Backbone and side chain NMR assignment of the heme-nitric oxide/oxygen binding (H-NOX) domain from Nostoc punctiforme.

Soluble guanylate cyclase (sGC) is considered as the primary NO receptor across several known eukaryotes. The main interest regarding the biological role and its function, focuses on the H-NOX domain of the ß1 subunit. This domain in its active form bears a ferrous b type heme as prosthetic group, which facilitates the binding of NO and other diatomic gases. The key point that still needs to be answered is how the protein selectively binds the NO and how the redox state of heme and coordination determines H-NOX active state upon binding of diatomic gases. H-NOX domain is present in the genomes of both prokaryotes and eukaryotes, either as a stand-alone protein domain or as a partner of a larger polypeptide. The biological functions of these signaling modules for a wide range of genomes, diverge considerably along with their ligand binding properties. In this direction, we examine the prokaryotic H-NOX protein domain from Nostoc punctiforme (Npun H-NOX). Herein, we first report the almost complete NMR backbone and side-chain resonance assignment (1H, 13C, 15 N) of Npun H-NOX domain together with the NMR chemical shift-based prediction of the domain's secondary structure elements.

The pseudokinase domain in receptor guanylyl cyclases.

Cyclic GMP is produced by enzymes called guanylyl cyclases, of which the membrane-associated forms contain an intracellular pseudokinase domain that allosterically regulates the C-terminal guanylyl cyclase domain. Ligand binding to the extracellular domain of these single transmembrane-spanning domain receptors elicits an increase in cGMP levels in the cell. The pseudokinase domain (or kinase-homology domain) in these receptors appears to be critical for ligand-mediated activation. While the pseudokinase domain does not possess kinase activity, biochemical evidence indicates that the domain can bind ATP and thereby allosterically regulate the catalytic activity of these receptors. The pseudokinase domain also appears to be the site of interaction of regulatory proteins, as seen in the retinal guanylyl cyclases that are involved in visual signal transduction. In the absence of structural information on the pseudokinase-guanylyl cyclase domain organization of any member of this family of receptors, biochemical evidence has provided clues to the physical interaction of the pseudokinase and guanylyl cyclase domain. An α-helical linker region between the pseudokinase domain and the guanylyl cyclase domain regulates the basal activity of these receptors in the absence of a stimulatory ligand and is important for stabilizing the structure of the pseudokinase domain that can bind ATP. Here, we present an overview of salient features of ATP-mediated regulation of receptor guanylyl cyclases and describe biochemical approaches that allow a clearer understanding of the intricate interplay between the pseudokinase domain and catalytic domain in these proteins.

First 3D-Structural Data of Full-Length Guanylyl Cyclase 1 in Rod-Outer-Segment Preparations of Bovine Retina by Cross-Linking/Mass Spectrometry.

The rod-outer-segment guanylyl cyclase 1 (ROS-GC1) is a key transmembrane protein for retinal phototransduction. Mutations of ROS-GC1 correlate with different retinal diseases that often lead to blindness. No structural data are available for ROS-GC1 so far. We performed a 3D-structural analysis of native ROS-GC1 from bovine retina by cross-linking/mass spectrometry (XL-MS) and computational modeling. Absolute quantification and activity measurements of native ROS-GC1 were performed by MS-based assays directly in bovine retina samples. Our data present the first 3D-structural analysis of active, full-length ROS-GC1 derived from bovine retina. We propose a novel domain organization for the intracellular domain ROS-GC1. Our XL-MS data of native ROS-GC1 from rod-outer-segment preparations of bovine retina agree with a dimeric architecture. Our integrated approach can serve as a blueprint for conducting 3D-structural studies of membrane proteins in their native environment.

Possible dual contribution of a novel GUCY2D mutation in the development of retinal degeneration in a consanguineous population.

Molecular characterization of novel mutations in Leber Congenital Amaurosis (LCA) disease improves the disease diagnosis and contributes to the development of preventive and therapeutic approaches. We studied an isolated inbred population in Iran with a high prevalence of retinal degeneration with clinical variability. The clinical examinations were performed on eight patients belonging to three consanguineous families. The identical-by-descent (IBD) mapping technique was employed to identify the shared loci in patients. Subsequently, Sanger sequencing of the GUCY2D gene, in silico analysis, as well as segregation study were conducted. The whole-exome sequencing method was applied for negative cases of GUCY2D mutation, followed by segregation study in suspected variants among families. A novel deletion mutation in the GUCY2D gene can explain the emergence of LCA-1 in most patients but not all. Besides, a heterozygous variant of uncertain significance (VUS) was observed in the BEST1 gene in some healthy and participant patients. These results further support inter/intra-familial clinical heterogeneity in retinal dystrophy and suggest that screening the GUCY2D gene would be needed for the diagnosis of LCA in Iranian people living in the central regions. The variant in the BEST1 gene might be considered a benign heterozygous variant; however, we hypothesized a possible double heterozygosity in both GUCY2D and BEST1 genes that may cause the pathogenesis of cone-rod dystrophy-6 (CRD-6) disease. This would propose a new scenario for the pathogenesis of a monogenic disorder such as CRD-6 disease in which other genetic elements may be involved in the development of the disease.

The guanylate cyclase C agonist linaclotide ameliorates the gut-cardio-renal axis in an adenine-induced mouse model of chronic kidney disease.

BACKGROUND: Cardiorenal syndrome is a major cause of mortality in patients with chronic kidney disease (CKD). However, the involvement of detrimental humoral mediators in the pathogenesis of cardiorenal syndrome is still controversial. Trimethylamine-N-oxide (TMAO), a hepatic metabolic product of trimethylamine generated from dietary phosphatidylcholine or carnitine derived by the gut microbiota, has been linked directly with progression of cardiovascular disease and renal dysfunction. Thus, targeting TMAO may be a novel strategy for the prevention of cardiovascular disease and chronic kidney disease. METHODS: Linaclotide, a guanylate cyclase C agonist, was administered to adenine-induced renal failure (RF) mice and changes in renal function and levels of gut-derived uremic toxins, as well as the gut microbiota community, were analyzed using metabolomic and metagenomic methods to reveal its cardiorenal effect. RESULTS: Linaclotide decreased the plasma levels of TMAO at a clinically used low dose of 10 µg/kg in the adenine-induced RF mouse model. At a high concentration of 100 µg/kg, linaclotide clearly improved renal function and reduced the levels of various uremic toxins. A reduction in TMAO levels following linaclotide treatment was also observed in a choline-fed pro-atherosclerotic model. Linaclotide ameliorated renal inflammation and fibrosis and cardiac fibrosis, as well as decreased the expression of collagen I, transforming growth factor-ß, galectin-3 (Gal-3) and ST2 genes. Plasma levels of Gal-3 and ST2 were also reduced. Because exposure of cardiomyocytes to TMAO increased fibronectin expression, these data suggest that linaclotide reduced the levels of TMAO and various uremic toxins and may result in not only renal, but also cardiac, fibrosis. F4/80-positive macrophages were abundant in small intestinal crypts in RF mice, and this increased expression was decreased by linaclotide. Reduced colonic claudin-1 levels were also restored by linaclotide, suggesting that linaclotide ameliorated the 'leaky gut' in RF mice. Metagenomic analysis revealed that the microbial order Clostridiales could be responsible for the change in TMAO levels. CONCLUSION: Linaclotide reduced TMAO and uremic toxin levels and could be a powerful tool for the prevention and control of the cardiorenal syndrome by modification of the gut-cardio-renal axis.

IRAK3 modulates downstream innate immune signalling through its guanylate cyclase activity.

Interleukin-1 receptor associated kinase 3 (IRAK3) is a cytoplasmic homeostatic mediator of inflammatory responses and is potentially useful as a prognostic marker in inflammation. IRAK3 inhibits signalling cascades downstream of myddosome complexes associated with toll like receptors. IRAK3 contains a death domain that interacts with other IRAK family members, a pseudokinase domain and a C-terminus domain involved with tumour necrosis factor receptor associated factor 6 (TRAF6). Previous bioinformatic studies revealed that IRAK3 contained a guanylate cyclase centre in its pseudokinase domain but its role in IRAK3 action is unresolved. We demonstrate that wildtype IRAK3 is capable of producing cGMP. Furthermore, we show that a specific point mutation in the guanylate cyclase centre reduced cGMP production. Cells containing toll like receptor 4 and a nuclear factor kappa-light-chain-enhancer of activated B cells (NFĸB) reporter system were transfected with IRAK3 or mutant IRAK3 proteins. Cell-permeable cGMP treatment of untransfected control cells suppresses downstream signalling through modulation of the NFĸB in the presence of lipopolysaccharides. Cells transfected with wildtype IRAK3 also suppress lipopolysaccharide induced NFĸB activity in the absence of exogenous cGMP. Lipopolysaccharide induced NFĸB activity was not suppressed in cells transfected with the IRAK3 mutant with reduced cGMP-generating capacity. Whereas in the presence of exogenously applied cell-permeable cGMP the IRAK3 mutant was able to retain its function by suppressing lipopolysaccharide induced NFĸB activity. Furthermore, increasing the amount of membrane permeable cGMP did not affect IRAK3's ability to reduce NFĸB activity. These results suggest that cGMP generated by IRAK3 may be involved in regulatory function of the protein where the presence of cGMP may selectively affect downstream signalling pathway(s) by modulating binding and/or activity of nearby proteins that interact in the inflammatory signalling cascade.

Coordinated regulation of scaffold opening and enzymatic activity during CARD11 signaling.

The activation of key signaling pathways downstream of antigen receptor engagement is critically required for normal lymphocyte activation during the adaptive immune response. CARD11 is a multidomain signaling scaffold protein required for antigen receptor signaling to NF-κB, c-Jun N-terminal kinase, and mTOR. Germline mutations in the CARD11 gene result in at least four types of primary immunodeficiency, and somatic CARD11 gain-of-function mutations drive constitutive NF-κB activity in diffuse large B cell lymphoma and other lymphoid cancers. In response to antigen receptor triggering, CARD11 transitions from a closed, inactive state to an open, active scaffold that recruits multiple signaling partners into a complex to relay downstream signaling. However, how this signal-induced CARD11 conversion occurs remains poorly understood. Here we investigate the role of Inducible Element 1 (IE1), a short regulatory element in the CARD11 Inhibitory Domain, in the CARD11 signaling cycle. We find that IE1 controls the signal-dependent Opening Step that makes CARD11 accessible to the binding of cofactors, including Bcl10, MALT1, and the HOIP catalytic subunit of the linear ubiquitin chain assembly complex. Surprisingly, we find that IE1 is also required at an independent step for the maximal activation of HOIP and MALT1 enzymatic activity after cofactor recruitment to CARD11. This role of IE1 reveals that there is an Enzymatic Activation Step in the CARD11 signaling cycle that is distinct from the Cofactor Association Step. Our results indicate that CARD11 has evolved to actively coordinate scaffold opening and the induction of enzymatic activity among recruited cofactors during antigen receptor signaling.

An unusual and vital protein with guanylate cyclase and P4-ATPase domains in a pathogenic protist.

cGMP signaling is one of the master regulators of diverse functions in eukaryotes; however, its architecture and functioning in protozoans remain poorly understood. Herein, we report an exclusive guanylate cyclase coupled with N-terminal P4-ATPase in a common parasitic protist, Toxoplasma gondii This bulky protein (477-kD), termed TgATPaseP-GC to fairly reflect its envisaged multifunctionality, localizes in the plasma membrane at the apical pole of the parasite, whereas the corresponding cGMP-dependent protein kinase (TgPKG) is distributed in the cytomembranes. TgATPaseP-GC is refractory to genetic deletion, and its CRISPR/Cas9-assisted disruption aborts the lytic cycle of T. gondii Besides, Cre/loxP-mediated knockdown of TgATPaseP-GC reduced the synthesis of cGMP and inhibited the parasite growth due to impairments in the motility-dependent egress and invasion events. Equally, repression of TgPKG by a similar strategy recapitulated phenotypes of the TgATPaseP-GC-depleted mutant. Notably, despite a temporally restricted function, TgATPaseP-GC is expressed constitutively throughout the lytic cycle, entailing a post-translational regulation of cGMP signaling. Not least, the occurrence of TgATPaseP-GC orthologs in several other alveolates implies a divergent functional repurposing of cGMP signaling in protozoans, and offers an excellent drug target against the parasitic protists.

The pseudokinase domains of guanylyl cyclase-A and -B allosterically increase the affinity of their catalytic domains for substrate.

Natriuretic peptides regulate multiple physiologic systems by activating transmembrane receptors containing intracellular guanylyl cyclase domains, such as GC-A and GC-B, also known as Npr1 and Npr2, respectively. Both enzymes contain an intracellular, phosphorylated pseudokinase domain (PKD) critical for activation of the C-terminal cGMP-synthesizing guanylyl cyclase domain. Because ATP allosterically activates GC-A and GC-B, we investigated how ATP binding to the PKD influenced guanylyl cyclase activity. Molecular modeling indicated that all the residues of the ATP-binding site of the prototypical kinase PKA, except the catalytic aspartate, are conserved in the PKDs of GC-A and GC-B. Kinase-inactivating alanine substitutions for the invariant lysine in subdomain II or the aspartate in the DYG-loop of GC-A and GC-B failed to decrease enzyme phosphate content, consistent with the PKDs lacking kinase activity. In contrast, both mutations reduced enzyme activation by blocking the ability of ATP to decrease the Michaelis constant without affecting peptide-dependent activation. The analogous lysine-to-alanine substitution in a glutamate-substituted phosphomimetic mutant form of GC-B also reduced enzyme activity, consistent with ATP stimulating guanylyl cyclase activity through an allosteric, phosphorylation-independent mechanism. Mutations designed to rigidify the conserved regulatory or catalytic spines within the PKDs increased guanylyl cyclase activity, increased sensitivity to natriuretic peptide, or reduced the Michaelis constant in the absence of ATP, consistent with ATP binding stabilizing the PKD in a conformation analogous to that of catalytically active kinases. We conclude that allosteric mechanisms evolutionarily conserved in the PKDs promote the catalytic activation of transmembrane guanylyl cyclases.

Rhodopsin-cyclases for photocontrol of cGMP/cAMP and 2.3 Å structure of the adenylyl cyclase domain.

The cyclic nucleotides cAMP and cGMP are important second messengers that orchestrate fundamental cellular responses. Here, we present the characterization of the rhodopsin-guanylyl cyclase from Catenaria anguillulae (CaRhGC), which produces cGMP in response to green light with a light to dark activity ratio >1000. After light excitation the putative signaling state forms with τ = 31 ms and decays with τ = 570 ms. Mutations (up to 6) within the nucleotide binding site generate rhodopsin-adenylyl cyclases (CaRhACs) of which the double mutated YFP-CaRhAC (E497K/C566D) is the most suitable for rapid cAMP production in neurons. Furthermore, the crystal structure of the ligand-bound AC domain (2.25 Å) reveals detailed information about the nucleotide binding mode within this recently discovered class of enzyme rhodopsin. Both YFP-CaRhGC and YFP-CaRhAC are favorable optogenetic tools for non-invasive, cell-selective, and spatio-temporally precise modulation of cAMP/cGMP with light.

The E3 Ubiquitin Ligase RNF7 Negatively Regulates CARD14/CARMA2sh Signaling.

The three CARD-containing MAGUK (CARMA) proteins function as scaffolding molecules that regulate activation of the pro-inflammatory transcription factor NF-κB. Recently, mutations in CARMA2 have been linked to psoriasis susceptibility due to their acquired altered capacity to activate NF-κB. By means of two-hybrid screening with yeast, we identified RING finger protein 7 (RNF7) as an interactor of CARMA2. We present evidence that RNF7 functions as a negative regulator of the NF-κB-activating capacity of CARMA2. Mechanistically, RNF7 influences CARMA2 signaling by regulating the ubiquitination state of MALT1 and the NF-κB-regulatory molecule NEMO. Interestingly, CARMA2short (CARMA2sh) mutants associated with psoriasis susceptibility escape the negative control exerted by RNF7. In conclusion, our findings identify a new mechanism through which the ability of CARMA2 to activate NF-κB is regulated, which could have significant implications for our understanding of why mutations of this protein trigger human psoriasis.

Synthesis and degradation of cAMP in Giardia lamblia: possible role and characterization of a nucleotidyl cyclase with a single cyclase homology domain.

Despite its importance in the regulation of growth and differentiation processes of a variety of organisms, the mechanism of synthesis and degradation of cAMP (cyclic AMP) has not yet been described in Giardia lamblia In this work, we measured significant quantities of cAMP in trophozoites of G. lamblia incubated in vitro and later detected how it increases during the first hours of encystation, and how it then returns to basal levels at 24 h. Through an analysis of the genome of G. lamblia, we found sequences of three putative enzymes - one phosphodiesterase (gPDE) and two nucleotidyl cyclases (gNC1 and gNC2) - that should be responsible for the regulation of cAMP in G. lamblia Later, an RT-PCR assay confirmed that these three genes are expressed in trophozoites. The bioinformatic analysis indicated that gPDE is a transmembrane protein of 154 kDa, with a single catalytic domain in the C-terminal end; gNC1 is predicted to be a transmembrane protein of 74 kDa, with only one class III cyclase homology domain (CHD) at the C-terminal end; and gNC2 should be a transmembrane protein of 246 kDa, with two class III CHDs. Finally, we cloned and enriched the catalytic domain of gNC1 (gNC1cd) from bacteria. After that, we confirmed that gNC1cd has adenylyl cyclase (AC) activity. This enzymatic activity depends on the presence of Mn2+ and Ca2+, but no significant activity was displayed in the presence of Mg2+ Additionally, the AC activity of gNC1cd is competitively inhibited with GTP, so it is highly possible that gNC1 has guanylyl cyclase activity as well.

Structural and Functional Impacts of ER Coactivator Sequential Recruitment.

Nuclear receptors recruit multiple coactivators sequentially to activate transcription. This "ordered" recruitment allows different coactivator activities to engage the nuclear receptor complex at different steps of transcription. Estrogen receptor (ER) recruits steroid receptor coactivator-3 (SRC-3) primary coactivator and secondary coactivators, p300/CBP and CARM1. CARM1 recruitment lags behind the binding of SRC-3 and p300 to ER. Combining cryo-electron microscopy (cryo-EM) structure analysis and biochemical approaches, we demonstrate that there is a close crosstalk between early- and late-recruited coactivators. The sequential recruitment of CARM1 not only adds a protein arginine methyltransferase activity to the ER-coactivator complex, it also alters the structural organization of the pre-existing ERE/ERα/SRC-3/p300 complex. It induces a p300 conformational change and significantly increases p300 HAT activity on histone H3K18 residues, which, in turn, promotes CARM1 methylation activity on H3R17 residues to enhance transcriptional activity. This study reveals a structural role for a coactivator sequential recruitment and biochemical process in ER-mediated transcription.

Substrate specificity determinants of class III nucleotidyl cyclases.

The two second messengers in signalling, cyclic AMP and cyclic GMP, are produced by adenylyl and guanylyl cyclases respectively. Recognition and discrimination of the substrates ATP and GTP by the nucleotidyl cyclases are vital in these reactions. Various apo-, substrate- or inhibitor-bound forms of adenylyl cyclase (AC) structures from transmembrane and soluble ACs have revealed the catalytic mechanism of ATP cyclization reaction. Previously reported structures of guanylyl cyclases represent ligand-free forms and inactive open states of the enzymes and thus do not provide information regarding the exact mode of substrate binding. The structures we present here of the cyclase homology domain of a class III AC from Mycobacterium avium (Ma1120) and its mutant in complex with ATP and GTP in the presence of calcium ion, provide the structural basis for substrate selection by the nucleotidyl cyclases at the atomic level. Precise nature of the enzyme-substrate interactions, novel modes of substrate binding and the ability of the binding pocket to accommodate diverse conformations of the substrates have been revealed by the present crystallographic analysis. This is the first report to provide structures of both the nucleotide substrates bound to a nucleotidyl cyclase. DATABASE: Coordinates and structure factors have been deposited in the Protein Data Bank with accession numbers: 5D15 (Ma1120CHD +ATP.Ca2+ ), 5D0E (Ma1120CHD +GTP.Ca2+ ), 5D0H (Ma1120CHD (KDA→EGY)+ATP.Ca2+ ), 5D0G (Ma1120CHD (KDA→EGY)+GTP.Ca2+ ). ENZYMES: Adenylyl cyclase (EC number: 4.6.1.1).

Novel GUCY2D mutation causes phenotypic variability of Leber congenital amaurosis in a large kindred.

BACKGROUND: Leber congenital amaurosis (LCA) is a severe retinal degenerative disease that manifests as blindness or poor vision in infancy. The purpose of this study was to clinically characterize and identify the cause of disease in a large inbred Bedouin Israeli tribe with LCA. METHODS: Thirty individuals of a single kindred, including eight affected with LCA, were recruited for this study. Patients' clinical data and electroretinography (ERG) findings were collected. Molecular analysis included homozygosity mapping with polymorphic markers and Sanger sequencing of candidate genes. RESULTS: Of the eight affected individuals of the kindred, nystagmus was documented in five subjects and keratoconus in three. Cataract was found in 5 of 16 eyes. Photopic and scotopic ERG performed in 5 patients were extinguished. All affected subjects were nearly blind, their visual acuity ranged between finger counting and uncertain light perception. Assuming autosomal recessive heredity of a founder mutation, studies using polymorphic markers excluded homozygosity of affected individuals at the genomic loci of all previously known genes associated with LCA, except GUCY2D. Sequencing of GUCY2D identified a novel missense mutation (c.2129C>T; p.Ala710Val) resulting in substitution of alanine by valine at position 710 within the protein kinase domain of the retina-specific enzyme guanylate cyclase 1 (GC1) encoded by GUCY2D. Molecular modeling implied that the mutation changes the conformation of the regulatory segment within the kinase styk-domain of GC1 and causes loss of its helical structure, likely inhibiting phosphorylation of threonine residue within this segment, which is needed to activate the catalytic domain of the protein. CONCLUSIONS: This is the first documentation of the p.Ala710Val mutation in GC1 and the second ever described mutation in its protein kinase domain. Our findings enlarge the scope of genetic variability of LCA, highlight the phenotypic heterogeneity found amongst individuals harboring an identical LCA mutation, and possibly provide hope for gene therapy in patients with this congenital blinding disease. As the Bedouin kindred studied originates from Saudi Arabia, the mutation found might be an ancient founder mutation in that large community.

Lymphocyte signaling and activation by the CARMA1-BCL10-MALT1 signalosome.

The CARMA1-BCL10-MALT1 (CBM) signalosome triggers canonical NF-κB signaling and lymphocyte activation upon antigen-receptor stimulation. Genetic studies in mice and the analysis of human immune pathologies unveiled a critical role of the CBM complex in adaptive immune responses. Great progress has been made in elucidating the fundamental mechanisms that dictate CBM assembly and disassembly. By bridging proximal antigen-receptor signaling to downstream signaling pathways, the CBM complex exerts a crucial scaffolding function. Moreover, the MALT1 subunit confers a unique proteolytic activity that is key for lymphocyte activation. Deregulated 'chronic' CBM signaling drives constitutive NF-κB signaling and MALT1 activation, which contribute to the development of autoimmune and inflammatory diseases as well as lymphomagenesis. Thus, the processes that govern CBM activation and function are promising targets for the treatment of immune disorders. Here, we summarize the current knowledge on the functions and mechanisms of CBM signaling in lymphocytes and how CBM deregulations contribute to aberrant signaling in malignant lymphomas.

Insights into open/closed conformations of the catalytically active human guanylate kinase as investigated by small-angle X-ray scattering.

Bio-catalysis is the outcome of a subtle interplay between internal motions in enzymes and chemical kinetics. Small-angle X-ray scattering (SAXS) investigation of an enzyme's internal motions during catalysis offers an integral view of the protein's structural plasticity, dynamics, and function, which is useful for understanding allosteric effects and developing novel medicines. Guanylate kinase (GMPK) is an essential enzyme involved in the guanine nucleotide metabolism of unicellular and multicellular organisms. It is also required for the intracellular activation of numerous antiviral and anticancer purine nucleoside analog prodrugs. Catalytically active recombinant human GMPK (hGMPK) was purified for the first time and changes in the size and shape of open/closed hGMPK were tracked by SAXS. The binding of substrates (GMP + AMPPNP or Ap5G or GMP + ADP) resulted in the compaction of size and shape of hGMPK. The structural changes between open and completely closed hGMPK conformation were confirmed by observing differences in the hGMPK secondary structures with circular dichroism spectroscopy.

Negative Regulation of CARD11 Signaling and Lymphoma Cell Survival by the E3 Ubiquitin Ligase RNF181.

NF-κB activation downstream of antigen receptor engagement is a highly regulated event required for lymphocyte activation during the adaptive immune response. The pathway is often dysregulated in lymphoma, leading to constitutive NF-κB activity that supports the aberrant proliferation of transformed lymphocytes. To identify novel regulators of antigen receptor signaling to NF-κB, we developed bioluminescence resonance energy transfer-based interaction cloning (BRIC), a screening strategy that can detect protein-protein interactions in live mammalian cells in a high-throughput manner. Using this strategy, we identified the RING finger protein RNF181 as an interactor of CARD11, a key signaling scaffold in the antigen receptor pathway. We present evidence that RNF181 functions as an E3 ubiquitin ligase to inhibit antigen receptor signaling to NF-κB downstream of CARD11. The levels of the obligate signaling protein Bcl10 are reduced by RNF181 even prior to signaling, and Bcl10 can serve as a substrate for RNF181 E3 ligase activity in vitro. Furthermore, RNF181 limits the proliferation of human diffuse large B cell lymphoma cells that depend upon aberrant CARD11 signaling to NF-κB for growth and survival in culture. Our results define a new regulatory checkpoint that can modulate the output of CARD11 signaling to NF-κB in both normal and transformed lymphocytes.

Computational analyses and prediction of guanylin deleterious SNPs.

Human guanylin, coded by the GUCA2A gene, is a member of a peptide family that activates intestinal membrane guanylate cyclase, regulating electrolyte and water transport in intestinal and renal epithelia. Deregulation of guanylin peptide activity has been associated with colon adenocarcinoma, adenoma and intestinal polyps. Besides, it is known that mutations on guanylin receptors could be involved in meconium ileus. However, there are no previous works regarding the alterations driven by single nucleotide polymorphisms in guanylin peptides. A comprehensive in silico analysis of missense SNPs present in the GUCA2A gene was performed taking into account 16 prediction tools in order to select the deleterious variations for further evaluation by molecular dynamics simulations (50 ns). Molecular dynamics data suggest that the three out of five variants (Cys104Arg, Cys112Ser and Cys115Tyr) have undergone structural modifications in terms of flexibility, volume and/or solvation. In addition, two nonsense SNPs were identified, both preventing the formation of disulfide bonds and resulting in the synthesis of truncated proteins. In summary the structural analysis of missense SNPs is important to decrease the number of potential mutations to be in vitro evaluated for associating them with some genetic diseases. In addition, data reported here could lead to a better understanding of structural and functional aspects of guanylin peptides.

The Influence of Nitric Oxide on Soluble Guanylate Cyclase Regulation by Nucleotides: ROLE OF THE PSEUDOSYMMETRIC SITE.

Activation of soluble guanylate cyclase (sGC) by the signaling molecule nitric oxide (NO) leads to formation of the second messenger cGMP, which mediates numerous physiological processes. NO activates sGC by binding to the ferrous heme cofactor; the relative amount of NO with respect to sGC heme affects the enzyme activity. ATP can also influence the activity by binding to an allosteric site, most likely the pseudosymmetric site located in the catalytic domain. Here, the role of the pseudosymmetric site on nucleotide regulation was investigated by point mutations at this site. ATP inhibition kinetics of wild type and a pseudosymmetric site (α1-C594A/ß1-D477A) variant of sGC was determined at various levels of NO. Results obtained show that in the presence of less than 1 eq of NO, there appears to be less than complete activation and little change in the nucleotide binding parameters. The most dramatic effects are observed for the addition of excess NO, which results in an increase in the affinity of GTP at the catalytic site and full activation of sGC. The pseudosymmetric site mutation only affected nucleotide affinities in the presence of excess NO; there was a decrease in the affinity for ATP in both the allosteric and catalytic sites. These observations led to a new kinetic model for sGC activity in the presence of excess NO. This model revealed that the active and allosteric sites show cooperativity. This new comprehensive model gives a more accurate description of sGC regulation by NO and nucleotides in vivo.