The Structure of 2-Oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline Decarboxylase Provides Insights into the Mechanism of Uric Acid Degradation

The complete degradation of uric acid to (S)-allantoin, as recently elucidated, involves three enzymatic reactions. Inactivation by pseudogenization of the genes of the pathway occurred during hominoid evolution, resulting in a high concentration of urate in the blood and susceptibility to gout. Here, we describe the 1.8A resolution crystal structure of the homodimeric 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline decarboxylase, which catalyzes the last step in the urate degradation pathway, for both ligand-free enzyme and enzyme in complex with the substrate analogs (R)-allantoin and guanine. Each monomer comprises ten alpha-helices, grouped into two domains and assembled in a novel fold. The structure and the mutational analysis of the active site have allowed us to identify some residues that are essential for catalysis, among which His-67 and Glu-87 appear to play a particularly significant role. Glu-87 may facilitate the exit of the carboxylate group because of electrostatic repulsion that destabilizes the ground state of the substrate, whereas His-67 is likely to be involved in a protonation step leading to the stereoselective formation of the (S)-allantoin enantiomer as reaction product. The structural and functional characterization of 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline decarboxylase can provide useful information in view of the potential use of this enzyme in the enzymatic therapy of gout

Transthyretin binding heterogeneity and antiamyloidogenic activity of natural polyphenols and their metabolites

Transthyretin (TTR) is an amyloidogenic protein, the amyloidogenic potential of which is enhanced by a number of specific point mutations. The ability to inhibit TTR fibrillogenesis is known for several classes of compounds, including natural polyphenols, which protect the native state of TTR by specifically interacting with its thyroxine binding sites. Comparative analyses of the interaction and of the ability to protect the TTR native state for polyphenols, both stilbenoids and flavonoids, and some of their main metabolites have been carried out. A main finding of this investigation was the highly preferential binding of resveratrol and thyroxine, both characterized by negative binding cooperativity, to distinct sites in TTR, consistent with the data of x-ray analysis of TTR in complex with both ligands. Although revealing the ability of the two thyroxine binding sites of TTR to discriminate between different ligands, this feature has allowed us to evaluate the interactions of polyphenols with both resveratrol and thyroxine preferential binding sites, by using resveratrol and radiolabeled T4 as probes. Among flavonoids, genistein and apigenin were able to effectively displace resveratrol from its preferential binding site, whereas genistein also showed the ability to interact, albeit weakly, with the preferential thyroxine binding site. Several glucuronidated polyphenol metabolites did not exhibit significant competition for resveratrol and thyroxine preferential binding sites and lacked the ability to stabilize TTR. However, resveratrol-3-O-sulfate was able to significantly protect the protein native state. A rationale for the in vitro properties found for polyphenol metabolites was provided by x-ray analysis of their complexes with TTR

Logical Identification of an Allantoinase Analog (puuE) Recruited from Polysaccharide Deacetylases

The hydrolytic cleavage of the hydantoin ring of allantoin, catalyzed by allantoinase, is required for the utilization of the nitrogen present in purine-derived compounds. The allantoinase gene (DAL1), however, is missing in many completely sequenced organisms able to use allantoin as a nitrogen source. Here we show that an alternative allantoinase gene (puuE) can be precisely identified by analyzing its logic relationship with three other genes of the pathway. The novel allantoinase is annotated in structure and sequence data bases as polysaccharide deacetylase for its homology with enzymes that catalyze hydrolytic reactions on chitin or peptidoglycan substrates. The recombinant PuuE protein from Pseudomonas fluorescens exhibits metal-independent allantoinase activity and stereospecificity for the S enantiomer of allantoin. The crystal structures of the protein and of protein-inhibitor complexes reveal an overall similarity with the polysaccharide deacetylase beta/alpha barrel and remarkable differences in oligomeric assembly and active site geometry. The conserved Asp-His-His metal-binding triad is replaced by Glu-His-Trp, a configuration that is distinctive of PuuE proteins within the protein family. An extra domain at the top of the barrel offers a scaffold for protein tetramerization and forms a small substrate-binding cleft by hiding the large binding groove of polysaccharide deacetylases. Substrate positioning at the active site suggests an acid/base mechanism of catalysis in which only one member of the catalytic pair of polysaccharide deacetylases has been conserved. These data provide a structural rationale for the shifting of substrate specificity that occurred during evolution

Distinctive binding and structural properties of piscine transthyretin

AbstractThe thyroid hormone binding protein transthyretin (TTR) forms a macromolecular complex with the retinol-specific carrier retinol binding protein (RBP) in the blood of higher vertebrates. Piscine TTR is shown here to exhibit high binding affinity for L-thyroxine and negligible affinity for RBP. The 1.56 Å resolution X-ray structure of sea bream TTR, compared with that of human TTR, reveals a high degree of conservation of the thyroid hormone binding sites. In contrast, some amino acid differences in discrete regions of sea bream TTR appear to be responsible for the lack of protein–protein recognition, providing evidence for the crucial role played by a limited number of residues in the interaction between RBP and TTR. Overall, this study makes it possible to draw conclusions on evolutionary relationships for RBPs and TTRs of phylogenetically distant vertebrates

Ligand binding and structural analysis of a human putative cellular retinol-binding protein.

Three cellular retinol-binding protein (CRBP) types (CRBP I, II, and III) with distinct tissue distributions and retinoid binding properties have been structurally characterized thus far. A human binding protein, whose mRNA is expressed primarily in kidney, heart, and transverse colon, is shown here to be a CRBP family member (human CRBP IV), according to amino acid sequence, phylogenetic analysis, gene structure organization, and x-ray structural analysis. Retinol binding to CRBP IV leads to an absorption spectrum distinct from a typical holo-CRBP spectrum and is characterized by an affinity (K(d) = approximately 200 nm) lower than those for CRBP I, II, and III, as established in direct and competitive binding assays. As revealed by mutagenic analysis, the presence in CRBP IV of His(108) in place of Gln(108) is not responsible for the unusual holo-CRBP IV spectrum. The 2-A resolution crystal structure of human apo-CRBP IV is very similar to those of other structurally characterized CRBPs. The side chain of Tyr(60) is present within the binding cavity of the apoprotein and might affect the interaction with the retinol molecule. These results indicate that human CRBP IV belongs to a clearly distinct CRBP subfamily and suggest a relatively different mode of retinol binding for this binding protein

The Ile-84-->Ser amino acid substitution in transthyretin interferes with the interaction with plasma retinol-binding protein.

In plasma the thyroid hormone-binding protein transthyretin (TTR) forms a tight complex with the specific retinol carrier retinol-binding protein (RBP). The Ile-84–>Ser mutation and several other point mutations in TTR are associated with familial amyloidotic polyneuropathy, which is characterized by extracellular depositions of amyloid fibrils mainly consisting of mutated TTRs. The interactions with human RBP of recombinant human normal and Ser-84 TTRs were investigated by monitoring the fluorescence anisotropy of RBP-bound retinol. A nearly negligible affinity of the recombinant Ser-84 TTR for RBP was found. This result indicates the participation of a region on the outer surface of TTR that comprises Ile-84 in the recognition of RBP. In preliminary studies the Ser-84 TTR was the only one among several amyloidogenic variant TTRs to display negligible interaction with RBP. Therefore, in general a substantially altered binding of TTR to RBP is not associated with familial amyloidotic polyneuropathy. Instead, the altered binding of Ser-84 TTR to RBP appears to be responsible for an abnormal plasma transport of RBP. The recombinant normal TTR exhibits binding properties, in its interaction with human RBP, approximately similar to those of TTR purified from human plasma. Two independent and equivalent RBP binding sites on recombinant normal TTR are characterized by a dissociation constant of about 0.4 microM

Retinoids Regulate Survival and Antigen Presentation by Immature Dendritic Cells

Maturation of dendritic cells (DCs) is a critical step for the induction of an immune response. We have examined the role of retinoid nuclear receptor pathways in this process. Retinoids induce DC apoptosis, in the absence of inflammatory signals, through retinoic acid receptor (RAR)α/retinoic X receptor (RXR) heterodimers. In contrast, via a cross talk with inflammatory cytokines, retinoids increase DNA binding activity of nuclear factor κB in DCs, trigger membrane major histocompatibility complex class II and costimulatory molecule expression, induce the differentiation of immature DCs into mature DCs, and enhance antigen-specific T cell response. This maturation of DCs is mediated via a RXR-dependent/RAR-independent pathway and via an RARα/RXR pathway distinct from the one responsible for apoptosis. Apoptosis and activation, mediated through distinct nuclear retinoid receptor pathways, can be dissociated from each other with selective synthetic retinoids. We identify a novel cellular function for retinoids and suggest that selective retinoids might be of interest for controlling antigen presentation

The GLP-1 receptor agonists exenatide and liraglutide activate Glucose transport by an AMPK-dependent mechanism

Additional file 2: Figure S2. Effects of EXE on Glut-4 in cultured L6 myotubes. Myotubes were stimulated with 100 nmol/l EXE for 20 min or 48 h. Panel A shows qPCR of Glut-4 mRNA. In panel B is a representative western blot for Glut-4 and β-Actin (loading control). In panel C is a representative western blot for Glut-4 and β-IR (loading control) in plasma membrane (PM) extracts (Glut-4 translocation). For A and C panels, data are shown as fold increase over control ± SD of three independent experiments (*p < 0.001, vs Ctrl)

Proteomics Reveals Novel Oxidative and Glycolytic Mechanisms in Type 1 Diabetic Patients' Skin Which Are Normalized by Kidney-Pancreas Transplantation

Background: In type 1 diabetes (T1D) vascular complications such as accelerated atherosclerosis and diffused macro-/microangiopathy are linked to chronic hyperglycemia with a mechanism that is not yet well understood. End-stage renal disease (ESRD) worsens most diabetic complications, particularly, the risk of morbidity and mortality from cardiovascular disease is increased several fold. Methods and Findings: We evaluated protein regulation and expression in skin biopsies obtained from T1D patients with and without ESRD, to identify pathways of persistent cellular changes linked to diabetic vascular disease. We therefore examined pathways that may be normalized by restoration of normoglycemia with kidney-pancreas (KP) transplantation. Using proteomic and ultrastructural approaches, multiple alterations in the expression of proteins involved in oxidative stress (catalase, superoxide dismutase 1, Hsp27, Hsp60, ATP synthase δ chain, and flavin reductase), aerobic and anaerobic glycolysis (ACBP, pyruvate kinase muscle isozyme, and phosphoglycerate kinase 1), and intracellular signaling (stratifin-14-3-3, S100-calcyclin, cathepsin, and PPI rotamase) as well as endothelial vascular abnormalities were identified in T1D and T1D+ESRD patients. These abnormalities were reversed after KP transplant. Increased plasma levels of malondialdehyde were observed in T1D and T1D+ESRD patients, confirming increased oxidative stress which was normalized after KP transplant. Conclusions: Our data suggests persistent cellular changes of anti-oxidative machinery and of aerobic/anaerobic glycolysis are present in T1D and T1D+ESRD patients, and these abnormalities may play a key role in the pathogenesis of hyperglycemia-related vascular complications. Restoration of normoglycemia and removal of uremia with KP transplant can correct these abnormalities. Some of these identified pathways may become potential therapeutic targets for a new generation of drugs

Human Stiff-Person Syndrome IgG Induces Anxious Behavior in Rats

Background: Anxiety is a heterogeneous behavioral domain playing a role in a variety of neuropsychiatric diseases. While anxiety is the cardinal symptom in disorders such as panic disorder, co-morbid anxious behavior can occur in a variety of diseases. Stiff person syndrome (SPS) is a CNS disorder characterized by increased muscle tone and prominent agoraphobia and anxiety. Most patients have high-titer antibodies against glutamate decarboxylase (GAD) 65. The pathogenic role of these autoantibodies is unclear. Methodology/Principal Findings: We re-investigated a 53 year old woman with SPS and profound anxiety for GABA-A receptor binding in the amygdala with (11)C-flumazenil PET scan and studied the potential pathogenic role of purified IgG from her plasma filtrates containing high-titer antibodies against GAD 65. We passively transferred the IgG fraction intrathecally into rats and analyzed the effects using behavioral and in vivo electrophysiological methods. In cell culture, we measured the effect of patient IgG on GABA release from hippocampal neurons. Repetitive intrathecal application of purified patient IgG in rats resulted in an anxious phenotype resembling the core symptoms of the patient. Patient IgG selectively bound to rat amygdala, hippocampus, and frontal cortical areas. In cultured rat hippocampal neurons, patient IgG inhibited GABA release. In line with these experimental results, the GABA-A receptor binding potential was reduced in the patient’s amygdala/hippocampus complex. No motor abnormalities were found in recipient rats. Conclusion/Significance: The observations in rats after passive transfer lead us to propose that anxiety-like behavior can be induced in rats by passive transfer of IgG from a SPS patient positive for anti-GAD 65 antibodies. Anxiety, in this case, thus may be an antibody-mediated phenomenon with consecutive disturbance of GABAergic signaling in the amygdala region