RESUMO
Pyrazolo[3,4-d]pyrimidines represent an important class of heterocyclic compounds well-known for their anticancer activity exerted by the inhibition of eukaryotic protein kinases. Recently, pyrazolo[3,4-d]pyrimidines have become increasingly attractive for their potential antimicrobial properties. Here, we explored the activity of a library of in-house pyrazolo[3,4-d]pyrimidines, targeting human protein kinases, against Staphylococcus aureus and Escherichia coli and their interaction with ampicillin and kanamycin, representing important classes of clinically used antibiotics. Our results represent a first step towards the potential application of dual active pyrazolo[3,4-d]pyrimidine kinase inhibitors in the prevention and treatment of bacterial infections in cancer patients.
Assuntos
Antibacterianos/síntese química , Antibacterianos/farmacologia , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Pirazóis/síntese química , Pirazóis/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana , Filogenia , Domínios Proteicos , Inibidores de Proteínas Quinases/química , Proteínas Quinases/química , Pirazóis/química , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimentoRESUMO
Following recent success in genome-wide association studies, a critical focus of human genetics is to understand how genetic variation at implicated loci influences cellular and disease processes. Crohn's disease (CD) is associated with SNPs around IRGM, but coding-sequence variation has been excluded as a source of this association. We identified a common, 20-kb deletion polymorphism, immediately upstream of IRGM and in perfect linkage disequilibrium (r2 = 1.0) with the most strongly CD-associated SNP, that causes IRGM to segregate in the population with two distinct upstream sequences. The deletion (CD risk) and reference (CD protective) haplotypes of IRGM showed distinct expression patterns. Manipulation of IRGM expression levels modulated cellular autophagy of internalized bacteria, a process implicated in CD. These results suggest that the CD association at IRGM arises from an alteration in IRGM regulation that affects the efficacy of autophagy and identify a common deletion polymorphism as a likely causal variant.
Assuntos
Doença de Crohn/genética , Proteínas de Ligação ao GTP/genética , Polimorfismo de Nucleotídeo Único , Autofagia/genética , Linhagem Celular , Regulação da Expressão Gênica , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Deleção de SequênciaRESUMO
We present a genome-wide association study of ileal Crohn disease and two independent replication studies that identify several new regions of association to Crohn disease. Specifically, in addition to the previously established CARD15 and IL23R associations, we identified strong and significantly replicated associations (combined P < 10(-10)) with an intergenic region on 10q21.1 and a coding variant in ATG16L1, the latter of which was also recently reported by another group. We also report strong associations with independent replication to variation in the genomic regions encoding PHOX2B, NCF4 and a predicted gene on 16q24.1 (FAM92B). Finally, we demonstrate that ATG16L1 is expressed in intestinal epithelial cell lines and that functional knockdown of this gene abrogates autophagy of Salmonella typhimurium. Together, these findings suggest that autophagy and host cell responses to intracellular microbes are involved in the pathogenesis of Crohn disease.
Assuntos
Autofagia/fisiologia , Proteínas de Transporte/genética , Cromossomos Humanos Par 10/genética , Doença de Crohn/genética , Doença de Crohn/fisiopatologia , Predisposição Genética para Doença/genética , Animais , Proteínas Relacionadas à Autofagia , Proteínas de Transporte/metabolismo , Perfilação da Expressão Gênica , Células HeLa , Proteínas de Homeodomínio/genética , Humanos , Imuno-Histoquímica , Camundongos , NADPH Oxidases/genética , América do Norte , Polimorfismo de Nucleotídeo Único , Interferência de RNA , Fatores de Transcrição/genéticaRESUMO
In innate immune sensing, the detection of pathogen-associated molecular patterns by recognition receptors typically involve leucine-rich repeats (LRRs). We provide a categorization of 375 human LRR-containing proteins, almost half of which lack other identifiable functional domains. We clustered human LRR proteins by first assigning LRRs to LRR classes and then grouping the proteins based on these class assignments, revealing several of the resulting protein groups containing a large number of proteins with certain non-LRR functional domains. In particular, a statistically significant number of LRR proteins in the typical (T) and bacterial + typical (S+T) categories have transmembrane domains, whereas most of the LRR proteins in the cysteine-containing (CC) category contain an F-box domain (which mediates interactions with the E3 ubiquitin ligase complex). Furthermore, by examining the evolutionary profiles of the LRR proteins, we identified a subset of LRR proteins exhibiting strong conservation in fungi and an enrichment for "nucleic acid-binding" function. Expression analysis of LRR genes identifies a subset of pathogen-responsive genes in human primary macrophages infected with pathogenic bacteria. Using functional RNAi, we show that MFHAS1 regulates Toll-like receptor (TLR)-dependent signaling. By using protein interaction network analysis followed by functional RNAi, we identified LRSAM1 as a component of the antibacterial autophagic response.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Evolução Molecular , Imunidade Inata/genética , Proteínas Oncogênicas/metabolismo , Proteínas/genética , Proteínas/imunologia , Transdução de Sinais/genética , Análise por Conglomerados , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Imunidade Inata/imunologia , Proteínas de Repetições Ricas em Leucina , Macrófagos/metabolismo , Macrófagos/microbiologia , Proteínas/classificação , Interferência de RNA , Receptores Toll-Like/metabolismoRESUMO
Microrheology, the study of fluids on micron length-scales, promises to reveal insights into cellular biology, including mechanical biomarkers of disease and the interplay between biomechanics and cellular function. Here a minimally-invasive passive microrheology technique is applied to individual living cells by chemically binding a bead to the surface of a cell, and observing the mean squared displacement of the bead at timescales ranging from milliseconds to 100s of seconds. Measurements are repeated over the course of hours, and presented alongside analysis to quantify changes in the cells' low-frequency elastic modulus, G0', and the cell's dynamics over the time window â¼10-2 s to 10 s. An analogy to optical trapping allows verification of the invariant viscosity of HeLa S3 cells under control conditions and after cytoskeletal disruption. Stiffening of the cell is observed during cytoskeletal rearrangement in the control case, and cell softening when the actin cytoskeleton is disrupted by Latrunculin B. These data correlate with conventional understanding that integrin binding and recruitment triggers cytoskeletal rearrangement. This is, to our knowledge, the first time that cell stiffening has been measured during focal adhesion maturation, and the longest time over which such stiffening has been quantified by any means. STATEMENT OF SIGNIFICANCE: Here, we present an approach for studying mechanical properties of live cells without applying external forces or inserting tracers. Regulation of cellular biomechanics is crucial to healthy cell function. For the first time in literature, we can non-invasively and passively quantify cell mechanics during interactions with functionalised surface. Our method can monitor the maturation of adhesion sites on the surface of individual live cells without disrupting the cell mechanics by applying forces to the cell. We observe a stiffening response in cells over tens of minutes after a bead chemically binds. This stiffening reduces the deformation rate of the cytoskeleton, although the internal force generation increases. Our method has potential for applications to study mechanics during cell-surface and cell-vesicle interactions.
Assuntos
Citoesqueleto , Pinças Ópticas , Citoesqueleto/metabolismo , Membrana Celular/metabolismo , Módulo de Elasticidade , Citoesqueleto de ActinaRESUMO
Control over intracellular release of therapeutic compounds incorporated into nano-carriers will open new possibilities for targeted treatments of various diseases including cancer, and viral and bacterial infections. Here we report our study on mechanoresponsive nano-sized liposomes which, following internalization by cells, achieve intracellular delivery of encapsulated cargo on application of external ultrasound stimulus. This is demonstrated in a bespoke cell reporter system designed to assess free drug in cytoplasm. Biophysical analyses show that drug release is attributable to the action of a mechanoresponsive spiropyran-based compound embedded in the liposomal lipid membrane. Exposure to external ultrasound stimulus results in opening of the molecular structure of the embedded spiropyran, a consequent increase in liposomal lipid membrane fluidity, and size-dependent release of encapsulated model drugs, all pointing to lipid bilayer perturbation. The study hence illustrates feasibility of the proposed concept where intracellular drug release from mechanoresponsive liposomes can be triggered on demand by external ultrasound stimulus.
RESUMO
Ileal lesions in Crohn's disease (CD) patients are colonized by pathogenic adherent-invasive Escherichia coli (AIEC) able to invade and to replicate within intestinal epithelial cells. Recent genome-wide association studies have highlighted the autophagy pathway as being associated with CD risk. In the present study we investigated whether defects in autophagy enhance replication of commensal and pathogenic Escherichia coli and CD-associated AIEC. We show that functional autophagy limits intracellular AIEC replication and that a subpopulation of the intracellular bacteria is located within LC3-positive autophagosomes. In IRGM and ATG16L1 deficient cells intracellular AIEC LF82 bacteria have enhanced replication. Surprisingly autophagy deficiency did not interfere with the ability of intracellular bacteria to survive and/or replicate for any other E. coli strains tested, including non-pathogenic, environmental, commensal, or pathogenic strains involved in gastro enteritis. Together these findings demonstrate a central role for autophagy restraining Adherent-Invasive E. coli strains associated with ileal CD. AIEC infection in patients with polymorphisms in autophagy genes may have a significant impact on the outcome of intestinal inflammation.
Assuntos
Autofagia/fisiologia , Doença de Crohn/microbiologia , Escherichia coli/fisiologia , Animais , Autofagia/genética , Proteínas Relacionadas à Autofagia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Doença de Crohn/genética , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Células HeLa , Humanos , Immunoblotting , Mucosa Intestinal/microbiologia , Camundongos , Microscopia Confocal , Microscopia de Fluorescência , RNA Interferente PequenoRESUMO
Autophagy is a conserved cellular process required for the removal of defective organelles, protein aggregates, and intracellular pathogens. We used a network analysis strategy to identify novel human autophagy components based upon the yeast interactome centered on the core yeast autophagy proteins. This revealed the potential involvement of 14 novel mammalian genes in autophagy, several of which have known or predicted roles in membrane organization or dynamics. We selected one of these membrane interactors, FNBP1L (formin binding protein 1-like), an F-BAR-containing protein (also termed Toca-1), for further study based upon a predicted interaction with ATG3. We confirmed the FNBP1L/ATG3 interaction biochemically and mapped the FNBP1L domains responsible. Using a functional RNA interference approach, we determined that FNBP1L is essential for autophagy of the intracellular pathogen Salmonella enterica serovar Typhimurium and show that the autophagy process serves to restrict the growth of intracellular bacteria. However, FNBP1L appears dispensable for other forms of autophagy induced by serum starvation or rapamycin. We present a model where FNBP1L is essential for autophagy of intracellular pathogens and identify FNBP1L as a differentially used molecule in specific autophagic contexts. By using network biology to derive functional biological information, we demonstrate the utility of integrated genomics to novel molecule discovery in autophagy.
Assuntos
Autofagia/imunologia , Proteínas de Transporte/imunologia , Proteínas de Transporte/metabolismo , Saccharomyces cerevisiae/imunologia , Saccharomyces cerevisiae/metabolismo , Proteínas Relacionadas à Autofagia , Proteínas de Transporte/genética , Linhagem Celular , Biologia Computacional , Deleção de Genes , Regulação da Expressão Gênica , Humanos , Espaço Intracelular/imunologia , Ligação Proteica , RNA Interferente Pequeno/genética , Saccharomyces cerevisiae/genética , Salmonella typhimurium/imunologia , Especificidade por Substrato , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
PURPOSE OF REVIEW: The field of autophagy is rapidly expanding to encompass many important areas of cell biology, physiology and disease. Recent discoveries and tools allow the connection of the autophagy pathway to other cellular signals and processes, thus beginning a systematic approach to elucidation of autophagy components, functions and connections. RECENT FINDINGS: We outline recent discoveries illustrating the role of autophagy in Parkinson's disease, inflammatory bowel disease (IBD) and cancer. Recently important details of the mechanisms by which autophagy operates in these contexts have been elucidated. We illustrate how autophagy can be triggered by diverse stimuli and how cell fate is determined by the responses to many signals and stresses. We discuss the known links between autophagy and apoptosis and present a working model of the current interactions between autophagy components, apoptosis and cell cycle control at different stages of autophagic vesicle progression. SUMMARY: Autophagy represents not only an essential metabolic process, but a hub which responds to diverse stresses and signals to aid cell survival or control cell fate. There are currently many known links between autophagy and disease states, and the pace of discovery appears to be accelerating. Thus an understanding of autophagy is likely to be crucial to current and future approaches to therapy. Here we give a systems biology view of the autophagy field and how it is being connected to other pathways, such as apoptosis and responses to reactive oxygen damage.
Assuntos
Autofagia/fisiologia , Doenças Inflamatórias Intestinais/fisiopatologia , Hepatopatias/fisiopatologia , Neoplasias/fisiopatologia , Doença de Parkinson/fisiopatologia , Biologia de Sistemas , Animais , Autofagia/genética , Retículo Endoplasmático/fisiologia , Humanos , Espécies Reativas de Oxigênio , Transdução de SinaisRESUMO
Discs large homolog 1 (DLGH1), a founding member of the membrane-associated guanylate kinase family of proteins containing PostSynaptic Density-95/Discs large/Zona Occludens-1 domains, is an ortholog of the Drosophila tumor suppressor gene Discs large. In the mammalian embryo, DLGH1 is essential for normal urogenital morphogenesis and the development of skeletal and epithelial structures. Recent reports also indicate that DLGH1 may be a critical mediator of signals triggered by the antigen receptor complex in T lymphocytes by functioning as a scaffold coordinating the activities of T-cell receptor (TCR) signaling proteins at the immune synapse. However, it remains unclear if DLGH1 functions to enhance or attenuate signals emanating from the TCR. Here, we used Dlgh1 gene-targeted mice to determine the requirement for DLGH1 in T-cell development and activation. Strikingly, while all major subsets of T cells appear to undergo normal thymic development in the absence of DLGH1, peripheral lymph node Dlgh1(-/-) T cells show a hyper-proliferative response to TCR-induced stimulation. These data indicate that, consistent with the known function of Discs large proteins as tumor suppressors and attenuators of cell division, in T lymphocytes, DLGH1 functions as a negative regulator of TCR-induced proliferative responses.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Membrana/metabolismo , Linfócitos T/citologia , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Animais , Polaridade Celular , Proliferação de Células , Citocinas/biossíntese , Citoesqueleto/metabolismo , Proteínas de Ligação a DNA/deficiência , Proteína 1 Homóloga a Discs-Large , Feto/citologia , Regulação da Expressão Gênica , Guanilato Quinases , Fígado/citologia , Proteínas de Membrana/deficiência , Camundongos , Camundongos Knockout , Transporte Proteico , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Fase S , Transdução de SinaisRESUMO
The common gastrointestinal pathogens enteropathogenic Escherichia coli (EPEC) and Salmonella typhimurium both reorganize the gut epithelial cell actin cytoskeleton to mediate pathogenesis, utilizing mimicry of the host signaling apparatus. The PDZ domain-containing protein Shank3, is a large cytoskeletal scaffold protein with known functions in neuronal morphology and synaptic signaling, and is also capable of acting as a scaffolding adaptor during Ret tyrosine kinase signaling in epithelial cells. Using immunofluorescent and functional RNA-interference approaches we show that Shank3 is present in both EPEC- and S. typhimurium-induced actin rearrangements and is required for optimal EPEC pedestal formation. We propose that Shank3 is one of a number of host synaptic proteins likely to play key roles in bacteria-host interactions.
Assuntos
Actinas/metabolismo , Proteínas de Transporte/fisiologia , Proteínas do Citoesqueleto/fisiologia , Citoesqueleto/fisiologia , Escherichia coli Enteropatogênica/fisiologia , Salmonella typhimurium/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Animais , Aderência Bacteriana , Proteínas de Transporte/genética , Proteínas do Citoesqueleto/genética , Fibroblastos/metabolismo , Fibroblastos/microbiologia , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/fisiologia , Transdução de SinaisRESUMO
PDZ domain containing molecular scaffolds plays a central role in organizing synaptic junctions. Observations in Drosophila and mammalian cells have implicated that ubiquitination and endosomal trafficking, of molecular scaffolds are critical to the development and maintenance of cell-cell junctions and cell polarity. To elucidate if there is a connection between these pathways, we applied an integrative genomic strategy, which combined comparative genomics and proteomics with cell biological assays. Given the importance of ubiquitin in regulating endocytic processes, we first identified the subset of E3 ligases with conserved PDZ binding motifs. Among this subset, the MARCH family ubiquitin ligases account for the largest family and MARCH2 has been previously implicated in endosomal trafficking. Next, we tested in an unbiased fashion, if MARCH2 binds PDZ proteins in vivo using a modified tandem affinity purification strategy followed by mass spectrometry. Of note, DLG1 was co-purified from MARCH2, with subsequent confirmation that MARCH2 interacts with full-length DLG1 in a PDZ domain dependent manner. Furthermore, we demonstrated that MARCH2 co-localized with DLG1 at sites of cell-cell contact. In addition, loss of the MARCH2 PDZ binding motif led to loss of MARCH2 localization at cell-cell contact sites and MARCH2 appeared to localize away from cell-cell junctions. In in vivo ubiquitination assays we show that MARCH2 promotes DLG1 ubiquitination. Overall, these results suggest that PDZ ligands with E3 ligase activity may link PDZ domain containing tumor suppressors to endocytic pathways and cell polarity determination.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteínas de Transporte/fisiologia , Comunicação Celular/fisiologia , Proteínas de Membrana/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Polaridade Celular/fisiologia , Proteína 1 Homóloga a Discs-Large , Endocitose/fisiologia , Células Epiteliais/fisiologia , Células HeLa , Humanos , Domínios PDZ/fisiologia , Ligação Proteica/fisiologiaRESUMO
We present a high-content screen (HCS) for the simultaneous analysis of multiple phenotypes in HeLa cells expressing an autophagy reporter (mcherry-LC3) and one of 224 GFP-fused proteins from the Crohn's Disease (CD)-associated bacterium, Adherent Invasive E. coli (AIEC) strain LF82. Using automated confocal microscopy and image analysis (CellProfiler), we localised GFP fusions within cells, and monitored their effects upon autophagy (an important innate cellular defence mechanism), cellular and nuclear morphology, and the actin cytoskeleton. This data will provide an atlas for the localisation of 224 AIEC proteins within human cells, as well as a dataset to analyse their effects upon many aspects of host cell morphology. We also describe an open-source, automated, image-analysis workflow to identify bacterial effectors and their roles via the perturbations induced in reporter cell lines when candidate effectors are exogenously expressed.
Assuntos
Proteínas Relacionadas à Autofagia , Autofagia , Proteínas de Escherichia coli , Doença de Crohn/microbiologia , Escherichia coli , Proteínas de Fluorescência Verde , Células HeLa , HumanosAssuntos
Colite Ulcerativa/genética , Doença de Crohn/genética , Marcadores Genéticos , Testes Genéticos , Síndrome do Intestino Irritável/genética , Colite Ulcerativa/diagnóstico , Colite Ulcerativa/patologia , Colo/química , Colo/patologia , Doença de Crohn/diagnóstico , Doença de Crohn/patologia , DNA Complementar/análise , Diagnóstico Diferencial , Perfilação da Expressão Gênica/métodos , Predisposição Genética para Doença , Genótipo , Humanos , Mucosa Intestinal/química , Mucosa Intestinal/patologia , Síndrome do Intestino Irritável/diagnóstico , Síndrome do Intestino Irritável/patologia , Fenótipo , Valor Preditivo dos Testes , RNA/análise , Reprodutibilidade dos Testes , Fatores de RiscoRESUMO
The mucosal immune system has evolved to protect the host against the establishment of infections at or through the mucosal surfaces of the body. Protective immunity must be activated to specific pathogenic agents or their products but inappropriate immune responses to food/environmental antigens must be avoided. Thus, the mucosal immune system is under tight regulation. Pathogenic bacteria and their products can be exploited as specific probes of mucosal immune responses. Bacterial enterotoxins such as cholera toxin are potent mucosal immunogens and adjuvants that activate both mucosal and systemic immune responses. Infection models involving microorganisms such as Citrobacter rodentium can also be used to investigate the consequences of mucosal colonisation that lead to immune disfunction.
Assuntos
Citrobacter freundii/patogenicidade , Infecções por Enterobacteriaceae/imunologia , Imunidade nas Mucosas , Mucosa/microbiologia , Animais , Modelos Animais de Doenças , Humanos , CamundongosAssuntos
Autofagia , Doença de Crohn/patologia , Trato Gastrointestinal/citologia , Homeostase/fisiologia , Proteínas Relacionadas à Autofagia , Proteínas de Transporte/fisiologia , Doença de Crohn/genética , Proteínas de Ligação ao GTP/fisiologia , Trato Gastrointestinal/imunologia , Trato Gastrointestinal/fisiologia , Predisposição Genética para Doença , HumanosRESUMO
Several species of pathogenic bacteria replicate within an intracellular vacuolar niche. Bacteria that escape into the cytosol are captured by the autophagic pathway and targeted for lysosomal degradation, representing a defense against bacterial exploitation of the host cytosol. Autophagic capture of Salmonella Typhimurium occurs predominantly via generation of a polyubiquitin signal around cytosolic bacteria, binding of adaptor proteins, and recruitment of autophagic machinery. However, the components mediating bacterial target selection and ubiquitination remain obscure. We identify LRSAM1 as the E3 ligase responsible for anti-Salmonella autophagy-associated ubiquitination. LRSAM1 localizes to several intracellular bacterial pathogens and generates the bacteria-associated ubiquitin signal; these functions require LRSAM1's leucine-rich repeat and RING domains, respectively. Using cells from LRSAM1-deficient individuals, we confirm that LRSAM1 is required for ubiquitination associated with intracellular bacteria but dispensable for ubiquitination of aggregated proteins. LRSAM1 is therefore a bacterial recognition protein and ubiquitin ligase that defends the cytoplasm from invasive pathogens.
Assuntos
Autofagia , Salmonella typhimurium/imunologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Técnicas de Inativação de Genes , Células HeLa , Humanos , Estrutura Terciária de Proteína , Ubiquitina-Proteína Ligases/genéticaRESUMO
BACKGROUND: Toll-like receptors (TLRs) are expressed by a variety of cells, including intestinal epithelia. However, the full spectrum of regulators modulating innate responses via TLRs has not been delineated. Tribbles (Trib) have been identified as a highly conserved family of kinase-like proteins. We sought to clarify the role of Trib2 in the TLR signaling pathway. METHODS: Trib2 mRNA and protein levels were analyzed by quantitative polymerase chain reaction (PCR) and western blot, respectively. Immunohistochemical staining was used to determine the expression of Trib2 in human tissue. Involvement of Trib2 in nuclear factor kappa B (NF-κB) pathways was assessed in epithelial cells by NF-κB reporter assay. Proteins that interacted with Trib2 were identified by mass spectrometry and confirmed by immunoprecipitation. The domain essential for Trib2 function was mapped using truncated constructs. RESULTS: Trib2 expression is decreased in active inflamed tissue from patients with inflammatory bowel disease (IBD). Trib2 is expressed in human and mouse colonic epithelium as well as immune cells, and its expression in epithelium is inducible in a ligand-dependent manner by TLR5 ligand stimulation. Trib2 inhibits TLR5-mediated activation of NF-κB downstream of TRAF6. Trib2 selectively modulates mitogen-activated protein kinase (MAPK) pathways p38 and Jun N-terminal kinase (JNK) but not p44/p42 (ERK1/2). NF-κB2 (p100) was identified as a Trib2 binding partner in regulating the TLR5 signaling pathway that leads to inhibition of NF-κB activity. Residues 158-177 in the Trib2 kinase-like domain are required for Trib2 function. CONCLUSIONS: These observations indicate that Trib2 is a novel regulator in the TLR5 signaling pathway and altered expression of Trib2 may play a role in IBD.