus, Singapore grouper iridovirus (SGIV) and grouper nervous necrosis virus (GNNV). Recombinant Et-TRAF6 (rEt-TRAF6) was expressed in Escherichia BL21 (DE3) and purified for mouse anti-Et-TRAF6 serum preparation. Intracellular localization revealed that Et-TRAF6 is distributed in both cytoplasm and nucleus, and predominantly in the cytoplasm. These results together indicated that Et-TRAF6 might be involved in immune responses toward bacterial and virus challenges.
Asunto(s)
Lubina , Enfermedades de los Peces/microbiología , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Regulación de la Expresión Génica , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/genética , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Lubina/clasificación , Lubina/genética , Lubina/inmunología , Enfermedades de los Peces/virología , Proteínas de Peces/química , Proteínas de Peces/metabolismo , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/química , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/metabolismoRESUMEN
Eukaryotic epithelial cells form a sheet of contiguous cells, called epithelium, by means of the establishment of well-developed junctional complexes. These junctional complexes ensure the cell cohesion in the tissue and separate the plasma membrane into an apical and a basolateral compartment. This apicobasal polarity, which is crucial for both the architecture and the function of epithelia, is mainly maintained by tight junctions (TJS). Indeed, TJS weakening or loss disrupts the integrity of the epithelium, a process participating to the formation and progression of carcinomas. It has recently been shown that TRAF4, a protein dynamically localized in TJS and commonly overexpressed in carcinomas, plays a variety of functions in tumor progression. Here, we review recent data implicating TRAF4 in carcinogenesis. First, the conserved TRAF proteins family will be presented, and then the molecular mechanism addressing TRAF4 to TJS which involves lipid binding by the TRAF domain will be described. The various roles of TRAF4 in carcinogenesis will be discussed. Finally, we will highlight the ability of all TRAF proteins to bind lipids and discuss its potential functional relevance.
Asunto(s)
Carcinoma/patología , Proteínas de Neoplasias/fisiología , Factor 4 Asociado a Receptor de TNF/fisiología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sitios de Unión , Carcinoma/metabolismo , Compartimento Celular , Movimiento Celular , Polaridad Celular/fisiología , Secuencia Conservada , Progresión de la Enfermedad , Células Epiteliales/patología , Transición Epitelial-Mesenquimal , Humanos , Lípidos de la Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Familia de Multigenes , Fosfatos de Fosfatidilinositol/metabolismo , Conformación Proteica , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de Señal/fisiología , Factor 4 Asociado a Receptor de TNF/genética , Uniones Estrechas/fisiología , Factor de Crecimiento Transformador beta/fisiología , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/química , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/genética , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/fisiologíaRESUMEN
TRAF3 interacting protein 2 (TRAF3IP2) is important for immune responses to pathogens, inflammatory signals and autoimmunity in mammals. In the present study, we collected 19 mammalian TRAF3IP2 sequences and investigated the various types of selection pressure acting on them. Maximum likelihood estimations of nonsynonymous (dN) to synonymous (dS) substitution (dN/dS) ratios for the aligned coding sequences indicated that, as a whole, TRAF3IP2 has been subject to purifying selection. However, the N-terminus of the protein has been subject to higher selection pressure than the C-terminal domain. While eight amino acid residues within the N-terminus appear to have evolved under positive selection, no evidence for such selection was found in the C-terminus. The positively selected residues, which fall outside the currently known functional sites within TRAF3IP2, may have novel functions. The different selection pressures acting on the N- and C-terminal regions are consistent with their protein structures: the C-terminal structure is an ordered structure, whereas the N-terminus is disordered. Taken together with the results of previous studies, it is plausible that positive selection on the N-terminus of TRAF3IP2 may have occurred by competitive coevolution between mammalian hosts and viruses.
Asunto(s)
Inmunidad/genética , Mamíferos/genética , Selección Genética , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/genética , Proteínas Adaptadoras Transductoras de Señales , Animales , Bovinos , Evolución Molecular , Cobayas , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , Mamíferos/inmunología , Ratones , Modelos Moleculares , Conejos , Ratas , Transducción de Señal/genética , Transducción de Señal/inmunología , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/químicaRESUMEN
Members of the tumor necrosis factor receptor superfamily play key roles in innate and adaptive immunity. Here, we review recent structural studies in the intracellular signal transduction of these receptors. A central theme revealed from these structural studies is that upon ligand binding, multiple intracellular proteins form higher-order signaling machines to transduce and amplify receptor activation information to different cellular fates, including NF-κB activation, apoptosis, and programmed necrosis. These studies open a new vista for understanding the biophysical principles in these signaling cascades.
Asunto(s)
FN-kappa B/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Transducción de Señal , Apoptosis , Activación Enzimática , Humanos , Quinasa I-kappa B/química , Quinasa I-kappa B/metabolismo , Necrosis , Estructura Terciaria de Proteína , Receptores del Factor de Necrosis Tumoral/química , Receptores del Factor de Necrosis Tumoral/genética , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/química , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Receptor fas/metabolismoRESUMEN
TRAF3 is a highly versatile regulator that negatively regulates JNK and alternative nuclear factor-κB signalling, but positively controls type I interferon production. To investigate TRAF3 function in innate immune responses among invertebrate especially mollusk, we characterized TRAF3 (PfTRAF3) from pearl oyster Pinctada fucata, one of the most important bivalve mollusks for seawater pearl production. PfTRAF3 cDNA is 2261 bp with an open reading frame of 1623 bp encoding a putative protein of 541 amino acids. The deduced PfTRAF3 contains a RING finger domain, two TRAF domains with zinc finger domains and a conserved C-terminal meprin and TRAF homology (MATH) domain. Comparison and phylogenetic analysis revealed that PfTRAF3 from mollusk shared a higher identity with Ciona intestinalis TRAF3 from urochordata, Branchiostoma belcheri TRAF3 from cephalochordate, and even TRAF3 from vertebrate than with insect homologues. Furthermore, gene expression analyses suggested that PfTRAF3 was involved in the immune response to Vibrio alginolyticus.
Asunto(s)
Pinctada/genética , Pinctada/metabolismo , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/genética , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , China , Clonación Molecular , ADN Complementario/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Inmunidad Innata , Datos de Secuencia Molecular , Especificidad de Órganos , Filogenia , Pinctada/inmunología , Pinctada/microbiología , ARN Mensajero/análisis , Reacción en Cadena en Tiempo Real de la Polimerasa , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/química , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/inmunología , Vibrio alginolyticus/fisiologíaRESUMEN
Transforming growth factor-ß (TGF-ß) principally relays its effects through the Smad pathway however, accumulating evidence indicate that alternative signaling routes are also employed by this pleiotropic cytokine. For instance recently, we have demonstrated that ligand occupied TGF-ß receptors can directly trigger the TRAF6-TAK1 signaling module, resulting in MAP kinase activation. Here we report identification of the adaptor molecule TTRAP as a novel component of this non-canonical TGF-ß pathway. We show that the protein associates with TGF-ß receptors and components of the TRAF6-TAK1 signaling module, resulting in differential regulation of TGF-ß activated p38 and NF-κB responses. Modulation of cellular TTRAP level affects cell viability in the presence of TGF-ß, suggesting that the protein is an important component of the TGF-ß induced apoptotic process.
Asunto(s)
Quinasas Quinasa Quinasa PAM/metabolismo , Transducción de Señal , Factor 6 Asociado a Receptor de TNF/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/metabolismo , Animales , Apoptosis/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Células HEK293 , Humanos , Quinasas Quinasa Quinasa PAM/química , Ratones , Estabilidad Proteica/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Factor 6 Asociado a Receptor de TNF/química , Factor de Crecimiento Transformador beta1/farmacología , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/química , Ubiquitinación/efectos de los fármacosRESUMEN
SEF/IL-17R/CIKS/ACT1 homology (SEFIR) domain containing proteins, which include the IL-17 receptors and an adaptor protein Act1, have essential roles in vertebrate immunity. However, the molecular mechanisms of Act1 function remain largely unexplored. In this article, we employed an evolutionary analysis to discover novel structural and functional properties of Act1. Firstly, we have found that the previously identified helix-loop-helix and Ufd2-box domains in human Act1 have relatively recent evolutionary origins in higher vertebrates. Zebrafish Act1, which lacks these domains, is unable to induce JNK phosphorylation and activate cytokine expression when expressed in human cells. Secondly, we have established that Act1-like proteins contain DEATH-domains in basal animals, such as Hydra and primitive chordates, but lack this domain in vertebrates. Finally, we have shown that Act1-TRAF6 interactions are conserved throughout vertebrate evolution: Act1 derived from zebrafish can bind to TRAF6 and activate NF-κB in human cells. Moreover, we have identified a novel highly conserved motif at the amino-terminus of Act1, which is critical for binding to TRAF6 and activating NF-κB-dependent gene expression. We propose a model of evolutionary changes in Act1-mediated signalling, which contributes to a better understanding of evolution of the vertebrate immunity.
Asunto(s)
Evolución Molecular , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/química , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/metabolismo , Vertebrados/inmunología , Secuencias de Aminoácidos/genética , Secuencia de Aminoácidos , Animales , Regulación de la Expresión Génica , Células HEK293 , Humanos , Modelos Biológicos , Datos de Secuencia Molecular , Unión Proteica/genética , Receptores de Interleucina-17/genética , Receptores de Interleucina-17/metabolismo , Alineación de Secuencia , Transducción de Señal , Especificidad de la Especie , Factor 6 Asociado a Receptor de TNF/metabolismo , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/genética , Vertebrados/genética , Vertebrados/metabolismoRESUMEN
NF-κBs are a family of transcription factors that control a number of essential cellular functions including immune responses, cell proliferation and antiapoptosis. NF-κB activities are tightly regulated through upstream signaling molecules and downstream feedback loops. In this review, structural discoveries in the NF-κB pathway are presented. With the structure information, the following questions may be addressed: (1) How do NF-κBs activate their target genes? (2) How do IκBs inhibit NF-κB activities in the steady state? (3) How do upstream signaling molecules activate the NF-κB pathway? and (4) How do the feedback loops shut down the NF-κB pathway to avoid constitutive NF-κB activation?
Asunto(s)
FN-kappa B/química , ADN/química , ADN/metabolismo , Humanos , Quinasa I-kappa B/química , Quinasa I-kappa B/metabolismo , FN-kappa B/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Transducción de Señal , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/química , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/metabolismoRESUMEN
Restriction of long-distance movement of several potyviruses in Arabidopsis (Arabidopsis thaliana) is controlled by at least three dominant restricted TEV movement (RTM) genes, named RTM1, RTM2, and RTM3. RTM1 encodes a protein belonging to the jacalin family, and RTM2 encodes a protein that has similarities to small heat shock proteins. In this article, we describe the positional cloning of RTM3, which encodes a protein belonging to an undescribed protein family of 29 members that has a meprin and TRAF homology (MATH) domain in its amino-terminal region and a coiled-coil domain at its carboxy-terminal end. Involvement in the RTM resistance system is the first biological function experimentally identified for a member of this new gene family in plants. Our analyses showed that the coiled-coil domain is not only highly conserved between RTM3-homologous MATH-containing proteins but also in proteins lacking a MATH domain. The cluster organization of the RTM3 homologs in the Arabidopsis genome suggests the role of duplication events in shaping the evolutionary history of this gene family, including the possibility of deletion or duplication of one or the other domain. Protein-protein interaction experiments revealed RTM3 self-interaction as well as an RTM1-RTM3 interaction. However, no interaction has been detected involving RTM2 or the potyviral coat protein previously shown to be the determinant necessary to overcome the RTM resistance. Taken together, these observations strongly suggest the RTM proteins might form a multiprotein complex in the resistance mechanism to block the long-distance movement of potyviruses.