Targeting dexamethasone to Kupffer cells: effects on liver inflammation and fibrosis in rats.

Kupffer cells (KC) play an important role in the pathogenesis of inflammatory liver diseases leading to fibrosis. Anti-inflammatory drugs are only effective when administered at high doses that may cause side effects. Therefore, dexamethasone coupled to mannosylated albumin (Dexa(5)-Man(10)-HSA) was designed by us to selectively deliver this anti-inflammatory drug to the KC. The effectiveness of Dexa(5)-Man(10)-HSA was studied both in organ cultures and fibrosis induced by bile duct ligation (BDL) in rats. Dexa(5)-Man(10)-HSA accumulated in livers of both healthy and fibrotic rats (67% +/- 5% and 70% +/- 9% of the dose, respectively) and uptake was found almost exclusively in KC. Active dexamethasone was liberated from its carrier, because Dexa(5)-Man(10)-HSA could effectively inhibit nitric oxide (NO) and tumor necrosis factor alpha (TNF-alpha) release in endotoxin-activated liver slices. In vivo, however, this was associated with increased collagen I and III depositions and enhanced tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA expression. This was accompanied by a decreased influx of reactive oxygen species (ROS) producing cells in the livers of BDL animals treated with Dexa(5)-Man(10)-HSA as compared with untreated BDL rats. Dexa(5)-Man(10)-HSA treatment also replenished the depleted glycogen stores in hepatocytes of BDL livers. In conclusion, our studies showed selective delivery of dexamethasone to KC with Dexa(5)-Man(10)-HSA. This conjugate reduced intrahepatic ROS in vivo and TNF-alpha production in vitro and prevented glycogen depletion in vivo, indicating effective pharmacologic targeting. Dexa(5)-Man(10)-HSA, however, also accelerated fibrogenesis, which was paralleled by TIMP-1 mRNA induction. Targeting of dexamethasone to KC provides evidence for a dual role of this cell type in fibrogenesis of BDL rats.

Characterization of the beta-lactam binding site of penicillin acylase of Escherichia coli by structural and site-directed mutagenesis studies.

The binding of penicillin to penicillin acylase was studied by X-ray crystallography. The structure of the enzyme-substrate complex was determined after soaking crystals of an inactive betaN241A penicillin acylase mutant with penicillin G. Binding of the substrate induces a conformational change, in which the side chains of alphaF146 and alphaR145 move away from the active site, which allows the enzyme to accommodate penicillin G. In the resulting structure, the beta-lactam binding site is formed by the side chains of alphaF146 and betaF71, which have van der Waals interactions with the thiazolidine ring of penicillin G and the side chain of alphaR145 that is connected to the carboxylate group of the ligand by means of hydrogen bonding via two water molecules. The backbone oxygen of betaQ23 forms a hydrogen bond with the carbonyl oxygen of the phenylacetic acid moiety through a bridging water molecule. Kinetic studies revealed that the site-directed mutants alphaF146Y, alphaF146A and alphaF146L all show significant changes in their interaction with the beta-lactam substrates as compared with the wild type. The alphaF146Y mutant had the same affinity for 6-aminopenicillanic acid as the wild-type enzyme, but was not able to synthesize penicillin G from phenylacetamide and 6-aminopenicillanic acid. The alphaF146L and alphaF146A enzymes had a 3-5-fold decreased affinity for 6-aminopenicillanic acid, but synthesized penicillin G more efficiently than the wild type. The combined results of the structural and kinetic studies show the importance of alphaF146 in the beta-lactam binding site and provide leads for engineering mutants with improved synthetic properties.

Molecular cloning and analysis of the gene encoding the thermostable penicillin G acylase from Alcaligenes faecalis.

Alcaligenes faecalis penicillin G acylase is more stable than the Escherichia coli enzyme. The activity of the A. faecalis enzyme was not affected by incubation at 50 degrees C for 20 min, whereas more than 50% of the E. coli enzyme was irreversibly inactivated by the same treatment. To study the molecular basis of this higher stability, the A. faecalis enzyme was isolated and its gene was cloned and sequenced. The gene encodes a polypeptide that is characteristic of periplasmic penicillin G acylase (signal peptide-alpha subunit-spacer-beta subunit). Purification, N-terminal amino acid analysis, and molecular mass determination of the penicillin G acylase showed that the alpha and beta subunits have molecular masses of 23.0 and 62.7 kDa, respectively. The length of the spacer is 37 amino acids. Amino acid sequence alignment demonstrated significant homology with the penicillin G acylase from E. coli A unique feature of the A. faecalis enzyme is the presence of two cysteines that form a disulfide bridge. The stability of the A. faecalis penicillin G acylase, but not that of the E. coli enzyme, which has no cysteines, was decreased by a reductant. Thus, the improved thermostability is attributed to the presence of the disulfide bridge.

Preliminary X-ray study of naproxen esterase from Bacillus subtilis.

Single crystals of naproxen esterase from Bacillus subtilis have been obtained from PEG6000 solutions at pH 8.0 by liquid-liquid diffusion while applying a temperature gradient from 4 degrees C to room temperature over a period of four weeks. The crystals belong to the trigonal space group P3(1)21 or P3(2)21 with a = b = 47.59 A and c = 212.91 A. The asymmetric part of the unit cell contains one protein molecule with M(r) = 33,771. The crystals diffract to at least 3.0 A resolution and are suitable for an X-ray structure analysis.

Crystal structure of the reduced form of p-hydroxybenzoate hydroxylase refined at 2.3 A resolution.

The crystal structure of the reduced form of the enzyme p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens, complexed with its substrate p-hydroxybenzoate, has been obtained by protein X-ray crystallography. Crystals of the reduced form were prepared by soaking crystals of the oxidized enzyme-substrate complex in deaerated mother liquor containing 300-400 mM NADPH. A rapid bleaching of the crystals indicated the reduction of the enzyme-bound FAD by NADPH. This was confirmed by single crystal spectroscopy. X-ray data to 2.3 A were collected on oscillation films using a rotating anode generator as an X-ray source. After data processing and reduction, restrained least squares refinement using the 1.9 A structure of the oxidized enzyme-substrate complex as a starting model, yielded a crystallographic R-factor of 14.8% for 11,394 reflections. The final model of the reduced complex contains 3,098 protein atoms, the FAD molecule, the substrate p-hydroxybenzoate and 322 solvent molecules. The structures of the oxidized and reduced forms of the enzyme-substrate complex were found to be very similar. The root-mean-square discrepancy for all atoms between both structures was 0.38 A. The flavin ring is almost completely planar in the final model, although it was allowed to bend or twist during refinement. The observed angle between the benzene and the pyrimidine ring is 2 degrees. This value should be compared with observed values of 10 degrees for the oxidized enzyme-substrate complex and 19 degrees for the enzyme-product complex. The position of the substrate is virtually unaltered with respect to its position in the oxidized enzyme. No trace of a bound NADP+ or NADPH molecule was found.

Crystal structure of the high-alkaline serine protease PB92 from Bacillus alcalophilus.

The crystal structure of a serine protease from the alkalophilic strain Bacillus alcalophilus PB92 has been determined by X-ray diffraction at 1.75 A resolution. The structure has been solved by molecular replacement using the atomic model of subtilisin Carlsberg. The model of the PB92 protease has been refined to an R-factor of 14.0% and contains 1882 protein atoms, two calcium ions and 188 water molecules. The overall folding of the polypeptide chain closely resembles that of the subtilisins. Furthermore, almost all of the secondary structure elements found in subtilisin Carlsberg are also present in the PB92 protease. The major differences between the two structures are located around the deletion regions (residues 37 and 158-161 in subtilisin Carlsberg) and in two loops which are known to be the most variable parts of subtilisin structures. Flexibility of one of these loops (residues 126-130 in the PB92 protease) is believed to account for the induced-fit mechanism of substrate binding.

Protein engineering of the high-alkaline serine protease PB92 from Bacillus alcalophilus: functional and structural consequences of mutation at the S4 substrate binding pocket.

Serine endoproteases such as trypsins and subtilisins are known to have an extended substrate binding region that interacts with residues P6 to P3' of a substrate. In order to investigate the structural and functional effects of replacing residues at the S4 substrate binding pocket, the serine protease from the alkalophilic Bacillus strain PB92, which shows homology with the subtilisins, was mutated at positions 102 and 126-128. Substitution of Val102 by Trp results in a 12-fold increase in activity towards succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (sAAPFpNA). An X-ray structure analysis of the V102W mutant shows that the Trp side chain occupies a hydrophobic pocket at the surface of the molecule leaving a narrow crevice for the P4 residue of a substrate. Better binding of sAAPFpNA by the mutant compared with the wild type protein as indicated by the kinetic data might be due to the hydrophobic interaction of Ala P4 of the substrate with the introduced Trp102 side chain. The observed difference in binding of sAAPFpNA by protease PB92 and thermitase, both of which possess a Trp at position 102, is probably related to the amino acid substitutions at positions 105 and 126 (in the protease PB92 numbering). Kinetic data for the variants obtained by random mutation of residues Ser126, Pro127 and Ser128 reveal that the activity towards sAAPFpNA increases when a hydrophobic residue is introduced at position 126.(ABSTRACT TRUNCATED AT 250 WORDS)

The coenzyme analogue adenosine 5-diphosphoribose displaces FAD in the active site of p-hydroxybenzoate hydroxylase. An x-ray crystallographic investigation.

p-Hydroxybenzoate hydroxylase (PHBH) is an NADPH-dependent enzyme. To locate the NADPH binding site, the enzyme was crystallized under anaerobic conditions in the presence of the substrate p-hydroxybenzoate, the coenzyme analogue adenosine 5-diphosphoribose (ADPR), and sodium dithionite. This yielded colorless crystals that were suitable for X-ray analysis. Diffraction data were collected up to 2.7-A resolution. A difference Fourier between data from these colorless crystals and data from yellow crystals of the enzyme-substrate complex showed that in the colorless crystals the flavin ring was absent. The adenosine 5'-diphosphate moiety, which is the common part between FAD and ADPR, was still present. After restrained least-squares refinement of the enzyme-substrate complex with the riboflavin omitted from the model, additional electron density appeared near the pyrophosphate, which indicated the presence of an ADPR molecule in the FAD binding site of PHBH. The complete ADPR molecule was fitted to the electron density, and subsequent least-squares refinement resulted in a final R factor of 16.8%. Replacement of bound FAD by ADPR was confirmed by equilibrium dialysis, where it was shown that ADPR can effectively remove FAD from the enzyme under mild conditions in 0.1 M potassium phosphate buffer, pH 8.0. The empty pocket left by the flavin ring is filled by solvent, leaving the architecture of the active site and the binding of the substrate largely unaffected.

The influence of purification and protein heterogeneity on the crystallization of p-hydroxybenzoate hydroxylase.

The structure of the enzyme p-hydroxybenzoate hydroxylase was determined to a resolution of 0.25 nm [Wierenga et al. (1979) J. Mol. Biol. 131, 53-73] with crystals belonging to space group C222(1). Subsequently it was impossible to repeat the growth of this crystal form and only poor quality tetragonal crystals could be obtained. We have thoroughly investigated this problem and found that Cibacron-blue-purified enzyme appears to be heterogeneous with respect to aggregation state and Cys-116 oxidation. Most importantly, it could be firmly established that C222(1) crystals can only be grown from purely dimeric p-hydroxybenzoate hydroxylase possessing an intact SH group. Ion-exchange chromatography on DEAE-Sepharose can successfully remove those forms of the enzyme which impede successful crystallization. Sulfite and dithiothreitol improve crystallization by dissociating the enzyme oligomers into dimers; sulfite especially gives excellent results.

Crystal structure of p-hydroxybenzoate hydroxylase complexed with its reaction product 3,4-dihydroxybenzoate.

Crystals of the flavin-containing enzyme p-hydroxybenzoate hydroxylase (PHBHase) complexed with its reaction product were investigated in order to obtain insight into the catalytic cycle of this enzyme involving two substrates and two cofactors. PHBHase was crystallized initially with its substrate, p-hydroxybenzoate and the substrate was then converted into the product 3,4-dihydroxybenzoate by allowing the catalytic reaction to proceed in the crystals. In addition, crystals were soaked in mother liquor containing a high concentration of this product. Data up to 2.3 A (1 A = 0.1 nm) were collected by the oscillation method and the structure of the enzyme product complex was refined by alternate restrained least-squares procedures and model building by computer graphics techniques. A total of 273 solvent molecules could be located, four of them being presumably sulfate ions. The R-factor for 14,339 reflections between 6.0 A and 2.3 A is 19.3%. The 3-hydroxyl group of the product introduced by the enzyme is clearly visible in the electron density, showing unambiguously which carbon atom of the substrate is hydroxylated. A clear picture of the hydroxylation site is obtained. The plane of the product is rotated 21 degrees with respect to the plane of the substrate in the current model of enzyme-substrate complex. The 4-hydroxyl group of the product is hydrogen bonded to the hydroxyl group of Tyr201, its carboxyl group is interacting with the side-chains of Tyr222, Arg214 and Ser212, while the newly introduced 3-hydroxyl group makes a hydrogen bond with the backbone carbonyl oxygen of Pro293.

Comparison of the structure of turtle pancreatic ribonuclease with those of mammalian ribonucleases.

There are 33 invariant amino acid positions out of 132 positions in 42 investigated sequences of ribonucleases from a number of mammalian species and a reptile (snapping turtle, Chelydra serpentina). These invariant residues are unequally distributed over 3 different parts of the molecule. The lobe of the S-protein part of the molecule, which lacks one disulfide bridge and has two shortened loops in turtle ribonuclease, has the lowest percentage of invariant residues, although the active-site residue His 119 is located in this part.

Crystallization and preliminary X-ray investigation of lipoamide dehydrogenase from Azotobacter vinelandii.

The FAD-containing enzyme lipoamide dehydrogenase (EC 1.6.4.3. NADH: lipoamide oxidoreductase) of Azotobacter vinelandii has been crystallized from polyethylene glycol solutions. The space group is P2(1)2(1)2(1) with one dimer in the asymmetric unit. The cell dimensions are: a = 64.2, b = 83.8, c = 193 A. X-ray reflections extend to at least 2.2 A resolution.

Reassembly of wall domains of Roman-snail (Helix pomatia) beta-haemocyanin.

beta-Haemocyanin molecules consist of 20 very large polypeptide chains. These chains are composed of eight structural domains. So-called 'collar' domains can be removed by trypsinolysis of the native cylindrical molecule, resulting in an association of the remaining hollow cylinders into large tubular polymers. Dissociation of the tubular polymers gives one single- and four multi-domain fragments. The role of these fragments in the reassembly process of these tubular polymers was investigated. The two-domain fragment could form tubular polymers. The other domain fragments were not able to form tubular polymers unless in the presence of the two-domain fragment. Tubular polymers with enlarged diameter and ribbon-like structures were observed in the reassembly products when the one-domain fragment was omitted.

Structural and functional aspects of collar domains of Helix pomatia beta c-hemocyanin.

Digestion of beta c-hemocyanin yielded tubular polymers as well as so-called collars. Analysis of the collar fraction revealed that it consisted of two fragments, one having a relative molecular mass of approx. 125000 and the other a relative molecular mass of approx. 65000. They were separated using ion-exchange chromatography. The large fragment dissociated around pH 9.5 into two components having a molecular mass of approx. 65000. Both fragments bound oxygen and displayed heterotropic interactions. The large fragment bound oxygen with a Hill coefficient less than unity under certain conditions. The fragments differed also in amino acid composition, sugar content, spectral parameters, association-dissociation phenomena and ageing. by comparison with fragments obtained after limited proteolysis of tenth molecules, their location in the polypeptide chain was established.