Hepatitis B virus X protein induces TNF-alpha expression via down-regulation of selenoprotein P in human hepatoma cell line,HepG2

Human hepatitis B virus X protein (HBx) is associated with the induction of oxidative stress, which is considered significant in the development of liver damage. In this study, we investigated the molecular mechanisms by which HBx induced lipid peroxidation and tumor necrosis factor-alpha (TNF-alpha) expression through regulation of selenoprotein P (SeP) expression in the human hepatoma cell line, HepG2. Forced expression of HBx significantly down-regulated the expression of SeP mRNA and protein in both the cell lysates and the culture medium. Lipid peroxidation increased 2.5-fold when expression of the SeP protein was blocked with a SeP antisense vector. Also, HBx transfection increased lipid peroxidation by 3.0-fold, whereas the hepatitis B virus core protein (HBc) had no significant effects. The induction of lipid peroxidation due to the block in SeP protein expression or treatment with ferric chloride (FeCl(3)) up-regulated the expression levels of TNF-alpha mRNA and protein. The pattern of HBx-induced lipid peroxidation and TNF-alpha up-regulation was reversed by SeP introduction. These results suggest that HBx induces lipid peroxidation via down-regulation of SeP expression, resulting in increased expression of TNF-alpha in the human hepatoma cell line, HepG2.     

A captured folding intermediate involved in dimerization and domain-swapping of GB1

Immunoglobulin binding domain B1 of streptococcal protein G (GB1), a small (56 residues), stable, single domain protein, is one of the most extensively used model systems in the area of protein folding and design. The recently determined NMR structure of a quadruple mutant (HS#124F26A, L5V/F30V/Y33F/A34F) revealed a domain-swapped dimer that dissociated into a partially folded, monomeric species at low micromolar protein concentrations. Here, we have characterized this monomeric, partially folded species by NMR and show that extensive conformational heterogeneity for a substantial portion of the polypeptide chain exists. Exchange between the conformers within the monomer ensemble on the microsecond to millisecond timescale renders the majority of backbone amide resonances broadened beyond detection. Despite these extensive temporal and spatial fluctuations, the overall architecture of the monomeric mutant protein resembles that of wild-type GB1 and not the monomer unit of the domain-swapped dimer.     

Site-directed immobilization of antibody onto solid surfaces for the construction of immunochip

The performance of an immuno-analytical system can be assessed in terms of its analytical sensitivity,i.e., the detection limit of an analyte, which is determined by the amount of analyte molecules bound to the capture antibody that has been immobilized onto a solid surface. To increase the number of the binding complexes, we have investigated a site-directed immobilization of an antibody that has the ability to resolve a current problem associated with a random arrangement of the insolubilized immunoglobulin. The binding molecules were chemically reduced to produce thiol groups that were limited at the hinge region, and then, the reduced products were coupled to biotin. This biotinylated antibody was bound to a streptavidincoated surface via the streptavidin-biotin reaction. This method can control the orientation of the antibody molecules present on a solid surface and also can significantly reduce the possibility of steric hindrance in the antigen-antibody reactions. In a two-site immunoassay, the introduction of the site-directly immobilized antibody as the capture enhanced the sensitivity of analyte detection approximately 10 times compared to that of the antibody randomly coupled to biotin. Such a novel approach would offer a protocol of antibody immobilization in order for the possibility of constructing a high performance immunochip.     

Sequential phosphorylation and multisite interactions characterize specific target recognition by the FHA domain of Ki67

The forkhead-associated (FHA) domain of human Ki67 interacts with the human nucleolar protein hNIFK, recognizing a 44-residue fragment, hNIFK226-269, phosphorylated at Thr234. Here we show that high-affinity binding requires sequential phosphorylation by two kinases, CDK1 and GSK3, yielding pThr238, pThr234 and pSer230. We have determined the solution structure of Ki67FHA in complex with the triply phosphorylated peptide hNIFK226-269(3P), revealing not only local recognition of pThr234 but also the extension of the beta-sheet of the FHA domain by the addition of a beta-strand of hNIFK. The structure of an FHA domain in complex with a biologically relevant binding partner provides insights into ligand specificity and potentially links the cancer marker protein Ki67 to a signaling pathway associated with cell fate specification.     

1H NMR structural characterization of a recombinant kringle 2 domain from human tissue-type plasminogen activator

The kringle 2 domain of human tissue-type plasminogen activator (t-PA) has been characterized via 1H NMR spectroscopy at 300 and 620 MHz. The experiments were performed on the isolated domain obtained by expression of the 174-263 portion of t-PA in Escherichia coli [Cleary et al. (1989) Biochemistry 28, 1884-1891]. The spectrum of t-PA kringle 2 is characteristic of a globular structure and shows overall similarity to that of the plasminogen (PGN) kringle 4. Spectral comparison with human and bovine PGN kringle 4 identifies side-chain resonances from Leu46, which afford a fingerprint of kringle folding, and from most of the aromatic ring spin systems. Assignment of signals arising from the His13, His48a, and His64 side chains, which are unique to t-PA kringle 2, was assisted by the availability of a His64----Tyr mutant. Ligand-binding studies confirm that t-PA kringle 2 binds L-lysine with an association constant Ka approximately 11.9 mM-1. The data indicate that homologous or conserved residues relative to those that compose the lysine-binding sites of PGN kringles 1 and 4 are involved in the binding of L-lysine to t-PA kringle 2. These include Tyr36 and, within the kringle inner loop, Trp62, His64, Trp72, and Tyr74. Acid/base titration of aromatic singlets in the presence of L-lysine yields pKa* approximately 6.25 and approximately 4.41 for His13 and His64, respectively, and shows that the His48a imidazole group does not protonate down to pH* approximately 4.3. Thus, the His48a and His64 side chains are in solvent-shielded locations. As observed for the PGN kringles, the Trp62 indole group titrates with pKa* approximately 4.60, which indicates proximity of the side chain to a titratable carboxyl group, most likely that of Asp57 at the binding site. Several labile NH protons of t-PA kringle 2 exhibit retarded H-exchange kinetics, requiring more than a week in 2H2O for full deuteration in the presence of L-lysine at 37 degrees C. This reveals that kringle 2 is endowed with a compact, dynamically stable conformation. Proton Overhauser experiments in 1H2O, centered on well-resolved NH resonances between 9.8 and 12 ppm, identify signals arising from the His48a imidazole NH3 proton and the three Trp indole NH1 protons. A strong dipolar interaction was observed among the Trp25 indole NH1, the Tyr50 amide NH, and the His48a imidazole CH2 protons, which affords evidence for an aromatic cluster in t-PA kringle 2 similar to that found at the hydrophobic kernel of PGN kringles.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ligand specificity of human plasminogen kringle 4.

The ligand specificity of the human plasminogen kringle 4 was characterized in terms of ligand size, aromatic/aliphatic character, and ionic charge distribution. The binding of the following ligands was investigated via 1H NMR spectroscopy, and their equilibrium association constants (Ka) were determined: (1) p-aminomethylbenzoic acid (Ka approximately 4.8 mM-1), (2) benzylamine (Ka approximately 0.2 mM-1), (3) l-aminohexane (Ka approximately 0.07 mM-1), (4) 7-aminoheptanoic acid (Ka approximately 6.6 mM-1), (5) 5-aminopentanoic acid (Ka approximately 16 mM-1), (6) N alpha-acetyl-L-arginine (Ka approximately 0.3 mM-1), and (7) N alpha-acetyl-L-arginine methyl ester (Ka approximately 0.08 mM-1). Benzamidine and L-arginine do not bind measurably to kringle 4. We have also established that 1-hexanoic acid and 4-methylbenzoic acid do not interact significantly with kringle 4 (Ka less than 0.05 mM-1). The Trp62 resonances were found to be quite sensitive to aromatic ligands as well as to aliphatic ligand length. Phe64 is similarly sensitive to the ligand aromatic/aliphatic character and chain length and to the identity of the ligand anionic group. His31 and His33 do not respond significantly to variations in ligand structure, although they are perturbed by aromatic and aliphatic effectors. The perturbations induced by the arginine derivatives on these residues show that these compounds interact with the lysine-binding site (LBS) of kringle 4. The LBS was further characterized using 2D NMR studies of a kringle 4/trans-(aminomethyl)cyclohexanecarboxylic acid (AMCHA) complex. A complete assignment of the AMCHA spectrum in the bound state was achieved. This enabled the unambiguous identification of intermolecular contact points between the central AMCHA protons and Trp62 and Trp72. A model based on the X-ray crystallographic structure of kringle 4, incorporating these constraints, has been derived.     

Phospholipase A2 engineering. Structural and functional roles of highly conserved active site residues tyrosine-52 and tyrosine-73.

Site-directed mutagenesis was used to probe the structural and functional roles of two highly conserved residues, Tyr-52 and Tyr-73, in interfacial catalysis by bovine pancreatic phospholipase A2 (PLA2, overproduced in Escherichia coli). According to crystal structures, the side chains of these two active site residues form H-bonds with the carboxylate of the catalytic residue Asp-99. Replacement of either or both Tyr residues by Phe resulted in only very small changes in catalytic rates, which suggests that the hydrogen bonds are not essential for catalysis by PLA2. Substitution of either Tyr residue by nonaromatic amino acids resulted in substantial decreases in the apparent kcat toward 1,2-dioctanoyl-sn-glycero-3-phosphocholine (DC8PC) micelles and the v(o) (turnover number at maximal substrate concentration, i.e., mole fraction = 1) toward 1,2-dimyristoyl-sn-glycero-3-phosphomethanol (DC14PM) vesicles in scooting mode kinetics [Berg, O. G., Yu, B.-Z., Rogers, J., & Jain, M. K. (1991) Biochemistry 30, 7283-7297]. The Y52V mutant was further analyzed in detail by scooting mode kinetics: the E to E* equilibrium was examined by fluorescence; the dissociation constants of E*S, E*P, and E*I (KS*, KP*, and KI*, respectively) in the presence of Ca2+ were measured by protection of histidine-48 modification and by difference UV spectroscopy; the Michaelis constant KM* was calculated from initial rates of hydrolysis in the absence and presence of competitive inhibitors; and the turnover number under saturating conditions (kcat, which is a theoretical value since the enzyme may not be saturated at the interface) was calculated from the vo and KM* values. The results indicated little perturbation in the interfacial binding step (E to E*) but ca. 10-fold increases in KS*, KP*, KI*, and KM* and a less than 10-fold decrease in kcat. Such changes in the function of Y52V are not due to global conformational changes since the proton NMR properties of Y52V closely resemble those of wild-type PLA2; instead, it is likely to be caused by perturbed enzyme-substrate interactions at the active site. Tyr-73 appears to play an important structural role. The conformational stability of all Tyr-73 mutants decreased by 4-5 kcal/mol relative to that of the wild-type PLA2. The proton NMR properties of Y73A suggested significant conformational changes and substantially increased conformational flexibility. These detailed structural and functional analyses represent a major advancement in the structure-function study of an enzyme involved in interfacial catalysis.     

Structural analysis of phospholipase A2 from functional perspective. 1. Functionally relevant solution structure and roles of the hydrogen-bonding network

Bovine pancreatic phospholipase A2 (PLA2), a small (13.8 kDa) Ca2+-dependent lipolytic enzyme, is rich in functional and structural character. In an effort to examine its detailed structure-function relationship, we determined its solution structure by multidimensional nuclear magnetic resonance (NMR) spectroscopy at a functionally relevant pH. An ensemble of 20 structures generated has an average root-mean-square deviation (RMSD) of 0.62 +/- 0.08 A for backbone (N, Calpha, C) atoms and 0.98 +/- 0.09 A for all heavy atoms. The overall structure shows several notable differences from the crystal structure: the first three residues at the N-terminus, the calcium-binding loop (Y25-T36), and the surface loop (V63-N72) appear to be flexible; the alpha-helical conformation of helix B (E17-F22) is absent; helix D appears to be shorter (D59-V63 instead of D59-D66); and the hydrogen-bonding network is less defined. These differences were analyzed in relation to the function of PLA2. We then further examined the H-bonding network, because its functional role or even its existence in solution has been in dispute recently. Our results show that part of the H-bonding network (the portion away from N-terminus) clearly exists in solution, as evidenced by direct observation (at 11.1 ppm) of a strong H-bond between Y73 and D99 and an implicated interaction between D99 and H48. Analyses of a series of mutants indicated that the existence of the Y73.D99 H-bond correlates directly with the conformational stability of the mutant. Loss of this H-bond results in a loss of 2-3 kcal/mol in the conformational stability of PLA2. The unequivocal identification and demonstration of the structural importance of a specific hydrogen bond, and the magnitude of its contribution to conformational stability, are uncommon to the best of our knowledge. Our results also suggest that, while the D99.H48 catalytic diad is the key catalytic machinery of PLA2, it also helps to maintain conformational integrity.     

Structural analysis of phospholipase A2 from functional perspective. 2. Characterization of a molten globule-like state induced by site-specific mutagenesis

Previous NMR studies have shown that many phospholipase A2 (PLA2, from bovine pancreas, overexpressed in Escherichia coli) mutants display some properties reminiscent of a molten globule state. Further NMR analyses for some of the mutants indicated that formation of the "molten globule-like state" is a pH-dependent phenomenon. The mutants I9Y and I9F showed perturbed NMR properties throughout the pH range studied, while the mutants H48A and C44A/C105A displayed native-like spectra at neutral pH but molten globule-like ones under acidic conditions, with a "transition pH" around 4. On the other hand, wild-type PLA2 exhibits exceptional pH stability and turns into a similar molten globule-like state only under highly acidic conditions such as 1 M HCl. The H48A mutant was used to rigorously establish the property of the molten globule-like state of PLA2 mutants. The results of far-UV CD, near-UV CD, and ANS-binding fluorescence suggest that H48A retains native-like secondary structures but loses tertiary structure during the conformational transition. However, the tertiary structure is not completely lost, as evidenced by the retention of some long-range NOEs in two-dimensional NOESY spectra. The conclusion was further substantiated by three-dimensional NOESY-HSQC experiments on a 15N-labeled H48A sample. It was revealed that the molten globule-like state at mildly acidic pH retained some rigid tertiary structure, which consisted of partial alpha-helix II (Y52-L58), alpha-helix III (D59-V63), beta-wing (S74-S85) and partial alpha-helix IV (A90-N97). These residual tertiary structures grouped in half of the protein could be attributed to stabilization by some of the disulfide bonds. The extreme sensitivity of the PLA2 structure to site-directed mutagenesis is unprecedented. It is interesting to note that most of the functional residues (the active site, the hydrophobic channel, the interfacial binding site, and the calcium-binding loop) are located in the remainder of the protein, which is well disrupted in tertiary interactions.