Mutagenicity of electrophilic N-acyloxy-N-alkoxyamides.

N-acyloxy-N-alkoxybenzamides are mutagenic in TA100 without the need for metabolic activation with S9. Electronic effects of substituents on both the benzamide ring in N-acetoxy-N-butoxybenzamides or the benzyloxy ring in N-acetoxy-N-benzyloxybenzamides do not influence mutagenicity levels. For N-benzoyloxy-N-benzyloxybenzamides, mutagenicity levels are inversely related to the electron-withdrawing effect of substituents on the benzoyloxy leaving group. Since reactivities increase with increasing electron-withdrawing effects, mutagenicity correlates with stability rather than reactivity of these mutagens. Hydrophobicity is the dominant factor controlling mutagenicity levels and data for all mutagens correlate with computed logP values with a lower dependence (h=0.22) than that recorded for indirect mutagens (h=1.0), except where a sterically demanding p-tert-butyl substituent or a naphthyl group is present. N-acetoxy-N-butoxynaphthamide exhibits a much higher level of mutagenicity than predicted by its logP value and activity may be ascribed to an intercalative binding process with DNA rather than straightforward hydrophobic binding in the major or minor groove. Since these are direct-acting mutagens, structural factors influence binding and reactivity towards DNA.

Misusing informed consent: a critique of limitations on research subjects' access to genetic research results.

Express denials of access to genetic research results are being drafted into consent instruments. Some commentators suggest that the principle of beneficence can justify such a denial of access. This paper provides an ethical and legal critique of the use of consent instruments to disclaim responsibility for on-going disclosure by genetic researchers. Currently, the law of torts provides only weak protection for on-going disclosure for research subjects. The most substantive rights are to be found in the law of fiduciary obligations. The author concludes that, notwithstanding arguments to the contrary, there should be a presumption of disclosure in genetic research, unless the research subject elects otherwise. The author outlines one possible exception to this general presumption.

Assignments and structure determination of the catalytic domain of human fibroblast collagenase using 3D double and triple resonance NMR spectroscopy.

We report here the backbone 1HN, 15N, 13C alpha, 13CO, and 1H alpha NMR assignments for the catalytic domain of human fibroblast collagenase (HFC). Three independent assignment pathways (matching 1H, 13C alpha, and 13CO resonances) were used to establish sequential connections. The connections using 13C alpha resonances were obtained from HNCOCA and HNCA experiments; 13CO connections were obtained from HNCO and HNCACO experiments. The sequential proton assignment pathway was established from a 3D (1H/15N) NOESY-HSQC experiment. Amino acid typing was accomplished using 13C and 15N chemical shifts, specific labeling of 15N-Leu, and spin pattern recognition from DQF-COSY. The secondary structure was determined by analyzing the 3D (1H/15N) NOESY-HSQC. A preliminary NMR structure calculation of HFC was found to be in agreement with recent X-ray structures of human fibroblast collagenase and human neutrophil collagenase as well as similar to recent NMR structures of a highly homologous protein, stromelysin. All three helices were located; a five-stranded beta-sheet (four parallel strands, one antiparallel strand) was also determined. beta-Sheet regions were identified by cross-strand d alpha N and d NN connections and by strong intraresidue d alpha N correlations, and were corroborated by observing slow amide proton exchange. Chemical shift changes in a selectively 15N-labeled sample suggest that substantial structural changes occur in the active site cleft on the binding of an inhibitor.

Production, purification, and crystallization of human interleukin-1 beta converting enzyme derived from an Escherichia coli expression system.

Interleukin-1 beta converting enzyme (ICE) is a cysteine protease that catalyzes the conversion of the inactive precursor form of IL-1 beta to an active mature form. The mature form of IL-1 beta is involved in mediating inflammatory responses and in the progression of autoimmune diseases. We recently reported on the production of active human ICE in insect cells using the baculovirus expression system (Wang XM et al., 1994, Gene 145:273-277). Because the levels of expression achieved with this system were limiting for the purpose of performing detailed biochemical and biophysical studies, we examined the production of ICE in Escherichia coli. By using a tac promoter-based expression system and fusion to thioredoxin we were able to recover high levels of active ICE protein. The expressed protein, which was distributed between the soluble and insoluble fractions, was purified to homogeneity from both fractions using a combination of classical and affinity chromatography. Comparisons of ICE derived from both fractions indicated that they were comparable in their specific activities, subunit composition, and sensitivities to specific ICE inhibitors. The combined yields of ICE obtained from the soluble and insoluble fractions was close to 1 mg/L of induced culture. Recombinant human ICE was crystallized in the presence of a specific ICE inhibitor in a form suitable for X-ray crystallographic analysis. This readily available source of ICE will facilitate the further characterization of this novel and important protease.

Production of selenomethionine-labeled recombinant human neutrophil collagenase in Escherichia coli.

Molecular analogs of amino acids can be incorporated into proteins. The amino acid analog selenomethionine (SeMet) has been shown to be efficiently incorporated into the proteins of growing Escherichia coli. SeMet-containing proteins are known to produce sufficiently strong anomalous scatter permitting the solution of the selenomethionyl crystal structure by multiwavelength anomalous diffraction (MAD) techniques. The recombinant protein chosen for these studies is mature, truncated neutrophil collagenase (rmNC-t). The rmNC-t protein is a monomer of 163 amino acid residues featuring one active site and two Met residues. We developed a T7 polymerase expression system allowing incorporation of SeMet into rmNC-t protein produced in E. coli. Substitution of Met with SeMet was accomplished by culturing E. coli DL41(DE3), a SeMet-tolerant strain with metA lesion, in a defined medium containing SeMet as the sole source of Met. The SeMet-labeled rmNC-t was isolated from inclusion bodies by solubilizing in urea, purified by anion column chromatography, and then refolded in the presence of Ca2+ and Zn2+. Analysis of SeMet-labeled rmNC-t demonstrated that Met replacement was 100%. Enzymatic characterization revealed no obvious differences in activity or inhibitor binding between rmNC-t and the SeMet-labeled product. We have produced pure, active SeMet-labeled rmNC-t in sufficient quantities for macromolecular crystallography studies.

Gene expression, purification and characterization of recombinant human neutrophil collagenase.

Human neutrophil collagenase (HNC) is a member of a family of matrix metalloproteinases (MMP). HNC is capable of cleaving all three alpha-chains of types I, II and III collagens. In rheumatoid and osteo-arthritis, MMP members have been implicated in the pathology associated with these diseases due to the accelerated breakdown of the extracellular matrix of articular cartilage. A cDNA coding for the HNC catalytic domain (lacking both the propeptide and C-terminal fragments) was sub-cloned into the pETlla prokaryotic expression vector. The cloned fragment encodes a protein that extends from amino acids (aa) Met100 through Gly262 of the full-length proenzyme, which as a result, would not require proteolytic or chemical activation. The HNC construct was expressed in Escherichia coli and recombinant mature, truncated neutrophil collagenase (re-mNC-t) was produced at high levels (approx. 30% of total bacterial protein). The re-mNC-t protein was extracted from inclusion bodies by solubilization in 6 M urea, followed by ion-exchange chromatography. The protein was refolded to an active conformation in the presence of Ca2+ and Zn2+. A final purification step on size-exclusion chromatography yielded 30 mg per liter of active re-mNC-t with minor autodegradative products. Alternatively, hydroxamate affinity chromatography was used to obtain pure, non-degraded re-mNC-t (20-25 mg per liter). The catalytic activity of re-mNC-t was abolished by known MMP inhibitors and the Ki measurement against actinonin was similar to that of HNC prepared from human blood.

1.56 A structure of mature truncated human fibroblast collagenase.

The X-ray crystal structure of a 19 kDa active fragment of human fibroblast collagenase has been determined by the multiple isomorphous replacement method and refined at 1.56 A resolution to an R-factor of 17.4%. The current structure includes a bound hydroxamate inhibitor, 88 waters and three metal atoms (two zincs and a calcium). The overall topology of the enzyme, comprised of a five stranded beta-sheet and three alpha-helices, is similar to the thermolysin-like metalloproteinases. There are some important differences between the collagenase and thermolysin families of enzymes. The active site zinc ligands are all histidines (His-218, His-222, and His-228). The presence of a second zinc ion in a structural role is a unique feature of the matrix metalloproteinases. The binding properties of the active site cleft are more dependent on the main chain conformation of the enzyme (and substrate) compared with thermolysin. A mechanism of action for peptide cleavage similar to that of thermolysin is proposed for fibroblast collagenase.

Structure of human neutrophil collagenase reveals large S1' specificity pocket.

The crystal structure of the catalytic domain of human neutrophil collagenase complexed with a peptide transition state analogue has been determined to a resolution of 2.1 A. The structure of the neutrophil enzyme, when compared with the three dimensional structure of the corresponding human fibroblast collagenase, shows differences in the first, S1', of the three enzyme specificity subsites on the carboxy-terminal side of the substrate scissile bond. The S1' pocket in the neutrophil collagenase is significantly larger than the equivalent site in the fibroblast enzyme, suggesting that the former enzyme has a broader range of possible substrates. Such differences also suggest approaches for the design of selective matrix metalloproteinase inhibitors.