Greater replication and differentiation of preadipocytes in inherited corticosteroid-binding globulin deficiency

Glucocorticoids are pivotal for adipose tissue development. Rodent studies suggest that corticosteroid-binding globulin (CBG) modulates glucocorticoid action in adipose tissue. In humans, both genetic CBG deficiency and suppressed CBG concentrations in hyperinsulinemic states are associated with obesity. We hypothesized that CBG deficiency in humans modulates the response of human preadipocytes to glucocorticoids, predisposing them to obesity. We compared normal preadipocytes with subcultured preadipocytes from an individual with the first ever described complete deficiency of CBG due to a homozygous null mutation. CBG-negative preadipocytes proliferated more rapidly and showed greater peroxisome proliferator-activated receptor-gamma-mediated differentiation than normal preadipocytes. CBG was not expressed in normal human preadipocytes. Glucocorticoid receptor number and binding characteristics and 11beta-hydroxysteroid dehydrogenase activity were similar for CBG-negative and normal preadipocytes. We propose that the increased proliferation and enhanced differentiation of CBG-negative preadipocytes may promote adipose tissue deposition and explain the obesity seen in individuals with genetic CBG deficiency. Furthermore, these observations may be relevant to obesity occurring with suppressed CBG concentrations associated with hyperinsulinemia.

Crystal structures of fusion proteins with large-affinity tags

The fusion of a protein of interest to a large-affinity tag, such as the maltose-binding protein (MBP), thioredoxin (TRX), or glutathione-S-transferase (GST), can be advantageous in terms of increased expression, enhanced solubility, protection from proteolysis, improved folding, and protein purification via affinity chromatography. Unfortunately, crystal growth is hindered by the conformational heterogeneity induced by the fusion tag, requiring that the tag is removed by a potentially problematic cleavage step. The first three crystal structures of fusion proteins with large-affinity tags have been reported recently. All three structures used a novel strategy to rigidly fuse the protein of interest to MBP via a short three- to five-amino acid spacer. This strategy has the potential to aid structure determination of proteins that present particular experimental challenges and are not conducive to more conventional crystallization strategies (e.g., membrane proteins). Structural genomics initiatives may also benefit from this approach as a way to crystallize problematic proteins of significant interest.

Quantitative description of the interaction between folate and the folate-binding protein from cow's milk

A detailed study has been carried out on the dependence of folate binding on the concentration of FBP (folate-binding protein) at pH 5.0, conditions selected to prevent complications arising from the pre-existing self-association of the acceptor. In contrast with the mandatory requirement that reversible interaction of ligand with a single acceptor site should exhibit a unique, rectangular hyperbolic binding curve, results obtained by ultrafiltration for the FBP-folate system required description in terms of (i) a sigmoidal relationship between concentrations of bound and free folate and (ii) an inverse dependence of affinity on FBP concentration. These findings have been attributed to the difficulties in determining the free ligand concentration in the FBP-folate mixtures for which reaction is essentially stoichiometric. This explanation also accounts for the similar published behaviour of the FBP-folate system at neutral pH, which had been attributed erroneously to acceptor self-association, a phenomenon incompatible with the experimental findings because of its prediction of a greater affinity for folate with increasing FBP concentration.

Increased site 1 affinity improves biopotency of porcine growth hormone - Evidence against diffusion dependent receptor dimerization

Based on phage display optimization studies with human growth hormone (GH), it is thought that the biopotency of GH cannot be increased. This is proposed to be a result of the affinity of the first receptor for hormone far exceeding that which is required to trap the hormone long enough to allow diffusion of the second receptor to form the ternary complex, which initiates signaling. We report here that despite similar site 1 kinetics to the hGH/hGH receptor interaction, the potency of porcine GH for its receptor can be increased up to 5-fold by substituting hGH residues involved in site 1 binding into pGH. Based on extensive mutations and BIAcore studies, we show that the higher potency and site 1 affinity of hGH for the pGHR is primarily a result of a decreased off-rate associated with residues in the extended loop between helices 1 and 2 that interact with the two key tryptophans Trp(104) and Trp(169) in the receptor binding hot spot. Our mutagenic analysis has also identified a second determinant (Lys(165)), which in addition to His(169), restricts the ability of non-primate hormones to activate hGH receptor. The increased biopotency of GH that we observe can be explained by a model for GH receptor activation where subunit alignment is critical for effective signaling.

Common E protein determinants for attenuation of glycosaminoglycan-binding variants of Japanese encephalitis and West Nile viruses

Natural isolates and laboratory strains of West Nile virus (WNV) and Japanese encephalitis virus (JEV) were attenuated for neuroinvasiveness in mouse models for flavivirus encephalitis by serial passage in human adenocarcinoma (SW13) cells. The passage variants displayed a small-plaque phenotype, augmented affinity for heparin-Sepharose, and a marked increase in specific infectivity for SW13 cells relative to the respective parental viruses, while the specific infectivity for Vero cells was not altered. Therefore, host cell adaptation of passage variants was most likely a consequence of altered receptor usage for virus attachment-entry with the involvement of cell surface glycosaminoglycans (GAG) in this process. In vivo blood clearance kinetics of the passage variants was markedly faster and viremia was reduced relative to the parental viruses, suggesting that affinity for GAG (ubiquitously present on cell surfaces and extracellular matrices) is a key determinant for the neuroinvasiveness of encephalitic flaviviruses. A difference in pathogenesis between WNV and JEV, which was reflected in more efficient growth in the spleen and liver of the WNV parent and passage variants, accounted for a less pronounced loss of neuroinvasiveness of GAG binding variants of WNV than JEV. Single gain-of-net-positive-charge amino acid changes at E protein residue 49, 138, 306, or 389/390, putatively positioned in two clusters on the virion surface, define molecular determinants for GAG binding and concomitant virulence attenuation that are shared by the JEV serotype flaviviruses.

Association between chronic fatigue syndrome and the corticosteroid-binding globulin gene ALA SER224 polymorphism

Chronic fatigue syndrome (CFS) is characterized by idiopathic fatigue of greater than 6 months' duration with postexertional exacerbation and many other symptoms. A trend toward relative hypocortisolism is described in CFS. Twin and family studies indicate a substantial genetic etiologic component to CFS. Recently, severe corticosteroid-binding globulin (CBG) gene mutations have been associated with CFS in isolated kindreds. Human leukocyte elastase, an enzyme important in CBG catabolism at inflammatory sites, is reported to be elevated in CFS. We hypothesized that CBG gene polymorphisms may act as a genetic risk factor for CFS. A total of 248 patients with CFS defined by Centers for Disease Control criteria, and 248 controls were recruited. Sequencing and restriction enzyme testing of the CBG gene coding region allowed detection of severe CBG gene mutations and a common exon 3 polymorphism (c.825G --> T, Ala-Ser(224)). Plasma CBG levels were measured in 125 CFS patients and 198 controls by radioimmunoassay. Total and free (calculated and measured) cortisol levels were ascertained in single samples between 8-10 a.m. The age of onset (mid 30s) and gender ratio (2.2:1, female:male) of the patients were similar to those reported in U.S. epidemiologic studies. A trend toward a preponderance of serine(224) homozygosity among the CFS patients was noted, compared with controls (chi(2) = 5.31, P = 0.07). Immunoreactive-CBG (IR-CBG) levels were higher in Serine/Alanine (Ser/Ala) than Ala/Ala subjects and higher again in Ser/Ser subjects, this effect was strongest in controls; Ser/Ser: 46.1 +/- 1.8 (n = 31, P = 0.03) vs. Ser/Ala: 42.4 +/- 1.0 (n = 56, P = 0.05) vs. Ala/Ala: 40.8 +/- 1.7 mug/mL (n = 21). Despite higher CBG levels, there was a nonsignificant trend toward lower total and free plasma cortisol in serine allele positive patients, total cortisol: Ser/Ser: 13.3 +/- 1.4 (n = 34) vs. Ser/Ala: 14.0 +/- 0.7 (n = 66) vs. Ala/Ala: 15.4 +/- 1.0 (n = 23). Homozygosity for the serine allele of the CBG gene may predispose to CFS, perhaps due to an effect on hypothalamic-pituitary-adrenal axis function related to altered CBG-cortisol transport function or immune-cortisol interactions.

Concurrent binding of anti-EphA3 antibody and ephrin-A5 amplifies EphA3 signaling and downstream responses: Potential as EphA3-specific tumor-targeting reagents

The Eph receptor tyrosine kinases and their membrane-bound ephrin ligands form a unique cell-cell contact-mediated system for controlling cell localization and organization. Their high expression in a wide variety of human tumors indicates a role in tumor progression, and relatively low Eph and ephrin levels in normal tissues make these proteins potential targets for anticancer therapies. The monoclonal antibody IIIA4, previously used to isolate EphA3, binds with subnanomolar affinity to a conformation-specific epitope within the ephrin-binding domain that is closely adjacent to the low-affinity ephrin-A5 heterotetramerization site. We show that similar to ephrin-A5, preclustered IIIA4 effectively triggers EphA3 activation, contraction of the cytoskeleton, and cell rounding. BIAcore analysis, immunoblot, and confocal microscopy of wild-type and mutant EphA3 with compromised ephrin-A5 or IIIA4-binding capacities indicate that IIIA4 binding triggers an EphA3 conformation which is permissive for the assembly of EphA3/ephrin-A5-type signaling clusters. Furthermore, unclustered IIIA4 and ephrin-A5 Fc applied in combination initiate greatly enhanced EphA3 signaling. Radiometal conjugates of ephrin-A5 and IIIA4 retain their affinity, and in mouse xenografts localize to, and are internalized rapidly into EphA3-positive, human tumors. These findings show the biological importance of EphA3/ ephrin-A5 interactions and that ephrin-A5 and IIIA4 have great potential as tumor targeting reagents.

beta 2 subunit contribution to 4/7 alpha-conotoxin binding to the nicotinic acetylcholine receptor

The structures of acetylcholine-binding protein ( AChBP) and nicotinic acetylcholine receptor ( nAChR) homology models have been used to interpret data from mutagenesis experiments at the nAChR. However, little is known about AChBP-derived structures as predictive tools. Molecular surface analysis of nAChR models has revealed a conserved cleft as the likely binding site for the 4/7 alpha-conotoxins. Here, we used an alpha 3 beta 2 model to identify beta 2 subunit residues in this cleft and investigated their influence on the binding of alpha-conotoxins MII, PnIA, and GID to the alpha 3 beta 2 nAChR by two-electrode voltage clamp analysis. Although a beta 2-L119Q mutation strongly reduced the affinity of all three alpha-conotoxins, beta 2-F117A, beta 2-V109A, and beta 2-V109G mutations selectively enhanced the binding of MII and GID. An increased activity of alpha-conotoxins GID and MII was also observed when the beta 2-F117A mutant was combined with the alpha 4 instead of the alpha 3 subunit. Investigation of A10L-PnIA indicated that high affinity binding to beta 2-F117A, beta 2-V109A, and beta 2-V109G mutants was conferred by amino acids with a long side chain in position 10 (PnIA numbering). Docking simulations of 4/7 alpha-conotoxin binding to the alpha 3 beta 2 model supported a direct interaction between mutated nAChR residues and alpha-conotoxin residues 6, 7, and 10. Taken together, these data provide evidence that the beta subunit contributes to alpha-conotoxin binding and selectivity and demonstrate that a small cleft leading to the agonist binding site is targeted by alpha-conotoxins to block the nAChR.

Artificial neural network modeling of phytoestrogen binding to estrogen receptors

Differential pathophysiological roles of estrogen receptors alpha (ERα) and beta (ERβ) are of particular interest for phytochemical screening. A QSAR incorporating theoretical descriptors was developed in the present study utilizing sequential multiple-output artificial neural networks. Significant steric, constitutional, topological and electronic descriptors were identified enabling ER affinity differentiation.

Comparison of the binding of 3-fluoromethyl-7-sulfonyl-1,2,3,4-tetrahydroisoquinolines with their isosteric sulfonamides to the active site of phenylethanolamine N-methyltransferase

3-Fluoromethyl-7-(N-substituted aminosulfonyl)-1,2,3,4-tetrahydroisoquinolines (14, 16, and 18-22) are highly potent and selective inhibitors of phenylethanolamine N-methyltransferase (PNMT). Molecular modeling studies with 3-fluoromethyl-7-(N-alkyl aminosulfonyl)-1,2,3,4-tetrahydroisoquinolines, such as 16, suggested that the sulfonamide -NH-could form a hydrogen bond with the side chain of Lys57. However, SAR studies and analysis of the crystal structure of human PNMT (hPNMT) in complex with 7 indicated that the sulfonamide oxygens, and not the sulfonamide -NH-, formed favorable interactions with the enzyme. Thus, we hypothesized that replacement of the sulfonamide -NH-with a methylene group could result in compounds that would retain potency at PNMT and that would have increased lipophilicity, thus increasing the likelihood they will cross the blood brain barrier. A series of 3-fluoromethyl-7-sulfonyl-1,2,3,4-tetrahydroisoquinolines (23-30) were synthesized and evaluated for their PNMT inhibitory potency and affinity for the R2-adrenoceptor. A comparison of these compounds with their isosteric sulfonamides (14, 16, and 18-22) showed that the sulfones were more lipophilic but less potent than their corresponding sulfonamides. Sulfone 24 (hPNMT K-i = 1.3 mu M) is the most potent compound in this series and is quite selective for PNMT versus the R2-adrenoceptor, but 24 is less potent than the corresponding sulfonamide, 16 (hPNMT K-i = 0.13 mu M). We also report the crystal structure of hPNMT in complex with sulfonamide 15, from which a potential hydrogen bond acceptor within the hPNMT active site has been identified, the main chain carbonyl oxygen of Asn39. The interaction of this residue with the sulfonamide -NH-is likely responsible for much of the enhanced inhibitory potency of the sulfonamides versus the sulfones.

Prediction of HLA-DQ3.2 beta ligands: evidence of multiple registers in class II binding peptides

Motivation: While processing of MHC class II antigens for presentation to helper T-cells is essential for normal immune response, it is also implicated in the pathogenesis of autoimmune disorders and hypersensitivity reactions. Sequence-based computational techniques for predicting HLA-DQ binding peptides have encountered limited success, with few prediction techniques developed using three-dimensional models. Methods: We describe a structure-based prediction model for modeling peptide-DQ3.2 beta complexes. We have developed a rapid and accurate protocol for docking candidate peptides into the DQ3.2 beta receptor and a scoring function to discriminate binders from the background. The scoring function was rigorously trained, tested and validated using experimentally verified DQ3.2 beta binding and non-binding peptides obtained from biochemical and functional studies. Results: Our model predicts DQ3.2 beta binding peptides with high accuracy [area under the receiver operating characteristic (ROC) curve A(ROC) > 0.90], compared with experimental data. We investigated the binding patterns of DQ3.2 beta peptides and illustrate that several registers exist within a candidate binding peptide. Further analysis reveals that peptides with multiple registers occur predominantly for high-affinity binders.

Site-specific labelling of proteins with a rigid lanthanide-binding tag

This paper describes a generic method for the site-specific attachment of lathanide complexes to proteins through a disulfide bond. The method is demonstrated by the attachment of a lanthanide-binding peptide tag to the single cysteine residue present in the N-terminal DNA-binding domain of the Echerichia coli arginine repressor. Complexes with Y3+, Tb3+, Dy3+, Ho3+, Er3+, Tm3+ and Yb3+ ions were formed and analysed by NMR spectroscopy. Large pseudocontact shifts and residual dipolar couplings were induced by the lanthanide-binding tag in the protein NMR spectrum, a result indicating that the tag was rigidly attached to the protein. The axial components of the magnetic susceptibility anisostropy tensors determined for the different lanthanide ions were similarly but not identically oriented. A single tag with a single protein attachment site can provide different pseudocontact shifts from different magnetic susceptibility tensors and thus provide valuable nondegenerate long-range structure information in the determination of 3D protein structures by NMR spectroscopy.

Insights to substrate binding and processing by West Nile Virus NS3 protease through combined modeling, protease mutagenesis, and kinetic studies

West Nile Virus is becoming a widespread pathogen, infecting people on at least four continents with no effective treatment for these infections or many of their associated pathologies. A key enzyme that is essential for viral replication is the viral protease NS2B-NS3, which is highly conserved among all flaviviruses. Using a combination of molecular fitting of substrates to the active site of the crystal structure of NS3,site-directed enzyme and cofactor mutagenesis, and kinetic studies on proteolytic processing of panels of short peptide substrates, we have identified important enzyme-substrate interactions that define substrate specificity for NS3 protease. In addition to better understanding the involvement of S2, S3, and S4 enzyme residues in substrate binding, a residue within cofactor NS2B has been found to strongly influence the preference of flavivirus proteases for lysine or arginine at P2 in substrates. Optimization of tetrapeptide substrates for enhanced protease affinity and processing efficiency has also provided important clues for developing inhibitors of West Nile Virus infection.

theta-defensins prevent HIV-1 Env-mediated fusion by binding gp41 and blocking 6-helix bundle formation

Retrocyclin-1, a 0-defensin, protects target cells from human immunodeficiency virus, type 1 (HIV-1) by preventing viral entry. To delineate its mechanism, we conducted fusion assays between susceptible target cells and effector cells that expressed HIV-1 Env. Retrocyclin-1 (4 mu M) completely blocked fusion mediated by HIV-1 Envs that used CXCR4 or CCR5 but had little effect on cell fusion mediated by HIV-2 and simian immunodeficiency virus Envs. Retrocyclin-1 inhibited HIV-1 Env-mediated fusion without impairing the lateral mobility of CD4, and it inhibited the fusion of CD4-deficient cells with cells bearing CD4-independent HIV-1 Env. Thus, it could act without cross-linking membrane proteins or inhibiting gp120-CD4 interactions. Retrocyclin-1 acted late in the HIV-1 Env fusion cascade but prior to 6-helix bundle formation. Surface plasmon resonance experiments revealed that retrocyclin bound the ectodomain of gp41 with high affinity in a glycan-independent manner and that it bound selectively to the gp41 C-terminal heptad repeat. Native-PAGE, enzyme-linked immunosorbent assay, and CD spectroscopic analyses all revealed that retrocyclin-1 prevented 6-helix bundle formation. This mode of action, although novel for an innate effector molecule, resembles the mechanism of peptidic entry inhibitors based on portions of the gp41 sequence.