A non-coding cancer mutation disrupting an HNF4α binding motif affects an enhancer regulating genes associated to the progression of liver cancer.

BACKGROUND: Somatic mutations in coding regions of the genome may result in non-functional proteins that can lead to cancer or other diseases, however cancer mutations in the non-coding regions have rarely been studied and the interpretation of their effects is difficult. Non-coding mutations might act by breaking or creating transcription factor binding motifs in promoters, enhancers or silencers resulting in altered expression of target gene(s). A high number of mutations have been reported in coding and non-coding regions in cells of liver cancer. Hepatocyte nuclear factor 4α is a transcription factor that regulates the expression of several genes in liver cells, while the motifs it binds are frequently mutated in promoters and enhancers in liver cancer. AIM: The aim of the study is to evaluate the genetic effects of a non-coding somatic mutation frequently observed in liver cancer. MATERIALS AND METHODS: We evaluated experimentally the effects of a somatic mutation frequently reported in liver cancer as a motif-breaker for the binding of hepatocyte nuclear factor 4α. The effects of the mutation on protein binding and enhancer activity were studied in HepG2 cells via electrophoresis mobility shift assay and dual luciferase reporter assays. We also studied genome-wide promoter-enhancer interactions performing targeted chromosome conformation capture in liver tissue to identify putative target genes whose expression could be altered by the mutation. RESULTS: We found that the mutation leads to reduced protein binding and a decrease in enhancer activity. The enhancer harboring the mutation interacts with the promoters of ANAPC13, MAP6D1 and MUC13, which have been implicated in liver cancer. CONCLUSIONS: The study highlights the importance of non-coding somatic mutations, vastly understudied, but likely to contribute to cancer development and progression.

'True' null allele detection in microsatellite loci: a comparison of methods, assessment of difficulties and survey of possible improvements.

Null alleles are alleles that for various reasons fail to amplify in a PCR assay. The presence of null alleles in microsatellite data is known to bias the genetic parameter estimates. Thus, efficient detection of null alleles is crucial, but the methods available for indirect null allele detection return inconsistent results. Here, our aim was to compare different methods for null allele detection, to explain their respective performance and to provide improvements. We applied several approaches to identify the 'true' null alleles based on the predictions made by five different methods, used either individually or in combination. First, we introduced simulated 'true' null alleles into 240 population data sets and applied the methods to measure their success in detecting the simulated null alleles. The single best-performing method was ML-NullFreq_frequency. Furthermore, we applied different noise reduction approaches to improve the results. For instance, by combining the results of several methods, we obtained more reliable results than using a single one. Rule-based classification was applied to identify population properties linked to the false discovery rate. Rules obtained from the classifier described which population genetic estimates and loci characteristics were linked to the success of each method. We have shown that by simulating 'true' null alleles into a population data set, we may define a null allele frequency threshold, related to a desired true or false discovery rate. Moreover, using such simulated data sets, the expected null allele homozygote frequency may be estimated independently of the equilibrium state of the population.

Reliability assessment of null allele detection: inconsistencies between and within different methods.

Microsatellite loci are widely used in population genetic studies, but the presence of null alleles may lead to biased results. Here, we assessed five methods that indirectly detect null alleles and found large inconsistencies among them. Our analysis was based on 20 microsatellite loci genotyped in a natural population of Microtus oeconomus sampled during 8 years, together with 1200 simulated populations without null alleles, but experiencing bottlenecks of varying duration and intensity, and 120 simulated populations with known null alleles. In the natural population, 29% of positive results were consistent between the methods in pairwise comparisons, and in the simulated data set, this proportion was 14%. The positive results were also inconsistent between different years in the natural population. In the null-allele-free simulated data set, the number of false positives increased with increased bottleneck intensity and duration. We also found a low concordance in null allele detection between the original simulated populations and their 20% random subsets. In the populations simulated to include null alleles, between 22% and 42% of true null alleles remained undetected, which highlighted that detection errors are not restricted to false positives. None of the evaluated methods clearly outperformed the others when both false-positive and false-negative rates were considered. Accepting only the positive results consistent between at least two methods should considerably reduce the false-positive rate, but this approach may increase the false-negative rate. Our study demonstrates the need for novel null allele detection methods that could be reliably applied to natural populations.

Metal-dependent self-assembly of protein tubes from Escherichia coli glutamine synthetase. Cu(2+) EPR studies of the ligation and stoichiometry of intermolecular metal binding sites.

Escherichia coli glutamine synthetase (GS) is a dodecameric assembly of identical subunits arranged as two back-to-back hexagonal rings. In the presence of divalent metal ions, the dodecamers "stack" along their six-fold axis of symmetry to yield elongated tubes. This self-assembly process provides a useful model for probing metal-dependent protein-protein interactions. However, no direct spectroscopic or structural data have confirmed the identity of the ligands to the shared metal ions in "stacked" GS. Here, 9-GHz Cu(2+) EPR studies have been used to probe the ligand structure and stoichiometry of the metal binding sites. The wild type protein, with N-terminal sequence (His-4)-X(3)-(Met-8)-X(3)-(His-12), exhibits a classic Cu(2+)-nitrogen spectrum, with g = 2.06 G, g = 2.24 G, and A = 19.3 x 10(-3) cm(-1). No superhyperfine structure is observed. The H4C mutant affords a spectrum that is the combination of two spectra at all stages of saturation. One of the overlapping spectra is nearly identical to the spectrum of wild type, and is due to His ligation. The second spectrum observed yields g = 2.28 and A = 17.1 x 10(-3) cm(-1). The linewidth and tensor values of the second component have been assigned to Cu(2+)-S ligation. In contrast, the H12C mutant exhibits an EPR spectrum at low Cu(2+) occupancy that is very similar to the second set of spectral features observed for H4C, and which is assigned to Cu(2+)-S ligation. No Cu(2+)-His ligation is apparent until the Cu(2+)/N-terminal helices ratio is >1.0. At saturation, the g = 2.00-2.06 region of the spectrum is essentially a mirror image of the spectrum obtained with H4C, and is due to overlapping Cu(2+)-N and Cu(2+)-S EPR spectra. The M8L and M8C mutants were also studied, in order to probe the role of position 8 in the N-terminal helix. Spectral parameters of these mutants are nearly identical to each other and to the wild type spectrum at saturating Cu(2+), suggesting that Met-8 does not act as a direct metal ligand. Together, the results provide the first direct evidence for a binuclear metal ion site between each N-terminal helix pair at the GS-GS interface, with both His-4 and His-12 providing metal ligands.

Stopped-flow kinetic analysis of the ligand-induced coil-helix transition in glutathione S-transferase A1-1: evidence for a persistent denatured state.

Structural studies have suggested that the glutathione S-transferase (GST) A1-1 isozyme contains a dynamic C-terminus which undergoes a ligand-dependent disorder-order transition and sequesters substrates within the active site. Here, the contribution of the C-terminus to the kinetics and thermodynamics of ligand binding and dissociation has been determined. Steady-state turnover rates of the wild type (WT) and a C-terminal truncated (Delta209-222) rGST A1-1 with ethacrynic acid (EA) were measured in the presence of variable concentrations of viscogen. The results indicate that a physical step involving segmental protein motion is at least partially rate limiting at temperatures between 10 and 40 degrees C for WT. Dissociation rates of the glutathione-ethacrynic acid product conjugate (GS-EA), determined by stopped-flow fluorescence, correspond to the steady-state turnover rates. In contrast, the chemical step governs the turnover reaction by Delta209-222, suggesting that the slow rate of product release for WT is controlled by the dynamics of the C-terminal coil-helix transition. In addition, the association reaction of WT rGST A1-1 with GS-EA established that the binding was biphasic and included ligand docking followed by slow isomerization of the enzyme-ligand complex. In contrast, binding of GS-EA to Delta209-222 was a monophasic, bimolecular reaction. These results indicate that the binding of GS-EA to WT rGST A1-1 proceeds via an induced fit mechanism, with a slow conformational step that corresponds to the coil-helix transition. However, the biphasic dissociation kinetics for the wild type, and the recovered kinetic parameters, suggest that a significant fraction of the [GST.GS-EA] complex ( approximately 15%) retains a persistent disordered state at equilibrium.

Strategies for protein-based nanofabrication: Ni2+-NTA as a chemical mask to control biologically imposed symmetry.

BACKGROUND: Technologies that improve control of protein orientation on surfaces or in solution, through designed molecular recognition, will expand the range of proteins that are useful for biosensors, molecular devices and biomaterials. A limitation of some proteins is their biologically imposed symmetry, which results in indistinguishable recognition surfaces. Here, we have explored methods for modifying the symmetry of an oligomeric protein that exhibits useful self-assembly properties. RESULTS: Escherichia coli glutamine synthetase (GS) contains 24 solvent-exposed histidines on two symmetry-related surfaces. These histidines drive a metal-dependent self-assembly of GS tubes. Immobilization of GS on the affinity resin Ni2+-NTA followed by on-column modification with diethyl pyrocarbonate affords asymmetrically modified GS that self-assembles only to the extent of 'short' dimeric GS tubes, as demonstrated by electron microscopy, dynamic light scattering and atomic force microscopy. The utility of Ni2+-NTA as a chemical mask was also demonstrated for asymmetric modification of engineered cysteines adjacent to the natural histidines. CONCLUSIONS: Current genetic methods do not provide distinguishable recognition elements on symmetry-related surfaces of biologically assembled proteins. Ni2+-NTA serves as a mask to control chemical modification in vitro of residues within symmetry-related pairs, on proteins containing functional His-tags. This strategy may be extended to modification of a wide range of amino acids with a myriad of reagents.

A procedure for assessing a contract testing facility: one sponsor's perspective.

The government agencies responsible for Good Laboratory Practices (GLP) place accountability for assuring compliance of studies intended for submission on the sponsor. An initial step toward meeting these legal obligations includes a comprehensive assessment of the candidate testing facility. Literature review has substantiated that the initial evaluation may be best accomplished through both technical and compliance-focused inspections (O'Brien-Pomerleau, 1991; Schroeder, 1989). Combined technical and compliance inspections have also been reported as an effective auditing approach for long-term studies (Hoover and Baldwin, 1984). However, for the purposes of the initial qualifying assessment inspection, personal experiences and published literature indicate that these two equally important evaluations are commonly conducted independently of one another (Scozzie, 1995). A collaborative approach utilized by Dow Corning Corporation in the preliminary assessment of a contract testing facility builds upon those methods just mentioned. Coupling this evaluation with a program of actively monitoring the testing facility's performance in study conduct provides continued assessment and has proven to be beneficial to all stakeholders (sponsor, testing facility, and regulatory agency). Details of the process practiced by this sponsor to assure that studies conducted by a contract testing facility meet GLP requirements are presented.

Synthesis and characterization of supramolecular protein aggregates: self-assembled, molecularly-ordered, tubes from electrostatic complementation of glutamine synthetase dodecamers.

Dodecameric Escherichia coli glutamine synthetase (GS) is formed from identical subunits arranged in face-to-face hexameric rings. In the presence of Zn2+ and other transition metal ions the individual dodecamers 'stack' to form protein tubes. Previous results have suggested that six binuclear intermolecular metal binding sites are generated at each dodecamer-dodecamer interface by juxtaposition of the N-terminal helices of each subunit adjacent to an analogous helix from a docked dodecamer. In principle, replacement of one of the metal binding sites within each pair of helices with charged amino acids could generate electrostatic interactions that would provide the basis for heterospecific protein-protein interactions. In turn, this would allow for ordered assembly of protein tubes with alternating, chemically distinguishable, components. This hypothesis was tested by replacement of one of the metalligating histidines (His12) with aspartic acid, arginine or cysteine. The H12C mutant was further elaborated by selective thiol modification, with either of the charged reagents 2-iodo-acetic acid or 2-chloro-acetamidine, which yield glutamate (H12C-IA) or arginine (H12C-CA) mimics at position 12. Light scattering and electron microscopy were used to monitor the 'stacking ability' of these variants in the presence of Zn2+. No, or few, GS 'tubes' were observed in solutions containing only H12D, H12R, H12C-CA or H12C-IA, in the presence or absence of Zn2+. In contrast, in mixtures containing H12C-CA and either H12D or H12C-IA, the complementary GS variants stack in the presence of 100 microM Zn2+, with apparent second order rate constants that are comparable to the wild type dodecamers. Fluorescence energy transfer experiments with fluorescein-labeled H12C-IA (donor) and rhodamine-labeled H12C-CA (acceptor) were performed and compared with the energy transfer efficiency with mixtures containing variable ratios of acceptor-labeled and donor-labeled wild type GS; the wild type mixtures provide a benchmark for the extent of energy transfer expected in random linear arrangements of donor and acceptor. The efficiency of metal-dependent energy transfer in mixtures containing the acceptor-labeled H12C-CA and the donor-labeled H12C-IA was 3.2-fold greater than expected for a random distribution of charged variants. Together, the results indicate that the charged variants provide a mechanism for heterospecific interaction between chemically distinguishable dodecamers that align in an ordered one-dimensional array.

Rational modulation of the catalytic activity of A1-1 glutathione S-transferase: evidence for incorporation of an on-face (pi...HO-Ar) hydrogen bond at tyrosine-9.

The alpha-, pi-, and mu-class glutathione S-transferases utilize a hydrogen bond between a conserved tyrosine and glutathione (GSH) to stabilize the nucleophilic thiolate anion, as Tyr-OH...-SG. This hydrogen bond is critical for efficient detoxication catalysis. The detailed structure of this hydrogen bond, however, is controlled by active site features which are not conserved across class boundaries. The alpha-class GST A1-1 has a cluster of aromatic residues on one side of the ring of the catalytic tyrosine, Tyr-9. Also, a hydrophobic Met-16 side chain is packed against the edge of the ring of Tyr-9. Molecular modeling and ab initio calculations suggested that substitution of Phe-220 with tyrosine could generate an aromatic on-face hydrogen bond (pi...HO-Ar) between the ring of Tyr-9 and the hydroxyl group of Tyr-220, and this would lower the pKa of enzyme-bound GSH. Therefore, Phe-220 was replaced by Tyr in the rat A1-1 isozyme. Also, Met-16 was replaced by Thr in order to investigate the effect of a hydrogen bond donor at the Tyr-9 ring edge. UV spectroscopic titration of GST.GSH and steady-state kinetic analysis indicate that substitution of Tyr at Phe-220 results in a decrease of the pKa of the cofactor, whereas substitution of Met-16 with Thr results in an increase of this pKa. Also, the pKa of Tyr-9 in the absence of substrates was determined directly by fluorescence titration. Substitutions F220Y and M16T resulted in a decrease of 0.5 pKa unit and an increase of 0.6 pKa unit, respectively. Together, these results indicate that a weak hydrogen bond between the engineered Tyr-220 side chain and the aromatic ring face of the catalytic Tyr-9 decreases the pKa of GSH and Tyr-9, and this alters the pH dependence of the enzymatic reaction.

Engineering the aggregation properties of dodecameric glutamine synthetase: a single amino acid substitution controls 'salting out'.

Escherichia coli glutamine synthetase (GS) is a dodecamer of identical subunits which are arranged as two face-to-face hexameric rings. In the presence of 10% ammonium sulfate, wild type GS exhibits a pH-dependent "salting out' with a pKa of 4.51. Electron micrographs indicate that the pH-dependent aggregation corresponds to a highly specific self-assembly of GS tubules, which result from stacking of individual dodecamers. This stacking of dodecamers is similar to the metal ion-induced GS tubule formation previously described. Site-directed mutagenesis experiments indicate that the N-terminal helix of each subunit is involved in the salting out reaction, as it is in the metal-induced stacking. A single substitution of alanine for His4 completely abolishes the (NH4)2SO4-induced aggregation. However, the H4C mutant protein does nearly completely precipitate under the same salting out conditions. Mutations at other residues within the helix have no effect on the stacking reaction. Differential catalytic activity of unadenylylated GS versus adenylylated GS has been used to determine whether wild type dodecamers "complement' the H4A mutant in the stacking reaction. The complementation experiments indicate that His4 residues on both sides of the dodecamer-dodecamer interfaces are not absolutely required for salting out, although the wild type dodecamers clearly stack preferentially with other wild type dodecamers. Approximately 20% of the protein precipitated from the mixtures containing the wild type GS and the H4A mutant is the mutant. The implications of these results for protein engineering are discussed.

Supramolecular self-assembly of Escherichia coli glutamine synthetase: characterization of dodecamer stacking and high order association.

Dodecameric glutamine synthetase (GS) from bacteria is formed from two face-to-face hexameric rings of identical subunits. These highly symmetrical aggregates from some bacteria, including Escherichia coli, "stack" in the presence of Zn2+ and other divalent ions to generate protein tubes (phase I) and subsequently associate side-to-side to yield "cables" and nonspecific aggregates (phase II). In order to understand the molecular mechanisms of recognition leading to this macromolecular self-assembly, the effects of solution conditions on the kinetics of these processes have been studied. These reactions have been monitored by changes in light scattering and by electron microscopy. Conditions have been established for isolation of phases I and II. At 0.04 mg of GS/mL, pH 7.0, 100 mM KCl, and 1 mM Mn2+, 25 degrees C, minimal side-to-side aggregation occurs, and the stacking reaction follows second-order kinetics, with respect to GS, at low extent of reaction. The second-order rate constants determined for phase I, initiated by Zn2+ or Co2+, demonstrate a pH optimum at 7.0-7.25, whereas phase II is favored at pHs below 6.5. The pH profile for the stacking reaction suggests that His residues are involved, and modification of 2-3 histidines/subunit with diethyl pyrocarbonate (DEPC) is sufficient to completely inhibit metal-dependent dodecamer stacking. The effect of ionic strength on GS stacking was also studied. Although hydrophobic interactions have previously been assumed to dominate this protein-protein association, both phase I and phase II of the assembly are inhibited by KCl and NaCl, suggesting that ionic interactions also play an essential role.(ABSTRACT TRUNCATED AT 250 WORDS)

Supramolecular self-assembly of glutamine synthetase: mutagenesis of a novel intermolecular metal binding site required for dodecamer stacking.

Dodecameric glutamine synthetase (GS) from Escherichia coli assembles into highly ordered supramolecular protein tubes in the presence of several divalent metal ions. The molecular mechanism for this metal-induced self-assembly of the E. coli GS has been studied by molecular modeling and site-directed mutagenesis. The X-ray crystal structure of the nearly identical Salmonella typhimurium GS has been used to construct a model of the "stacked" complex between two dodecamers. A complementary fit, based on steric constraints, reveals a possible interaction between the N-terminal helices from adjacent dodecamers. The amino acid side chains of His and Met residues within the helices from each of the subunits of one face of a dodecamer lie within approximately 3.5 A of the analogous side chains in the subunits from the adjacent dodecamer in the stacked complex. His-4, Met-8, and His-12 from adjacent helices provide potential ligands for a binuclear metal binding site. Replacement of each of these surface residues with aliphatic amino acids has negligible effects on the enzymatic activity, the regulation of activity via adenylylation, and gross dodecameric structure. However, the rate and extent of metal ion-mediated self-assembly of GS tubules are reduced to < 2% of the wild-type protein in the single mutants H4A, H12L, and H12D. The M8L mutant demonstrates a 3-fold decrease in the bimolecular rate constant for stacking, but electron microscopy indicates that this mutant does form stacked tubes. The cysteine-containing mutants H4C, M8C, and H12C were also constructed.(ABSTRACT TRUNCATED AT 250 WORDS)