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Gabriel Nuñez

Publications and source records attributed to Gabriel Nuñez.

At least 19 recordsLinked to original sources
PubMed2006

Crystal structure of the Nod1 caspase activation and recruitment domain.

Nod-like receptors (NLRs), Nod1 and Nod2 are cytosolic detectors of pathogen-associated molecular patterns (PAMPs). Nod1 is a three-domain protein, consisting of a caspase activation and recruitment domain (CARD), a nucleotide-binding oligomerization domain (NOD), and a leucine-rich repeat domain (LRR). The binding of PAMPs to the LRR results in the activation of signaling through homophilic CARD-CARD interactions. Several CARD structures have been determined, including a recent NMR structure of Nod1 CARD. In contrast to the reported NMR structure, the crystal structure reported here is a dimer, where the sixth helix is swapped between two monomers. While the overall structure is very similar to the known CARD structures, this is the first report of a homodimeric CARD structure. The ability of the CARD to exist in monomeric and dimeric forms suggests another level of regulation in the activation of NLR proteins.

Binding Sites↗
PubMed2006

CARMA3/Bcl10/MALT1-dependent NF-kappaB activation mediates angiotensin II-responsive inflammatory signaling in nonimmune cells.

Angiotensin II (Ang II) is a peptide hormone that, like many cytokines, acts as a proinflammatory agent and growth factor. After injury to the liver, the hormone assists in tissue repair by stimulating hepatocytes and hepatic stellate cells to synthesize extracellular matrix proteins and secrete secondary cytokines and by stimulating myofibroblasts to proliferate. However, under conditions of chronic liver injury, all of these effects conspire to promote pathologic liver fibrosis. Much of this effect of Ang II results from activation of the proinflammatory NF-kappaB transcription factor in response to stimulation of the type 1 Ang II receptor, a G protein-coupled receptor. Here, we characterize a previously undescribed signaling pathway mediating Ang II-dependent activation of NF-kappaB, which is composed of three principal proteins, CARMA3, Bcl10, and MALT1. Blocking the function of any of these proteins, through the use of either dominant-negative mutants, RNAi, or gene targeting, effectively abolishes Ang II-dependent NF-kappaB activation in hepatocytes. In addition, Bcl10(-/-) mice show defective hepatic cytokine production after Ang II treatment. Evidence also is presented that this pathway activates NF-kappaB through ubiquitination of IKKgamma, the regulatory subunit of the IkappaB kinase complex. These results elucidate a concrete series of molecular events that link ligand activation of the type 1 Ang II receptor to stimulation of the NF-kappaB transcription factor. These findings also uncover a function of the CARMA, Bcl10, and MALT1 proteins in cells outside the immune system.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Differential release and distribution of Nod1 and Nod2 immunostimulatory molecules among bacterial species and environments.

Nod1 and Nod2 are intracellular proteins that are involved in recognition of bacterial molecules and their genetic variations have been linked to several inflammatory diseases that are strongly affected by environmental factors. However, the distribution of Nod1- and Nod2-stimulatory molecules in different bacterial species and environments is unknown. Here we established a quantitative bioassay to screen and characterize Nod1- and Nod2-stimulatory activities in different environmental sites and bacterial species. Using this system, we found that common environments including foods and soils contain high levels of Nod1- and Nod2-stimulatory activities. Several Bacillus species were identified to possess the highest Nod1-stimulatory activity among soil bacteria. Unlike other immunostimulatory molecules, the higher level of Nod1-stimulatory activity was found in the culture supernatant and not in extracts from whole cell bacteria. Nod1-stimulatory molecules were highly stable at extreme pH and boiling conditions and were synthesized in an amidase- and sltY-independent manner. These results suggest a novel mechanism by which bacteria present in the environment stimulate the host immune system through Nod1.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Nucleotide-binding oligomerization domain-1 and epidermal growth factor receptor: critical regulators of beta-defensins during Helicobacter pylori infection.

Host-pathogen interactions that allow Helicobacter pylori to survive and persist in the stomach of susceptible individuals remain unclear. Human beta-defensins (hBDs), epithelial-derived antimicrobial peptides are critical components of host-defense at mucosal surfaces. The role of H. pylori-mediated NF-kappaB and epidermal growth factor receptor (EGFR) activation on beta-defensin expression was investigated. Transient transfection studies utilizing beta-defensin promoter constructs were conducted in gastric cells with contribution of individual signaling events evaluated by the addition of specific inhibitors, small interference nucleotide-binding oligomerization domain 1 (NOD1) RNA or plasmids encoding Vaccinia virus proteins that interrupt interleukin-1 and Toll-like receptor signaling. The role of individual MAPK pathways was further delineated in HEK-293 cells expressing conditional MAPK mutants. We found hBD2 expression exclusively dependent on the presence of the bacterial cag pathogenicity island, with NOD1 a critical host sensor. Impairment of murinebeta-defensin 4 (an orthologue of hBD2) expression in NOD1-deficient mice 7-days post-infection further confirmed the role of this cytoplasmic pattern-recognition receptor in eliciting host innate immunity. In contrast to hBD2, hBD3 expression was NOD1-independent but EGFR and ERK pathway-dependent. Importantly, Toll-like receptor signaling was not implicated in H. pylori-mediated hBD2 and hBD3 gene expression. The divergent signaling events governing hBD2 and hBD3 expression suggest temporal functional variation, such that hBD2 may contribute to antimicrobial barrier function during the inflammatory phase with hBD3 playing a greater role during the repair, wound healing phase of infection.

Animals↗
PubMed2005

A role for Erbin in the regulation of Nod2-dependent NF-kappaB signaling.

Nod2 is an intracellular sensor of a specific bacterial cell wall component, muramyl dipeptide, and activation of Nod2 stimulates an inflammatory response. Specific mutations of Nod2 have been associated with two inflammatory diseases, Crohn disease and Blau syndrome, and are thought to contribute to disease susceptibility through altering Nod2 signaling. Association of disease with inappropriate activation of Nod2 highlights the importance of proper regulation of Nod2 activity. However, little is known about specific regulation of the Nod2 pathway. We performed a biochemical screen to discover potential regulators of Nod2 and identified Erbin, a protein involved in cell polarity, receptor localization, and regulation of the mitogen-activated protein kinase pathway, as a novel Nod2-interacting protein. In our studies, we demonstrate specific interaction of Erbin and Nod2 both in vitro and in vivo and characterize the regions required for interaction in both proteins. We found that Nod2-dependent activation of NF-kappaB and cytokine secretion is inhibited by Erbin overexpression, whereas Erbin-/- mouse embryo fibroblasts show an increased sensitivity to muramyl dipeptide. These studies identify Erbin as a regulator of Nod2 signaling and demonstrate a novel role for Erbin in inflammatory responses.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed2005

A novel caspase-1/toll-like receptor 4-independent pathway of cell death induced by cytosolic Shigella in infected macrophages.

Shigella-induced macrophage cell death is an important step in the induction of acute inflammatory responses that ultimately lead to bacillary dysentery. Cell death was previously reported to be dependent upon the activation of caspase-1 via interaction with IpaB secreted by intracellular Shigella, but in this study, we show that Shigella infection of macrophages can also induce cell death independent of caspase-1 or IpaB activity. Time-lapse imaging and electron microscopic analyses indicated that caspase-1-dependent and -independent cell death is morphologically indistinguishable and that both resemble necrosis. Analyses of Shigella mutants or Escherichia coli using co-infection with Listeria suggested that a component common to Gram-negative bacteria is involved in inducing caspase-1-independent cell death. Further studies revealed that translocation of bacterial lipid A into the cytosol of macrophages potentially mediates cell death. Notably, cell death induced by cytosolic bacteria was TLR4-independent. These results identify a novel cell death pathway induced by intracellular Gram-negative bacteria that may play a role in microbial-host interactions and inflammatory responses.

Animals↗
PubMed2005

Nod2-dependent regulation of innate and adaptive immunity in the intestinal tract.

The gene encoding the Nod2 protein is frequently mutated in Crohn's disease (CD) patients, although the physiological function of Nod2 in the intestine remains elusive. Here we show that protective immunity mediated by Nod2 recognition of bacterial muramyl dipeptide is abolished in Nod2-deficient mice. These animals are susceptible to bacterial infection via the oral route but not through intravenous or peritoneal delivery. Nod2 is required for the expression of a subgroup of intestinal anti-microbial peptides, known as cryptdins. The Nod2 protein is thus a critical regulator of bacterial immunity within the intestine, providing a possible mechanism for Nod2 mutations in CD.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed2005

NOD-LRR proteins: role in host-microbial interactions and inflammatory disease.

Nods are cytosolic proteins that contain a nucleotide-binding oligomerization domain (NOD). These proteins include key regulators of apoptosis and pathogen resistance in mammals and plants. A large number of Nods contain leucine-rich repeats (LRRs), hence referred to as NOD-LRR proteins. Genetic variation in several NOD-LRR proteins, including human Nod2, Cryopyrin, and CIITA, as well as mouse Naip5, is associated with inflammatory disease or increased susceptibility to microbial infections. Nod1, Nod2, Cryopyrin, and Ipaf have been implicated in protective immune responses against pathogens. Together with Toll-like receptors, Nod1 and Nod2 appear to play important roles in innate and acquired immunity as sensors of bacterial components. Specifically, Nod1 and Nod2 participate in the signaling events triggered by host recognition of specific motifs in bacterial peptidoglycan and, upon activation, induce the production of proinflammatory mediators. Naip5 is involved in host resistance to Legionella pneumophila through cell autonomous mechanisms, whereas CIITA plays a critical role in antigen presentation and development of antigen-specific T lymphocytes. Thus, NOD-LRR proteins appear to be involved in a diverse array of processes required for host immune reactions against pathogens.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Critical role for DP5/Harakiri, a Bcl-2 homology domain 3-only Bcl-2 family member, in axotomy-induced neuronal cell death.

The survival of neurons is maintained primarily by neurotrophic factors that suppress the apoptotic program. Axotomy or removal of peripheral targets causes neuronal cell death, but the mechanisms involved in the induction of this type of cell death remain poorly understood. Here, we show that DP5/Harakiri, a Bcl-2 homology domain 3-only member of the Bcl-2 family, is induced in motoneurons after transection of the hypoglossal nerve in mice and in sympathetic neurons after nerve growth factor (NGF) withdrawal. To assess the role of DP5 in neuronal cell death, mutant mice deficient in DP5 were generated by gene targeting. DP5-/- mice were viable and exhibited normal postnatal development. Notably, motoneurons from DP5-/- mice were highly protected from cell death induced by resection of the hypoglossal nerve compared with motoneurons from DP5+/+ littermate mice. In addition, deficiency of DP5 in superior cervical ganglia (SCG) neurons resulted in delayed neuronal cell death triggered by NGF withdrawal. Analysis of SCG neurons from DP5-/- mice revealed increased preservation of mitochondrial membrane potential and reduced activation of caspase-3 compared with neurons from wild-type mice. These results indicate that DP5 plays an important role in neuronal cell death induced by axotomy and NGF deprivation through the regulation of mitochondrial function and caspase-3 activation.

Animals↗
PubMed2004

Battling enteroinvasive bacteria: Nod1 comes to the rescue.

Recognition of pathogenic bacteria by mammalian hosts is largely mediated by membrane-bound Toll-like receptors (TLRs). Recently, a family of cytosolic proteins, termed NODs, with homology to plant disease-resistance gene products has been implicated in sensing microbes within the cytosol. The role of NOD family members in host defense is largely unknown. However, a recent report revealed that Nod1 is a crucial sensor for certain enteroinvasive bacteria that avoid TLR signaling. This finding suggests that Nod1 plays an important role in the initial recognition of pathogenic bacteria at epithelial surfaces, such as the gut, where innate immune responses to commensal bacteria must be avoided.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Apoptosome formation and caspase activation: is it different in the heart?

Apoptosis is a form of cell death which utilizes energy resources to dismantle and remove cells in an orderly or programmed fashion. It plays an essential role in establishing normal embryonic development, maintaining adult tissue homeostasis and contributes to a variety of human diseases including certain pathological processes in the heart. Apoptosis is mediated by a distinct biochemical pathway that is conserved in multicellular organisms. Signaling for apoptosis is initiated from outside the cell (extrinsic or death receptor pathway) or from inside the cell (intrinsic or mitochondrial pathway). In both pathways, signaling results in the activation of a family of cysteine proteases, named caspases, that act in a proteolytic cascade to dismantle and remove the dying cell. The activation of the intrinsic death pathway involves the release of cytochrome c from the mitochondria and formation of the apoptosome, a catalytic multiprotein platform that activates caspase-9. There is evidence that the mitochondrial pathway is involved in ischemia-induced myocyte apoptosis in the heart. Diminished expression of pro-apoptotic factors and/or expression of certain inhibitors of the apoptosome may raise the threshold for apoptosis in long-lived post-mitotic cells including myocytes of the heart.

Animals↗
PubMed2004

Mindin the fort.

Explore the source record for details and available documents.

Animals↗
PubMed2003

Roles for homotypic interactions and transautophosphorylation in IkappaB kinase beta IKKbeta) activation [corrected].

The nuclear factor kappaB (NF-kappaB)/Rel family of transcription factors participates in a wide range of biological activities including inflammation, immunity, and apoptosis. NF-kappaB is kept inactive in the cytoplasm in unstimulated cells by virtue of the masking of its nuclear localization sequence by bound IkappaB protein. Cellular stimuli trigger the destruction of IkappaB proteins and the liberation of NF-kappaB to enter the nucleus and activate gene expression. A multisubunit IkappaB kinase complex (IKK) phosphorylates IkappaB proteins and mediates the activation of NF-kappaB by proinflammatory stimuli such as tumor necrosis factor alpha. Phosphorylation of IkappaB proteins triggers their polyubiquitination and their subsequent recognition and degradation by the proteasome. The IKK complex contains two catalytic subunits, IKKalpha and IKKbeta, and a noncatalytic subunit, NF-kappaB essential modifier/IKKgamma. IKK activation depends upon the phosphorylation of residues in the activation loop of IKKbeta and the subsequent activation of IKKbeta kinase activity. However, the events contributing to IKKbeta phosphorylation are not well understood. Here, we present evidence that the activation of IKKbeta depends on its ability to form homotypic interactions and to transautophosphorylate. We find that an intact leucine zipper in IKKbeta is necessary for homotypic interactions, kinase activation, and phosphorylation on its activation loop. Enforced oligomerization of an IKKbeta mutant defective in forming homotypic interactions restores kinase activation. Homotypic interactions allow IKKbeta molecules to transautophosphorylate one another on their activation loops. Finally, the oligomerization of IKKbeta is stimulated by tumor necrosis factor alpha in cultured cells. Our findings support a model whereby ligand-induced homotypic interactions between IKKbeta molecules result in IKKbeta phosphorylation and consequently IKK activation.

Cell Line↗
PubMed2003

An essential role for NOD1 in host recognition of bacterial peptidoglycan containing diaminopimelic acid.

Nucleotide-binding oligomerization domain protein 1 (NOD1) belongs to a family that includes multiple members with NOD and leucine-rich repeats in vertebrates and plants. NOD1 has been suggested to have a role in innate immune responses, but the mechanism involved remains unknown. Here we report that NOD1 mediates the recognition of peptidoglycan derived primarily from Gram-negative bacteria. Biochemical and functional analyses using highly purified and synthetic compounds indicate that the core structure recognized by NOD1 is a dipeptide, gamma-D-glutamyl-meso-diaminopimelic acid (iE-DAP). Murine macrophages deficient in NOD1 did not secrete cytokines in response to synthetic iE-DAP and did not prime the lipopolysaccharide response. Thus, NOD1 mediates selective recognition of bacteria through detection of iE-DAP-containing peptidoglycan.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Host recognition of bacterial muramyl dipeptide mediated through NOD2. Implications for Crohn's disease.

NOD2, a protein associated with susceptibility to Crohn's disease, confers responsiveness to bacterial preparations of lipopolysaccharide and peptidoglycan, but the precise moiety recognized remains elusive. Biochemical and functional analyses identified muramyl dipeptide (MurNAc-L-Ala-D-isoGln) derived from peptidoglycan as the essential structure in bacteria recognized by NOD2. Replacement of L-Ala for D-Ala or D-isoGln for L-isoGln eliminated the ability of muramyl dipeptide to stimulate NOD2, indicating stereoselective recognition. Muramyl dipeptide was recognized by NOD2 but not by TLR2 or co-expression of TLR2 with TLR1 or TLR6. NOD2 mutants associated with susceptibility to Crohn's disease were deficient in their recognition of muramyl dipeptide. Notably, peripheral blood mononuclear cells from individuals homozygous for the major disease-associated L1007fsinsC NOD2 mutation responded to lipopolysaccharide but not to synthetic muramyl dipeptide. Thus, NOD2 mediates the host response to bacterial muropeptides derived from peptidoglycan, an activity that is important for protection against Crohn's disease. Because muramyl dipeptide is the essential structure of peptidoglycan required for adjuvant activity, these results also have implications for understanding adjuvant function and effective vaccine development.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed2003

Crohn's disease and the NOD2 gene: a role for paneth cells.

BACKGROUND & AIMS: The NOD2 gene, which is strongly associated with susceptibility to Crohn's disease (CD) of the terminal ileum, interacts with bacterial lipopolysaccharide (LPS), inducing cellular activation. However, the mechanisms by which NOD2 mutations cause terminal ileitis are unknown, and NOD2 is expressed most highly by peripheral blood monocytes, which are distributed ubiquitously and readily respond to LPS via cell-surface receptors. Paneth cells on the other hand, are most numerous in the terminal ileum, are critically important in enteric antibacterial defense, and respond to LPS through as yet undefined pathways. We therefore determined if these specialized intestinal epithelial cells also expressed the NOD2 gene. METHODS: In situ hybridization, immunohistochemistry, and laser-capture microdissection were used to determine RNA and protein expression in tissue sections, and real-time reverse-transcription polymerase chain reaction (RT-PCR) was used to quantitate gene expression in intestinal epithelial cells and peripheral blood mononuclear cells. RESULTS: NOD2 was detected readily in monocytes, but not in mature macrophages in the lamina propria or within granulomas, and levels declined as monocytes differentiated into macrophages in vitro, so that Caco-2 cells expressed more NOD2 mRNA than macrophages. NOD2 mRNA was enriched in crypts compared with villi, and in situ, Paneth cells were the most prominent cells expressing NOD2 in normal and CD-affected intestinal tissue, where they also strongly expressed tumor necrosis factor alpha (TNFalpha) RNA. CONCLUSIONS: The NOD2 gene product is most abundant in Paneth cells in the terminal ileum, which could therefore play a critical and hitherto unrecognized role in the pathogenesis of NOD2-associated CD.

Caco-2 Cells↗
PubMed2003

Genetic variation and activity of mouse Nod2, a susceptibility gene for Crohn's disease.

Genetic variation in human Nod2 has been associated with susceptibility to Crohn's disease. The mouse Nod2 locus is located at chromosome 8 and composed of 12 exons, 11 of which encode the Nod2 protein. Sequence analysis of Nod2 from 45 different strains of Mus musculus and Mus spretus revealed extensive polymorphism involving all exons of Nod2. Of the 140 polymorphic sites identified, 68 were located in the coding region, of which 28 created amino acid substitutions in Nod2. Expression of mouse Nod2 activated NF-kappaB and conferred responsiveness to bacterial components, an activity that was deficient in mutants corresponding to those associated with susceptibility to Crohn's disease. These studies demonstrate a conserved role for Nod2 in the response to bacterial components and suggest that selective evolutionary pressure exerted by pathogens may have contributed to the high level of variability of Nod2 sequences in both humans and mice.

Amino Acid Sequence↗