Increases in free, unbound insulin-like growth factor I enhance insulin responsiveness in human hepatoma G2 cells in culture

Insulin-like growth factor-binding protein (IGFBP)-1 binds to insulin-like growth factor (IGF)-I and -II with high affinity and has been shown to modulate IGF-I actions in vivo and in vitro. The synthesis of IGFBP-1 is suppressed by insulin, and administration of IGFBP-1 to rats results in impaired glucose metabolism. A synthetic peptide (bp1-01) has been shown to have a high affinity and specificity for human IGFBP-1 and to inhibit IGF-I binding. The current studies were undertaken to determine if, after incubation of bp1-01 with IGF-I.IGFBP-1 complexes, anabolic and insulin-like effects of IGF-I could be detected in human hepatoma (HepG2) cell cultures and to determine the receptor subtype(s) through which these effects were mediated. Incubation of HepG2 cells with bp1-01 (200 nm) increased IGF-I-stimulated protein synthesis by 44% and glycogen synthesis by 170% compared with stimulation by IGF-I alone. Incubation with bp1-01 also enhanced IGF-I-stimulated tyrosine phosphorylation of the IGF-I/insulin hybrid receptor and insulin receptor substrate 1. Exposure of the cells to bp1-01 alone enhanced glycogen synthesis and phosphorylation of IGF-I/insulin hybrid receptors. This was not a direct effect of bp1-01 because it did not bind to the receptor and did not activate tyrosine kinase activity in the presence of an anti-IGF-I receptor antibody. The addition of bp1-01 (200 nm) plus insulin to HepG2 cell culture medium resulted in increased tyrosine phosphorylation of the hybrid receptor, insulin receptor substrate 1, and the glycogen synthesis response compared with the effects of insulin alone. This enhancement of hybrid receptor phosphorylation and glycogen synthesis by bp1-01 peptide was diminished by preincubation with an inhibitory antibody for the alpha subunit of IGF-I receptor (alphaIR3). bp1-01 stimulated the hybrid receptor phosphorylation response to IGF-I, and this effect was inhibited by prior incubation of the cells with alphaIR3. In conclusion, bp1-01 competes with IGF-I for binding to IGFBP-1, which leads to release of free IGF-I from IGF-I.IGFBP-1 complexes. This released IGF-I stimulates biologic actions that are mediated predominantly through the IGF-I/insulin hybrid receptor.     

Monomeric variants of IL-8: effects of side chain substitutions and solution conditions upon dimer formation.

IL-8 dimers have been observed in NMR and X-ray structures of the protein. We have engineered IL-8 monomers by mutations of residues throughout the dimer interface, which introduce hindrance determinants to dimerization. These IL-8 variants are shown by NMR to have wild-type monomer folding, but by ultracentrifugation to have a range of dimerization constants from microM to mM, as compared with a dimerization constant of about 10 microM for wild-type IL-8, under physiological salt and temperature conditions. The monomeric variants of IL-8 bind the erythrocyte chemokine receptor DARC, as well as the neutrophil IL-8 receptors CXCR1 and CXCR2 with affinities similar to that of wild-type IL-8. In addition, the monomeric variants were shown to have agonist activity, with similar potency to wild-type, in both Ca(2+)-flux assays on CXCR1 and CXCR2 transfected cells, and in chemotaxis assays on neutrophils. Thus, these variants confirm that monomeric IL-8 is functionally equivalent to wild-type in vitro assays. We have also investigated the effects of various solution conditions upon IL-8 dimer formation using analytical ultracentrifugation. At salt concentrations, temperatures, and pH conditions lower than physiological, the dimerization affinity of IL-8 is greatly enhanced. This suggests that, under some conditions, IL-8 dimer formation may occur at concentrations of IL-8 considerably lower than 10 microM, with consequences in vivo that are yet to be determined.     

Mutational analysis and protein engineering of receptor-binding determinants in human placental lactogen

Human placental lactogen (hPL) shares 85% sequence identity to human growth hormone (hGH) yet has some very different receptor-binding properties. For example, hPL binds 2300-fold weaker than hGH to the hGH receptor, yet these two hormones have similar affinities for prolactin receptors. We have expressed hPL in Escherichia coli, and we show that, like hGH, hPL requires zinc for tight binding to the extracellular domain of the human prolactin receptor (hPRLbp). In fact, hPL contains virtually the same receptor-binding determinants and zinc ligands (His-18, His-21, and Glu-174) that hGH uses for coordinating zinc in the hGH.hPRLbp complex. As with hGH, mutation of Glu-174 to Ala in hPL reduces the affinity for the hPRLbp by 1400-fold. We can increase the affinity of hPL by over 200-fold for the hGHbp by installing four hGH receptor determinants that are not conserved in hPL. By simultaneously introducing E174A, we produced a pentamutant whose binding affinity for the hGHbp is only 1.6-fold weaker than hGH, but whose binding affinity for the hPRLbp is weaker by greater than 1000-fold relative to wild-type hPL. Thus, we have identified an hPRLbp epitope in hPL, "recruited" an hGHbp epitope into hPL, and produced receptor selective analogs of hPL that are designed to bind tightly to either, neither, or both receptors. Such variants should be important molecular probes to link specific receptor-binding, activation, and biological events.     

Total alanine-scanning mutagenesis of insulin-like growth factor I (IGF-I) identifies differential binding epitopes for IGFBP-1 and IGFBP-3.

The bioavailability of insulin-like growth factor I (IGF-I) in the serum and tissues is controlled by members of the IGF binding protein family (IGFBP). These proteins form high-affinity complexes with IGF-I and thereby either inhibit or potentiate its mitogenic and metabolic effects. Thus, understanding the IGF-IGFBP interaction at the molecular level is crucial for attempts to modulate IGF-I activity in vivo. We have systematically investigated the binding contribution of each IGF-I amino acid side chain toward IGFBP-1 and IGFBP-3, combining alanine-scanning mutagenesis and monovalent phage display. Surprisingly, most IGF-I residues could be substituted by alanines, resulting in less than 5-fold affinity losses for IGFBP-3. In contrast, binding of IGFBP-1 was more sensitive to alanine substitutions in IGF-I. The glutamate and phenylalanine at positions 3 and 49 were identified as major specificity determinants for IGFBP-1: the corresponding alanine mutations, E3A and F49A, selectively decreased IGFBP-1 binding by 34- and 100-fold, whereas IGFBP-3 affinity was not affected or reduced maximally 4-fold. No side chain specificity determinant was found for IGFBP-3. Instead, our results suggest that the N-terminal backbone region of IGF-I is important for binding to IGFBP-3. The fact that the functional binding epitopes on IGF-I are overlapping but distinct for both binding proteins may be exploited to design binding protein-specific IGF variants.     

Maternal Genes and Facial Clefts in Offspring: A Comprehensive Search for Genetic Associations in Two Population-Based Cleft Studies from Scandinavia

Background

Fetal conditions can in principle be affected by the mother''s genotype working through the prenatal environment.

Methodology/Principal Findings

Genotypes for 1536 SNPs in 357 cleft candidate genes were available from a previous analysis in which we focused on fetal gene effects [1]. After data-cleaning, genotypes for 1315 SNPs in 334 autosomal genes were available for the current analysis of maternal gene effects. Two complementary statistical methods, TRIMM and HAPLIN, were used to detect multi-marker effects in population-based samples from Norway (562 case-parent and 592 control-parent triads) and Denmark (235 case-parent triads). We analyzed isolated cleft lip with or without cleft palate (iCL/P) and isolated cleft palate only (iCP) separately and assessed replication by looking for genes detected in both populations by both methods. In iCL/P, neither TRIMM nor HAPLIN detected more genes than expected by chance alone; furthermore, the selected genes were not replicated across the two methods. In iCP, however, FLNB was identified by both methods in both populations. Although HIC1 and ZNF189 did not fully satisfy our stringency criterion for replication, they were strongly associated with iCP in TRIMM analyses of the Norwegian triads.

Conclusion/Significance

Except for FLNB, HIC1 and ZNF189, maternal genes did not appear to influence the risk of clefting in our data. This is consistent with recent epidemiological findings showing no apparent difference between mother-to-offspring and father-to-offspring recurrence of clefts in these two populations. It is likely that fetal genes make the major genetic contribution to clefting risk in these populations, but we cannot rule out the possibility that maternal genes can affect risk through interactions with specific teratogens or fetal genes.     

Structure of a CXC chemokine-receptor fragment in complex with interleukin-8.

BACKGROUND: Interactions between CXC chemokines (e.g. interleukin-8, IL-8) and their receptors (e.g. CXCR-1) have a key role in host defense and disease by attracting and upregulating neutrophils to sites of inflammation. The transmembrane nature of the receptor impedes structure-based understanding of ligand interactions. Linear peptides based on the N-terminal, extracellular portion of the receptor CXCR-1 do bind to IL-8, however, and inhibit the binding of IL-8 to the full-length receptor. RESULTS: The NMR solution structure of the complex formed between IL-8 and one such receptor-based peptide indicates that a cleft between a loop and a beta hairpin constitute part of the receptor interaction surface on IL-8. Nine residues from the C terminus of the receptor peptide (corresponding to Pro21-Pro29 of CXCR-1) occupy the cleft in an extended fashion. Intermolecular contacts are mostly hydrophobic and sidechain mediated. CONCLUSIONS: The results offer the first details at an atomic level of the interaction between a chemokine and its receptor. Consideration of other biochemical data allow extrapolation to a model for the interaction of IL-8 with the full-length receptor. In this model, the heparin-binding residues of IL-8 are exposed, thereby allowing presentation of the chemokine from endothelial cell-surface glycosaminoglycans. This first glimpse of how IL-8 binds to its receptor provides a foundation for the structure-based design of chemokine antagonists.     

Haplotype-based genomic sequencing of a chromosomal polymorphism in the white-throated sparrow (Zonotrichia albicollis)

Inversion polymorphisms have been linked to a variety of fundamental biological and evolutionary processes. Yet few studies have used large-scale genomic sequencing to directly compare the haplotypes associated with the standard and inverted chromosome arrangements. Here we describe the targeted genomic sequencing and comparison of haplotypes representing alternative arrangements of a common inversion polymorphism linked to a suite of phenotypes in the white-throated sparrow (Zonotrichia albicollis). More than 7.4 Mb of genomic sequence was generated and assembled from both the standard (ZAL2) and inverted (ZAL2(m)) arrangements. Sequencing of a pair of inversion breakpoints led to the identification of a ZAL2-specific segmental duplication, as well as evidence of breakpoint reusage. Comparison of the haplotype-based sequence assemblies revealed low genetic differentiation outside versus inside the inversion indicative of historical patterns of gene flow and suppressed recombination between ZAL2 and ZAL2(m). Finally, despite ZAL2(m) being maintained in a near constant state of heterozygosity, no signatures of genetic degeneration were detected on this chromosome. Overall, these results provide important insights into the genomic attributes of an inversion polymorphism linked to mate choice and variation in social behavior.     

The Mnn2 Mannosyltransferase Family Modulates Mannoprotein Fibril Length,Immune Recognition and Virulence of Candida albicans

The fungal cell wall is the first point of interaction between an invading fungal pathogen and the host immune system. The outer layer of the cell wall is comprised of GPI anchored proteins, which are post-translationally modified by both N- and O-linked glycans. These glycans are important pathogen associated molecular patterns (PAMPs) recognised by the innate immune system. Glycan synthesis is mediated by a series of glycosyl transferases, located in the endoplasmic reticulum and Golgi apparatus. Mnn2 is responsible for the addition of the initial α1,2-mannose residue onto the α1,6-mannose backbone, forming the N-mannan outer chain branches. In Candida albicans, the MNN2 gene family is comprised of six members (MNN2, MNN21, MNN22, MNN23, MNN24 and MNN26). Using a series of single, double, triple, quintuple and sextuple mutants, we show, for the first time, that addition of α1,2-mannose is required for stabilisation of the α1,6-mannose backbone and hence regulates mannan fibril length. Sequential deletion of members of the MNN2 gene family resulted in the synthesis of lower molecular weight, less complex and more uniform N-glycans, with the sextuple mutant displaying only un-substituted α1,6-mannose. TEM images confirmed that the sextuple mutant was completely devoid of the outer mannan fibril layer, while deletion of two MNN2 orthologues resulted in short mannan fibrils. These changes in cell wall architecture correlated with decreased proinflammatory cytokine induction from monocytes and a decrease in fungal virulence in two animal models. Therefore, α1,2-mannose of N-mannan is important for both immune recognition and virulence of C. albicans.     

TIPRL Inhibits Protein Phosphatase 4 Activity and Promotes H2AX Phosphorylation in the DNA Damage Response

Despite advances in our understanding of protein kinase regulation in the DNA damage response, the mechanism that controls protein phosphatase activity in this pathway is unclear. Unlike kinases, the activity and specificity of serine/threonine phosphatases is governed largely by their associated proteins. Here we show that Tip41-like protein (TIPRL), an evolutionarily conserved binding protein for PP2A-family phosphatases, is a negative regulator of protein phosphatase 4 (PP4). Knockdown of TIPRL resulted in increased PP4 phosphatase activity and formation of the active PP4-C/PP4R2 complex known to dephosphorylate γ-H2AX. Thus, overexpression of TIPRL promotes phosphorylation of H2AX, and increases γ-H2AX positive foci in response to DNA damage, whereas knockdown of TIPRL inhibits γ-H2AX phosphorylation. In correlation with γ-H2AX levels, we found that TIPRL overexpression promotes cell death in response to genotoxic stress, and knockdown of TIPRL protects cells from genotoxic agents. Taken together, these data demonstrate that TIPRL inhibits PP4 activity to allow for H2AX phosphorylation and the subsequent DNA damage response.     

High-level expression of the simian virus 40 genes LP1, VP1 and VP2 as fusion proteins in Escherichia coli

The complete sequences of the SV40 agnogene (LP1) and the genes coding for the capsid proteins VP1 and VP2 have been cloned into Escherichia coli expression plasmids. High levels of expression were obtained when the SV40 genes were inserted into the coding sequence of the influenza virus NS1 gene, which has previously been expressed in E. coli. The NS1A-LP1 and NS1A-VP2 chimeric proteins consist of the 81 N-terminal residues of NS1 (designated as peptide NS1A) fused to the complete sequence of the corresponding SV40 protein. The NS1A-VP1 chimera consists of NS1A followed by a linker of nine arbitrary residues and the complete sequence of the SV40 major capsid protein. The observed levels of expression vary considerably among the three chimeric proteins, ranging from approx. 70 micrograms/ml in the case of NS1A-LP1 to approx. 5 micrograms/ml in the case of NS1A-VP2. Cyanogen bromide cleavage of the NS1A-LP1 fusion protein produces fragments with Mrs expected for isolated NS1A and LP1 peptides. A plasmid has also been constructed which expresses the NS1A peptide in high yield.