Inhibition of the receptor for advanced glycation endproducts (RAGE) protects pancreatic β-cells

Advanced glycation endproducts (AGEs) and the receptor for AGEs (RAGE) have been linked to the pathogenesis of diabetic complications, such as retinopathy, neuropathy, and nephropathy. AGEs may induce β-cell dysfunction and apoptosis, another complication of diabetes. However, the role of AGE-RAGE interaction in AGE-induced pancreatic β-cell failure has not been fully elucidated. In this study, we investigated whether AGE–RAGE interaction could mediate β-cell failure. We explored the potential mechanisms in insulin secreting (INS-1) cells from a pancreatic β-cell line, as well as primary rat islets. We found that glycated serum (GS) induced apoptosis in pancreatic β-cells in a dose- and time-dependent manner. Treatment with GS increased RAGE protein production in cultured INS-1 cells. GS treatment also decreased bcl-2 gene expression, followed by mitochondrial swelling, increased cytochrome c release, and caspase activation. RAGE antibody and knockdown of RAGE reversed the β-cell apoptosis and bcl-2 expression. Inhibition of RAGE prevented AGE-induced pancreatic β-cell apoptosis, but could not restore the function of glucose stimulated insulin secretion (GSIS) in rat islets. In summary, the results of the present study demonstrate that AGEs are integrally involved in RAGE-mediated apoptosis and impaired GSIS dysfunction in pancreatic β-cells. Inhibition of RAGE can effectively protect β-cells against AGE-induced apoptosis, but cannot reverse islet dysfunction in GSIS.     

The crystal structure of human α(1)-microglobulin reveals a potential haem-binding site

We describe the 2.3 ? (1 ?=0.1?nm) X-ray structure of α1m (α1-microglobulin), an abundant protein in human blood plasma, which reveals the β-barrel fold typical for lipocalins with a deep pocket lined by four loops at its open rim. Loop #1 harbours the residue Cys34 which is responsible for covalent cross-linking with plasma IgA. A single disulfide bond between Cys72 and Cys169 connects the C-terminal segment to the β-barrel, as in many other lipocalins. The exposed imidazole side chains of His122 and His123 in loop #4 give rise to a double Ni2+-binding site together with a crystallographic neighbour. The closest structural relatives of α1m are the complement protein component C8γ, the L-prostaglandin D synthase and lipocalin 15, three other structurally characterized members of the lipocalin family in humans that have only distant sequence similarity. In contrast with these, α1m is initially expressed as a bifunctional fusion protein with the protease inhibitor bikunin. Neither the electron density nor ESI-MS (electrospray ionization MS) provide evidence for a chromophore bound to the recombinant α1m, also known as 'yellow/brown lipocalin'. However, the three side chains of Lys92, Lys118 and Lys130 that were reported to be involved in covalent chromophore binding appear to be freely accessible to ligands accommodated in the hydrophobic pocket. A structural feature similar to the well-known Cys-Pro haem-binding motif indicates the presence of a haem-binding site within the loop region of α1m, which explains previous biochemical findings and supports a physiological role in haem scavenging, as well as redox-mediated detoxification.     

Identification of novel α7 nicotinic receptor ligands by in silico screening against the crystal structure of a chimeric α7 receptor ligand binding domain

A hierarchical in silico screening procedure using the crystal structure of an agonist bound chimeric α7/Ls-AChBP protein was successfully applied to both proprietary and commercial databases containing drug-like molecules. An overall hit rate of 26% (pK_i ?5.0) was obtained, with an even better hit rate of 35% for the commercial compound collection. Structurally novel and diverse ligands were identified. Binding studies with [³H]epibatidine on chimeric α7/5-HT₃ receptors yielded submicromolar inhibition constants for identified hits. Compared to a previous screening procedure that utilized the wild type Ls-AChBP crystal structure, the current study shows that the recently obtained α7/Ls-AChBP chimeric protein crystal structure is a better template for the identification of novel α7 receptor ligands.     

The crystal structure of a Thermus thermophilus tRNA(Gly) acceptor stem microhelix at 1.6 Å resolution

tRNAs are aminoacylated with the correct amino acid by the cognate aminoacyl-tRNA synthetase. The tRNA/synthetase systems can be divided into two classes: class I and class II. Within class I, the tRNA identity elements that enable the specificity consist of complex sequence and structure motifs, whereas in class II the identity elements are assured by few and simple determinants, which are mostly located in the tRNA acceptor stem. The tRNA(Gly)/glycyl-tRNA-synthetase (GlyRS) system is a special case regarding evolutionary aspects. There exist two different types of GlyRS, namely an archaebacterial/human type and an eubacterial type, reflecting the evolutionary divergence within this system. We previously reported the crystal structures of an Escherichia coli and of a human tRNA(Gly) acceptor stem microhelix. Here we present the crystal structure of a thermophilic tRNA(Gly) aminoacyl stem from Thermus thermophilus at 1.6? resolution and provide insight into the RNA geometry and hydration.     

Expression,purification and fluorine-18 radiolabeling of recombinant S100A4: a potential probe for molecular imaging of receptor for advanced glycation endproducts in vivo?

Data concerning the pathophysiological role of extracellular S100A4, a member of the multigenic family of Ca²⁺-modulated S100 proteins, and its interaction with the receptor for advanced glycation endproducts (RAGE) or other putative receptors in tumorigenesis, metastasis, and inflammatory processes in vivo are scarce. One reason is the shortage of suitable radiotracer methods. We report a novel methodology using recombinant human S100A4 as potential probe for molecular imaging and functional characterization of this interaction. Therefore, human S100A4 was cloned as GST fusion protein in the bacterial expression vector pGEX-6P-1 and expressed in E. coli strain BL21. Purified recombinant human S100A4 was radiolabeled with the positron emitter fluorine-18 (¹⁸F) by conjugation with N-succinimidyl-4-[¹⁸F]fluorobenzoate ([¹⁸F]SFB). The radioligand [¹⁸F]fluorobenzoyl-S100A4 (¹⁸F-S100A4) was used in cell binding experiments in RAGE-bearing human melanoma cells and endothelial cells in vitro, and in both biodistribution experiments and small animal positron emission tomography (PET) studies in normal rats in vivo. The cellular association and tissue-specific distribution of ¹⁸F-S100A4 in vitro and in vivo correlated well with the protein expression and anatomical localization of RAGE, e.g., in the vascular system and in lung. Compared to other S100 RAGE radioligands, the overall findings of this study indicate that extracellular S100A4 in vivo shows only a moderate interaction with RAGE and, furthermore, exhibits a substantially faster metabolic degradation. On the other hand, the approach allows the use of quantitative small animal PET and provides a novel probe to both delineate functional expression and differentiate multiligand interaction of RAGE under normal and pathophysiological conditions in rodent models of disease.     

The 1.9Å crystal structure of Prp20p from Saccharomyces cerevisiae and its binding properties to Gsp1p and histones

Prp20p is the homolog of mammalian RCC1 (regulator of chromosome condensation 1) in Saccharomyces cerevisiae, which acts as the guanine nucleotide exchange factor (GEF) for Gsp1p (yeast Ran). Prp20p plays multiple roles in mRNA metabolism, nucleocytoplasmic transport and mitosis regulation. Prp20p also functions as a linker between chromatin and nuclear pore complex (NPC) which regulates the NPC-mediated boundary activity (BA). Prp20p contains an N-terminal nuclear localization signal (NLS) and a typical RCC1-like domain (RLD). Here we present the 1.9? crystal structure of the RCC1-like domain of Prp20p, which exhibits a classical seven-bladed β-propeller. We also proved that the additional β-wedge in Prp20p is essential for the interaction between Prp20p and Gsp1p. Based on this structure, we built a complex model of Prp20p and Gsp1p which was optimized by molecular dynamics (MD) simulations. Our model reveals that Prp20p and RCC1 share similar Ran GTPase binding mode. In addition, we also studied the histone-binding property of Prp20p in vitro.     

Solution structure of the human CD4 (403–419) receptor peptide

The cytoplasmic part of CD4 is known to be essential for the interaction with the human immunodeficiency virus type 1 proteins Vpu and Nef. The 17 amino acid synthetic peptide CD4 (403–419) with the amino acid sequence of the membrane proximal part of the cytoplasmic domain of the human CD4 receptor was structurally investigated by circular dichroism and nuclear magnetic resonance spectroscopy. The average -helical content of the peptide could be estimated to be around 25%. Chemical shift index analysis and the connectivity pattern in nuclear Overhauser enhancement spectra located the -helical part of the peptide from Gln403 to Arg412. It may be speculated that this amphipathic -helix is the contact region with the Vpu and Nef proteins.The authors thank Prof. F.X. Schmid for help with the CD spectra.     

Characterization of the Japanese pufferfish (Takifugu rubripes) T-cell receptor α locus reveals a unique genomic organization

Polymerase chain reactions with degenerate V gene segment primers were used to isolate the putative T-cell receptor alpha-chain gene (TCRA) from Japanese pufferfish (Takifugu rubripes). The putative TCRA chain cDNA is composed of an N-terminus leader peptide followed by the variable region and the constant region. The variable portion of the TCRA gene is encoded by V and J gene segments separated in the germline. As in mammals, the V-J junction sequences are GC rich and highly diversified. Amino acid residues that are required to maintain the function and structural integrity of the TCRA polypeptide, including the conserved Trp-Tyr-Lys and Tyr-Tyr-Cys motifs in the V gene segments, the Lys-Leu-X-Phe-Gly-X-Gly-Thr-X-Leu motif in the J gene segment, the three cysteine residues in the constant region and the charged residues in the transmembrane region are all preserved in the pufferfish. These conserved features suggest that the TCRA gene families in fish and mammals have evolved from a common ancestor.     

The crystal structure of the α-neurexin-1 extracellular region reveals a hinge point for mediating synaptic adhesion and function

α- and β-neurexins (NRXNs) are transmembrane cell adhesion proteins that localize to presynaptic membranes in neurons and interact with the postsynaptic neuroligins (NLGNs). Their gene mutations are associated with the autism spectrum disorders. The extracellular region of α-NRXNs, containing nine independently folded domains, has structural complexity and unique functional characteristics, distinguishing it from the smaller β-NRXNs. We have solved the X-ray crystal structure of seven contiguous domains of the α-NRXN-1 extracellular region at 3.0 ? resolution. The structure reveals an arrangement where the N-terminal five domains adopt a more rigid linear conformation and the two C-terminal domains form a separate arm connected by a flexible hinge. In an extended conformation the molecule is suitably configured to accommodate a bound NLGN molecule, as supported by structural comparison and surface plasmon resonance. These studies provide the structural basis for a multifunctional synaptic adhesion complex mediated by α-NRXN-1.     

The crystal structure of human protein α1M reveals a chromophore-binding site and two putative protein–protein interfaces

Lipocalin α1-microglobulin (α1M) is a conserved glycoprotein present in plasma and in the interstitial fluids of all tissues. α1M is linked to a heterogeneous yellow–brown chromophore of unknown structure, and interacts with several target proteins, including α1-inhibitor-3, fibronectin, prothrombin and albumin. To date, there is little knowledge about the interaction sites between α1M and its partners. Here, we report the crystal structure of the human α1M. Due to the crystallization occurring in a low ionic strength solution, the unidentified chromophore with heavy electron density is observed at a hydrophobic inner tube of α1M. In addition, two conserved surface regions of α1M are proposed as putative protein–protein interface sites. Further study is needed to unravel the detailed information about the interaction between α1M and its partners.     

The extended conformation of the 2.9-Å crystal structure of the three-PASTA domain of a Ser/Thr kinase from the human pathogen Staphylococcus aureus

PASTA (penicillin-binding protein and serine/threonine kinase associated) modules are found in penicillin-binding proteins and bacterial serine/threonine kinases mainly from Gram-positive Firmicutes and Actinobacteria. They may act as extracellular sensors by binding peptidoglycan fragments. We report here the first crystal structure of a multiple-PASTA domain from Ser/Thr kinase, that of the protein serine/threonine kinase 1 (Stk1) from the Firmicute Staphylococcus aureus. The extended conformation of the three PASTA subunits differs strongly from the compact conformation observed in the two-PASTA domain of penicillin-binding protein PBP2x, whereas linear conformations were also reported for two-subunit fragments of the four-PASTA domain of the Actinobacteria Mycobacterium tuberculosis studied by liquid NMR. Thus, a stretched organization appears to be the signature of modular PASTA domains in Ser/Thr kinases. Signal transduction to the kinase domain is supposed to occur via dimerization and ligand binding. A conserved X-shaped crystallographic dimer stabilized by intermolecular interactions between the second PASTA subunits of each monomer is observed in the two crystal forms of Stk1 that we managed to crystallize. Extracellular PASTA domains are composed of at least two subunits, and this molecular assembly is a plausible candidate for the biological dimer. We have also performed docking experiments, which predict that the hinge regions of the PASTA domain can accommodate peptidoglycan. Finally, a three-dimensional homology molecular model of full-length Stk1 was generated, suggesting an interaction between the kinase domain and the cytoplasmic face of the plasma membrane via a eukaryotic-like juxtamembrane domain. A comprehensive activation mechanism for bacterial Ser/Thr kinases is proposed with the support of these structural data.     

Inhibition of fluorescent advanced glycation end products (AGEs) of human serum albumin upon incubation with 3-β-hydroxybutyrate

Advanced glycation end products (AGEs), which are the final products of glycation, have a major role in diabetic complication and neurodegenerative disorders. The 3-β-hydroxybutyrate (3BHB), a ketone body which is produced by the liver, can be detected in increased concentrations in individuals post fasting and prolonged exercises and in diabetic (type I) patients. In this study, the inhibitory effect of 3BHB on AGEs formation by glucose from the human serum albumin (HSA) was studied at physiological conditions after 35 days of incubation, using physical techniques such as circular dichroism and fluorescence spectroscopy, as well as differential scanning calorimetry (DSC). The fluorescence intensity measurements of glycated HSA by glucose (GHSA) in the presence of 3BHB indicate a decrease in AGEs formation. The DSC deconvolution profile results also confirm the protective role of 3BHB on incubated with glucose by preventing the enthalpy reduction of the HSA tail segment, compared with the deconvolution profile seen for incubated with glucose alone. The concentration of 3BHB used in this study is in accordance with the concentration detected in the body of individuals post fasting and prolonged exercises.     

Essential structure of opioid κ receptor agonist nalfurafine for binding to the κ receptor 2: synthesis of decahydro(iminoethano)phenanthrene derivatives and their pharmacologies

To clarify the essential structures of an opioid κ receptor selective agonist, nalfurafine, for binding to the κ receptor, we designed and synthesized some nalfurafine derivatives and the decahydro(iminoethano)phenanthrene derivatives with a cyclohexene moiety as a surrogate for the phenol ring. In addition to the 6-amide side chain and the 17-nitrogen substituted by a cyclopropylmethyl group, the 4,5-epoxy ring, phenolic hydroxy group, and angular hydroxy group played important roles in eliciting the binding properties of nalfurafine but these three moieties were not indispensable for binding to the κ receptor. Moreover, the phenol ring was also not essential for the binding to the κ receptor, and the cyclohexene moiety would play an important role in fixing the conformation of decahydro(iminoethano)phenanthrene derivatives to effectively raise the amide side chain, rendering a conformation that resembled the active one of nalfurafine.     

The search for a coordinated dialkyl disulphide: The crystal and molecular structure of (μ-ethanedithiolato)bis(tricarbonyliron)(FeFe)

Despite considerable effort, no compounds containing an alkyl disulphide linked to a single metalion have been isolated. The complex [{fFe(SCH₂CH₂- S)₂}₂]²⁻ is a strong reducing agent. [Fe₃(CO)₁₂] with 1,2,5,6-tetrathiacyclooctane yields [(SCH₂CH₂S){Fe- (CO)₃}₂], the structure of which has been determined by X-ray analysis.     

Synthesis and structure–activity relationships of N-aryl-piperidine derivatives as potent (partial) agonists for human histamine H3 receptor

4-((1H-Imidazol-4-yl)methyl)-1-aryl-piperazine and piperidine derivatives were designed and synthesized as candidate human histamine type 3 agonists. The piperazine derivatives were found to have low (or no) affinity for human histamine H3 receptor, whereas the piperidine derivatives showed moderate to high affinity, and their agonistic activity was greatly influenced by substituents on the aromatic ring. Among the piperidine-containing compounds, 17d and 17h were potent human histamine H3 receptor agonists with high selectivity over the closely related human H4 receptor. Our results indicate that appropriate conformational restriction, that is, by the piperidine spacer moiety, favors specific binding to the human histamine H3 receptor.     

The crystal structure of isopenicillin N synthase with δ-((L)-α-aminoadipoyl)-(L)-cysteinyl-(D)-methionine reveals thioether coordination to iron

Isopenicillin N synthase (IPNS) catalyses cyclization of δ-(l-α-aminoadipoyl)-l-cysteinyl-d-valine (ACV) to isopenicillin N (IPN), the central step in penicillin biosynthesis. Previous studies have shown that IPNS turns over a wide range of substrate analogues in which the valine residue of its natural substrate is replaced with other amino acids. IPNS accepts and oxidizes numerous substrates that bear hydrocarbon sidechains in this position, however the enzyme is less tolerant of analogues presenting polar functionality in place of the valinyl isopropyl group. We report a new ACV analogue δ-(l-α-aminoadipoyl)-l-cysteinyl-d-methionine (ACM), which incorporates a thioether in place of the valinyl sidechain. ACM has been synthesized using solution phase methods and crystallized with IPNS. A crystal structure has been elucidated for the IPNS:Fe(II):ACM complex at 1.40? resolution. This structure reveals that ACM binds in the IPNS active site such that the sulfur atom of the methionine thioether binds to iron in the oxygen binding site at a distance of 2.57?. The sulfur of the cysteinyl thiolate sits 2.36? from the metal.     

Which side of the π-macrocycle plane of (bacterio)chlorophylls is favored for binding of the fifth ligand?

Reported crystallographic data and calculated molecular models indicated that chlorophyll (Chl) a and bacteriochlorophyll (BChl) a tend to bind the fifth ligand on the side of the macrocycle where the C13²-(R)-methoxycarbonyl moiety protrudes (denoting the ‘back’ side). The crystal structures of 34 photosynthetic proteins possessing (B)Chl cofactors revealed that most of Chl a and BChl a (and b) are coordinated by any peptidyl residue (e.g., histydyl-imidazolyl group), peptidyl backbone or water from the ‘back’ side. Almost all the cofactors that bind a water molecule as the fifth ligand in these proteins have a ‘back’ configuration. Theoretical model calculations for methyl chlorophyllide a (MeChlid a) and methyl bacteriochlorophyllide a (MeBChlid a) bound to an imidazole molecule indicated that the ‘back’ side is energetically favored for the ligand binding. These results are consistent with the fact that ethyl chlorophyllide a (EtChlid a) dihydrate crystal consists of the ‘back’ complex. The modeling also showed that both removal and stereochemical inverse of the C13²-methoxycarbonyl group affect the relative stability between the ‘back’ and ‘face’ complexes. The effect of the C13²-moiety on the choice of the macrocycle side for the ligand binding is discussed in relation to the function of P700. This revised version was published online in June 2006 with corrections to the Cover Date.     

Solution structure of a synthetic peptide corresponding to a receptor binding region of FSH (hFSH-β 33–53)

The receptor binding surface of human follicle-stimulating hormone (hFSH) is mimicked by synthetic peptides corresponding to the hFSH-β chain amino acid sequences 33–53 [Santa-Coloma, T. A., Dattatreyamurty, D., and Reichert, L. E., Jr. (1990),Biochemistry 29, 1194–1200], 81–95 [Santa-Coloma, T. A., and Reichert, L. E., Jr. (1990),J. Biol. Chem. 265, 5037–5042], and the combined sequence (33–53)–(81–95) [Santa-Coloma, T. A., Crabb, J. W., and Reichert, L. E., Jr. (1991),Mol. Cell. Endocrinol. 78, 197–204]. These peptides have been shown to inhibit binding of hFSH to its receptor. Circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy were used to determine the structure of the first peptide in this series, the 21 amino acid peptide hFSH-β-(33–53), H₂N-YTRDLVYKDPARPKIQKTCTF-COOH. Analysis of CD data indicated the presence of approximately equal amounts of antiparallel β-pleated sheet, turns including a β-turn, “other” structures, and a small amount ofa-helix. The major characteristics of the structure were found to be relatively stable at acidicpH and the predominant effect of increased solvent polarity was a small increase ina-helical content. One- and two-dimensional NMR techniques were used to obtain full proton and carbon signal assignments in aqueous solution atpH 3.1. Analysis of NMR results confirmed the presence of the structural features revealed by CD analysis and provided a detailed picture of the secondary structural elements and global folding pattern in hFSH-β-(33–53). These features included an antiparallel β-sheet (residues 38–51 and 46–48), turns within residues 41–46, and 50–52 (a β-turn) and a small N-terminal helical region comprised of amino acids 34–36. One of the turns is facilitated by prolines 42 and 45. Proline-45 was constrained to thetrans conformation, whereas proline-42 favored thetrans conformer (～70%) over thecis (～30%). Two resonances were observed for the single alanine residue (A-43) sequentially proximal to P-42, but the rest of the structure was minimally affected by the isomerization at proline-42. The major population of molecules, containingtrans-42 andtrans-45 prolines, presented 120 NOEs. Distance geometry calculations with 140 distance constraints and energy minimization refinements were used to derive a moderately well-defined model of the peptide's structure. The hFSH-β-(33–53) structure has a highly polar surface composed of six cationic amino acid (arginie-35, lysine-40, arginine-44, lysine-46, glutamine-48, and lysine-49) and two anionic residues (aspartate-36 and aspartic acid-41). A hydrophobic region in the structure is composed of residues in the antiparallel β-sheet and β-turn which fold to produce a distorted “hairpin.” The structure of this domain, together with the protruding and positively charged region in the vicinity of residues 42–45, may mimic the surface of hFSH that binds to the receptor.