Identification of novel alleles associated with insulin resistance in childhood obesity using pooled-DNA genome-wide association study approach.

BACKGROUND: Recently, we witnessed great progress in the discovery of genetic variants associated with obesity and type 2 diabetes (T2D), especially in adults. Much less is known regarding genetic variants associated with insulin resistance (IR). We hypothesized that novel IR genes could be efficiently detected in a population of obese children and adolescents who may not exhibit comorbidities and other confounding factors. OBJECTIVES: This study aimed to determine whether a genome-wide association study (GWAS), using a DNA-pooling approach, could identify novel genes associated with IR. SUBJECTS: The pooled-DNA GWAS analysis included Slovenian obese children and adolescents with and without IR matched for body mass index, gender and age. A replication study was conducted in another independent cohort with or without IR. METHODS: For the pooled-DNA GWAS, we used HumanOmni5-Quad SNP array (Illumina). Allele frequency distributions were compared with modified t-tests and χ2-tests and ranked using PLINK. Top single nucleotide polymorphisms (SNPs) were validated using individual genotyping by high-resolution melting analysis and TaqMan assay. RESULTS: We identified five top-ranking SNPs from the pooled-DNA GWAS analysis within the ECE1, IL1R2, GNPDA1, HLA-J and PYGB loci. All except SNP rs9261108 (HLA-J locus) were confirmed in the validation phase using individual genotyping. The SNP rs2258617 within PYGB remained statistically significant for both recessive and additive models in both cohorts and in a merged analysis of both cohorts and present the strongest novel candidate gene for IR. CONCLUSION: We report for the first time a pooled-DNA GWAS approach to identify five novel SNPs or genes for IR in a paediatric population. The four loci confirmed in the second validation phase study warrant further studies, especially the strongest SNP rs2258617 within PYGB, and provide targets for further basic research of IR mechanisms and for the development of potential new IR and T2D therapies.

Designing the structure and folding pathway of modular topological bionanostructures.

Polypeptides and polynucleotides are programmable natural polymers whose linear sequence can be easily designed and synthesized by the cellular transcription/translation machinery. Nature primarily uses proteins as the molecular machines and nucleic acids as the medium for the manipulation of heritable information. A protein's tertiary structure and function is defined by multiple cooperative weak long-range interactions that have been optimized through evolution. DNA nanotechnology uses orthogonal pairwise interacting modules of complementary nucleic acids as a strategy to construct defined complex 3D structures. A similar approach has recently been applied to protein design, using orthogonal dimerizing coiled-coil segments as interacting modules. When concatenated into a single polypeptide chain, they self-assemble into the 3D structure defined by the topology of interacting modules within the chain. This approach allows the construction of geometric polypeptide scaffolds, bypassing the folding problem of compact proteins by relying on decoupled pairwise interactions. However, the folding pathway still needs to be optimized in order to allow rapid self-assembly under physiological conditions. Again the modularity of designed topological structures can be used to define the rules that guide the folding pathway of long polymers, such as DNA, based on the stability and topology of connected building modules. This approach opens the way towards incorporation of designed foldamers in biological systems and their functionalization.

Surface with antimicrobial activity obtained through silane coating with covalently bound polymyxin B.

Surfaces exhibiting antimicrobial activity were prepared for potential medical application. A polycationic lipopeptide polymyxin B was selected as the bioactive agent for covalent immobilization onto the surface. First, by using sol-gel technology the inert glass substrate was functionalized by a silane coating with epoxide rings to which the peptide was coupled by means of a catalyst. Preparation of the coating and presence of the peptide on the surface were followed by FTIR, XPS and AFM analyses. The obtained material showed antimicrobial effect indicating that in spite of immobilization the peptide has retained its bioactivity. The coated surface was able to reduce bacterial cell counts of the Gram-negative bacterium Escherichia coli by more than five orders of magnitude in 24 h of incubation. It can be concluded that bioactive coatings with covalently bound polycationic peptides have potential for application on medical devices where leakage into the surrounding is not allowed in order to prevent bacterial growth and biofilm formation.

Recognition of nucleic acids by Toll-like receptors and development of immunomodulatory drugs.

Microbial as well as endogenous nucleic acids are recognized by a group of endosomal Toll-like receptors TLR3, TLR7, TLR8 and TLR9. Recent discoveries significantly improved our understanding of molecular mechanism of their activation and their physiological role. Those include recognition of dsRNA through two nucleic acid binding sites of TLR3 ectodomain, activation of TLR9 by phosphodiester backbone of ssDNA, independent of the nucleotide sequence and phosphorothioate modified bonds, and the role of proteolysis in activation of TLR9. In addition, proteins that chaperone nucleic acids, such as HMGB1 or LL-37, have been described to mediate TLR activation. There is growing evidence that supports involvement of endosomal TLRs in a number of autoimmune diseases, suggesting a therapeutic potential of immunomodulatory endosomal TLR ligands. So far, inhibitory nucleic acids against TLR7, TLR8 and TLR9 as well as small compounds targeting downstream signal transduction of single or several endosomal TLRs have been reported. TLR-targeting drugs have been included in clinical trials as vaccine adjuvants or as therapeutic agents for the treatment of diseases, ranging from cancer, infections, asthma and allergy to autoimmune diseases.

Three-dimensional domain swapping in the folded and molten-globule states of cystatins, an amyloid-forming structural superfamily.

Cystatins, an amyloid-forming structural superfamily, form highly stable, domain-swapped dimers at physiological protein concentrations. In chicken cystatin, the active monomer is a kinetic trap en route to dimerization, and any changes in solution conditions or mutations that destabilize the folded state shorten the lifetime of the monomeric form. In such circumstances, amyloidogenesis will start from conditions where a domain-swapped dimer is the most prevalent species. Domain swapping occurs by a rearrangement of loop I, generating the new intermonomer interface between strands 2 and 3. The transition state for dimerization has a high level of hydrophobic group exposure, indicating that gross conformational perturbation is required for domain swapping to occur. Dimerization also occurs when chicken cystatin is in its reduced, molten-globule state, implying that the organization of secondary structure in this state mirrors that in the folded state and that domain swapping is not limited to the folded states of proteins. Although the interface between cystatin-fold units is poorly defined for cystatin A, the dimers are the appropriate size to account for the electron-dense regions in amyloid protofilaments.

Major differences in stability and dimerization properties of two chimeric mutants of human stefins.

Stefins A and B are cysteine proteinase inhibitors that have considerable sequence similarity but marked differences in their stability and folding properties. Two chimeric proteins were designed to shed light on these differences. The chimeric mutants have been expressed in Escherichia coli and have been isolated. The first, A37B, consists of 37 residues of stefin A, comprising the N-terminal and the alpha-helix, joined to 61 residues of stefin B; the second, A61B, consists of 61 N-terminal residues of stefin A, followed by 37 residues of stefin B. Spectroscopic properties of the chimeric proteins (absorption, CD, and NMR spectra), together with activity measurements, have confirmed that both have well-defined tertiary structure and are active as cysteine proteinase inhibitors. Characterization consisted of GuHCl denaturation, ANS binding as a function of pH, and monitoring of dimerization under partially denaturing conditions. The c(m) values are 1.3 M GuHCl for A61B as compared with 2.7 M GuHCl for stefin A, and 2.1 M GuHCl for A37B as compared with 1.4 M GuHCl for stefin B (all at pH 7.5, 25 degrees C). However (G degrees (N-U) is lower for both chimeric proteins (18 +/- 3 kJ/mol) than for the parent stefins (28 +/- 3 kJ/mol). In pH denaturation, unlike stefin B, neither chimeric mutant unfolds to I(N) below pH 5.4. At pH 3, where stefin B forms a molten globule and stefin A is native, both A37B and A61B show increased ANS fluorescence and aggregate visibly. Dimers at pre-denaturation conditions are observed in all the proteins under study, but they remain "trapped" only in stefin A.

Production of stable isotope enriched antimicrobial peptides in Escherichia coli: an application to the production of a 15N-enriched fragment of lactoferrin.

A method is described for the production of recombinant isotopically enriched peptides in E. coli. Peptides are produced in high yield as fusion proteins with ketosteroid isomerase which form insoluble inclusion bodies. This insoluble form allows easy purification, stabilizes the peptide against degradation and prevents bactericidal activity of the peptide. Cyanogen bromide cleavage released peptide which was conjugated with alkylamines to form lipopeptide. An important advantage of this system is that it allows production of peptides that are toxic to bacteria, which we have demonstrated on a dodecapeptide based on residues 21-31 of human bactericidal protein lactoferrin.

Bacterial expression and refolding of different fragments of human CD14.

We have investigated bacterial expression of several fragments of CD14, a human cellular receptor for lipopolysaccharides (LPS). Despite binding of CD14 to the LPS, a vital constituent of bacterial outer membrane, we have succeeded in producing full length recombinant hCD14 in E. coli. High level of production of CD14 resulted in deposits of aggregated CD14 in bacteria in form of inclusion bodies, which made production of this protein possible. We have also produced N-terminal fragments consisting of 134 and 152 residues, which comprise N-terminal domain with 2 and 3 leucine rich repeats, respectively and a fragment that contains only leucine rich repeats. Production of the N-terminal domain consisting of 69 residues could not be detected, probably due to the degradation of the produced protein within the bacteria. Full length CD14 and a fragment of 152 residues from inclusion bodies were refolded, while the 134 residues fragment and the one with 10 leucine-rich repeats could not be refolded. Those results confirm that the minimal folding unit of CD14 must include N-terminal domain and at least 3 leucine rich repeats.

Accessing the global minimum conformation of stefin A dimer by annealing under partially denaturing conditions.

Stefin A folds as a monomer under strongly native conditions. We have observed that under partially denaturing conditions in the temperature range from 74 to 93 degrees C it folds into a dimer, while it is monomeric above the melting temperature of 95 degrees C. Below 74 degrees C the dimer is trapped and it does not dissociate. The dimer is a folded and structured protein as judged by CD and NMR, nevertheless it is no more functional as an inhibitor of cysteine proteases. The monomer-dimer transition proceeds at a slow rate and the activation energy of dimerization at 99 kcal/mol is comparable to the unfolding enthalpy. A large and negative dimerization enthalpy of -111(+/- 8) kcal/mol was calculated from the temperature dependence of the dissociation constant. An irreversible pretransition at 10-15 deg. below the global unfolding temperature has been observed previously by DSC and can now be assigned to the monomer-dimer transition. Backbone resonances of all the dimer residues were assigned using 15N isotopically enriched protein. The dimer is symmetric and the chemical shift differences between the monomer and dimer are localized around the tripartite hydrophobic wedge, which otherwise interacts with cysteine proteases. Hydrogen exchange protection factors of the residues affected by dimer formation are higher in the dimer than in the monomer. The monomer to dimer transition is accompanied by a rapid exchange of all of the amide protons which are protected in the dimer, indicating that the transition state is unfolded to a large extent. Our results demonstrate that the native monomeric state of stefin A is actually metastable but is favored by the kinetics of folding. The substantial energy barrier which separates the monomer from the more stable dimer traps each state under native conditions.

Expression and refolding of functional fragments of the human lipopolysaccharide receptor CD14 in Escherichia coli and Pichia pastoris.

CD14 is a high-affinity cellular receptor specific for bacterial lipopolysaccharides (LPS), present in the bacterial cell wall. Binding of LPS to CD14 initiates the innate component of immune response and triggers a response that can lead to septic shock. In order to provide recombinant protein for the study of LPS-CD14 molecular interactions we have expressed human CD14 in Escherichia coli and Pichia pastoris. In bacteria, the protein was produced in high yield in the form of inclusion bodies. We have optimized the procedure for its refolding and generated correctly folded protein. A procedure to monitor the refolding efficiency by using conformation-specific anti-human CD14 monoclonal antibody has been established. A fragment of 152 amino acids of CD14 which retains the ability to bind LPS has been produced in a methylotrophic yeast, P. pastoris, expression system. The recombinant protein from yeast is glycosylated and secreted into the medium. The CD14 fragment was purified to homogeneity by immunoaffinity chromatography. Recombinant CD14 from both bacteria and yeast bind to LPS.

Mutational analysis of two stefin A epitopes.

Stefin A, an intracellular inhibitor of cysteine proteinases, is expressed most abundantly in epithelial cells and in cells of lymphatic origin. In order to study its role in normal and pathological conditions we have prepared and characterized monoclonal antibodies against recombinant stefin A. Two high affinity monoclonal antibodies (mAbs) (A22 and C52) were tested for binding to free and papain-complexed stefin A and to a chimeric inhibitor, consisting of 61 amino acid residues of stefin A and 37 carboxy-terminal residues of stefin B. mAb A22 recognized not only free stefin A but also stefin A in complex with papain. The mAbs were further tested for their cross-reactivity against stefin A and B isolated from different mammalian species. On the basis of sequence similarity and tertiary structure of human stefin A we have prepared three mutants - Glu33Lys, Asp61Gly and Asn62Tyr and their reactivity with the mAbs was tested. The binding affinities of mAb A22 for the Asp61Gly and Asn62Tyr mutants were significantly lower, indicating thatthe two amino acids are part of the stefin A epitope recognized by A22. The binding of both mAbs to the mutants Gly4Arg and Gly4Glu was comparable to wild-type stefin A.

Differences in the effects of TFE on the folding pathways of human stefins A and B.

Trifluoroethanol (TFE) has been used to probe differences in the stability of the native state and in the folding pathways of the homologous cysteine protein inhibitors, human stefin A and B. After complete unfolding in 4.5 mol/L GuHCl, stefin A refolded in 11% (vol/vol) TFE, 0.75 mol/L GuHCl, at pH 6.0 and 20 degrees C, with almost identical first-order rate constants of 4.1 s-1 and 5.5 s-1 for acquisition of the CD signal at 230 and 280 nm, respectively, rates that were markedly greater than the value of 0.11 s-1 observed by the same two probes when TFE was absent. The acceleration of the rates of refolding, monitored by tyrosine fluorescence, was maximal at 10% (vol/vol) TFE. Similar rates of refolding (6.2s-1 and 7.2 s-1 for ellipticity at 230 and 280 nm, respectively) were observed for stefin A denatured in 66% (vol/vol) TFE, pH 3.3, when refolding to the same final conditions. After complete unfolding in 3.45 mol/L GuHCl, stefin B refolded in 7% (vol/vol) TFE, 0.57 mol/L GuHCl, at pH 6.0 and 20 degrees C, with a rate constant for the change in ellipticity at 280 nm of 32.8 s-1; this rate was only twice that observed when TFE was absent. As a major point of distinction from stefin A, the refolding of stefin B in the presence of TFE showed an overshoot in the ellipticity at 230 nm to a value 10% greater than that in the native protein; this signal relaxed slowly (0.01 s-1) to the final native value, with little concomitant change in the near-ultraviolet CD signal; the majority of this changes in two faster phases. After denaturation in 42% (vol/vol) TFE, pH 3.3, the kinetics of refolding to the same final conditions exhibited the same rate-limiting step (0.01 s-1) but were faster initially. The results show that similarly to stefin A, stefin B forms its hydrophobic core and predominant part of the tertiary structure faster in the presence of TFE. The results imply that the alpha-helical intermediate of stefin B is highly structured. Proteins 1999;36:205-216.

On the mechanism of human stefin B folding: I. Comparison to homologous stefin A. Influence of pH and trifluoroethanol on the fast and slow folding phases.

The folding of human stefin B has been studied by several spectroscopic probes. Stopped-flow traces obtained by circular dichroism in the near and far UV, by tyrosine fluorescence, and by extrinsic probe ANS fluorescence are compared. Most (60+/-5%) of the native signal in the far UV circular dichroism (CD) appeared within 10 ms in an unresolved "burst" phase, which was followed by a fast phase (t = 83 ms) and a slow phase (t = 25s) with amplitudes of 30% and 10%, respectively. Similar fast and slow phases were also evident in the near UV CD, ANS fluorescence, and tyrosine fluorescence. By contrast, human stefin A, which has a very similar structure, exhibited only one kinetic phase of folding (t = 6s) detected by all the spectroscopic probes, which occurred subsequent to an initial "burst" phase observed by far UV CD. It is interesting that despite close structural similarity of both homologues they fold differently, and that the less stable human stefin B folds faster by an order of magnitude (comparing the non-proline limited phase). To gain more information on the stefin B folding mechanism, effects of pH and trifluoroethanol (TFE) on the fast and slow phases were investigated by several spectroscopic probes. If folding was performed in the presence of 7% of TFE, rate acceleration and difference in the mechanism were observed.

On the mechanism of human stefin B folding: II. Folding from GuHCl unfolded, TFE denatured, acid denatured, and acid intermediate states.

It has been shown that human stefin B exhibits molten globule intermediates when denatured by acid or GuHCl. In the presence of TFE, it transforms into a highly helical state. In our first study on its folding mechanism (Zerovnik et al., Proteins 32:296-303), the kinetics measured by circular dichroism (CD) and fluorescence were correlated. In the present work the kinetics of folding were monitored by tyrosine fluorescence, ANS fluorescence, and, for certain reactions, far ultraviolet (UV) CD. The folding was started from the unfolded state in 3.45 M GuHCl, the acid denatured state at pH 1.8+/-0.2, an acid molten globule intermediate I1 (pH 3.3+/-0.1, low salt), a more structured acid molten globule intermediate I2 (pH 3.3+/-0.1, 0.42 M NaCl), and the TFE state (pH 3.3+/-0.1, 42% TFE). It has been found that all denatured states, including GuHCl, TFE, acid denatured and acid molten globule intermediate I1, fold with the same kinetics, provided that the final conditions are identical. This does not apply to the second acid molten globule intermediate I2, which demonstrates a higher rate of folding by a factor of 270. Different energy of activation and pH dependence were found for folding from states I1 or I2.

pH-induced conformational transitions of the propeptide of human cathepsin L. A role for a molten globule state in zymogen activation.

Synthesis of proteases as inactive zymogens is a very important mechanism for the regulation of their activity. For lysosomal proteases proteolytic cleavage of the propeptide is triggered by the acidic pH. By using fluorescence, circular dichroism, and NMR spectroscopy, we show that upon decreasing the pH from 6.5 to 3 the propeptide of cathepsin L loses most of the tertiary structure, but almost none of the secondary structure is lost. Another partially structured intermediate, prone to aggregation, was identified between pH 6.5 and 4. The conformation, populated below pH 4, where the activation of cathepsin L occurs, is not completely unfolded and has the properties of molten globule, including characteristic binding of the 1-anilinonaphthalene-8-sulfonic acid. This pH unfolding of the propeptide parallels a decrease of its affinity for cathepsin L and suggests the mechanism for the acidic zymogen activation. Addition of anionic polysaccharides that activate cathepsin L already at pH 5.5 unfolds the tertiary structure of the propeptide at this pH. Propeptide of human cathepsin L which is able to fold independently represents an evolutionary intermediate in the emergence of novel inhibitors originating from the enzyme proregions.

Characterization of the equilibrium intermediates in acid denaturation of human stefin B.

Acid-induced denaturation of recombinant human stefin B was followed using circular dichroism (CD) and fluorimetry. By comparing different spectroscopic probes, a number of equilibrium intermediates were detected. In pH denaturation at very low salt concentration (0.03 M NaCl) four states can be distinguished: N - I(N) - I1 - U, where N is the native state, I(N) is a native-like intermediate, I1 is an acid intermediate state with properties of a molten globule and U is the unfolded state. State 1, exhibits no near-ultraviolet CD but has some residual far-ultraviolet CD. It differs from U in its ability to increase fluorescence of 1-anilino-naphthalene 8-sulfonate (ANS). In 0.42 M salt, the pH denaturation is three-state between the dimeric native state N2 and intermediates I(N2) and I2, which are also dimeric according to size-exclusion chromatography. The acid intermediate I2 is more structured than I1: it binds ANS to a lower extent an I1, its Tyr residues are protected from the solvent, it shows some near-ultraviolet CD and its far-ultraviolet CD is even more intense than that for the native state. 1H-NMR spectra confirmed the overall structural features of the acid intermediates. To obtain the enthalpies of unfolding, microcalorimetric measurements were performed under conditions where the acid intermediates are maximally populated (18 degrees C): state I(N) from pH 5.0 to 4.6, 0.03 M salt: state I1 below pH 3.8, 0.42 M salt; and state I1 in equilibrium with I(N) at pH 4.05, 0.03 M salt. Enthalpies of unfolding for states I(N) and I1 were comparable to those of the native state. The enthalpy of unfolding for state I1 could not be determined.

Simulation of the gel-fluid transition in a membrane composed of lipids with two connected acyl chains: application of a dimer-move step.

Phospholipids have been treated as dimers on a hexagonal lattice, and a move has been introduced that allows the dimers to move and change their orientation on the lattice. Simulations have been performed in which phospholipid chains have been treated as being either independent or infinitely coupled thermodynamically with regard to their conformational state. Both types of simulation have reproduced well experimental heat-capacity curves of dipalmitoyl phosphatidylcholine small unilamellar vesicles. Apart from a different gel-fluid interaction parameter and a different number of unlike nearest-neighbor contacts, most of the averages and thermodynamic quantities were essentially the same in the two types of simulation. These results indicate that the transition is not first order and validate those of previous Monte Carlo simulations that have neglected the dimeric nature of phospholipids in the sense that they show that for the thermotropic transition the approximation of phospholipids as monomers is valid.

1H, 15N and 13C resonance assignments and secondary structure of group II phospholipase A2 from Agkistrodon piscivorus piscivorus: presence of an amino-terminal helix in solution.

1H, 15N and 13C resonance assignments are presented for the group II phospholipase A2 (PLA2) from Agkistrodon piscivorus piscivorus. The secondary structure of the enzyme has been inferred from an analysis of coupling constants, interproton distances, chemical shifts, and kinetics of amide exchange. Overall, the secondary structure of this PLA2 is similar to the crystal structure of the homologous group II human nonpancreatic secretory phospholipase [Scott, D.L., White, S.P., Browning, J.L., Rosa, J.J., Gelb, M.H. and Sigler, P.B. (1991) Science, 254, 1007-1010]. In the group I enzyme from porcine pancreas, the amino-terminal helix becomes fully ordered in the ternary complex of enzyme, lipid micelles and inhibitor. The formation of this helix is thought to be important for the increase in activity of phospholipases on aggregated substrates [Van den Berg, B., Tessari, M., Boelens, R., Dijkman, R., De Haas, G. H., Kaptein, R. and Verheij, H.M. (1995) Nature Strct. Biol., 2, 402-406]. However, the group II enzyme from Agkistrodon piscivorus piscivorus possesses a defined and well-positioned amino-terminal helix in the absence of substrate. Therefore, there is a clear difference between the conformation group I and group II enzymes in solution. These conformational differences suggest that formation of the amino-terminal helix is a necessary, but not sufficient, step in interfacial activation of phospholipases.

The three-dimensional solution structure of human stefin A.

The three-dimensional solution structure of recombinant human stefin A has been determined by a simulated annealing protocol using a total of 1113 distance and angle constraints obtained from 1H and 15N HMR spectroscopy. The solution structure is represented by a family of 17 conformers with an average root-mean-square deviation relative to the mean structure of 0.44 A for backbone atoms and 0.94 A for all heavy atoms for the main body of the structure. The protein has a well-defined global fold consisting of five anti-parallel beta-strands wrapped around a central five-turn alpha-helix. There is considerable similarity between the structural features of free stefin A in solution and the X-ray structure of the homologous protein stefin B in its complex with papain, but there are also some important differences in the regions which are fundamental to proteinase binding. The differences consist primarily of two regions of high conformational heterogeneity in free stefin A which correspond in stefin B to two of the components of the tripartite wedge that docks into the active site of the target proteinase. These regions, which are shown to be mobile in solution, are the five N-terminal residues and the second binding loop. In the bound conformation of stefin B they form a turn and a short helix, respectively.

Compactness of the molten globule in comparison to unfolded states as observed by size-exclusion chromatography.

The volumes of elution of denatured states of four proteins at high urea (8 M) and ethylurea (6 M) concentration were determined. They were found equally unfolded in both solvents. The volumes of elution of the unfolded states were compared to those of the native states and of some molten globule intermediates. It has been shown that the protein proteinase inhibitor stefin B, exhibits 'molten globule'-like properties on acid denaturation. The high salt acidic intermediate (a molten globule) as well as the native state of stefin B eluted as dimers, at 18 degrees C. On thermal denaturation above 42 degrees C, the intermediate dissociated into compact monomers. The more stable stefin A, which is monomeric and does not transform into molten globule intermediates under similar perturbing conditions, was always used for comparison. The states of both, stefin A and B in 50% methanol were found to be monomeric and of native-like compactness.