Characterization of pulmonary CYP4B2, specific catalyst of methyl oxidation of 3-methylindole.

The selective toxicity of chemicals to lung tissues is predominantly mediated by the selective expression of certain pulmonary cytochrome P450 enzymes. This report describes the purification, cloning, and characterization of a unique enzyme, CYP4B2, from goat lung. The purified P450 enzyme was isolated by multistep ion exchange chromatography to electrophoretic homogeneity with an apparent molecular mass of 55,000 Da. Western blotting studies demonstrated that CYP4B enzymes were selectively expressed in lung tissues of rabbits, rats, and mice. Two cDNAs, CYP4B2 and CYP4B2v, were cloned from goat lung tissue. CYP4B2 was predicted to be 511 amino acids and approximately 82% similar to the four known CYP4B1 proteins. Concurrently, a variant of the known human CYP4B1 cDNA, that contained a S207 insertion, was cloned from human lung tissue. The modified recombinant goat CYP4B2 was expressed in Escherichia coli and the enzyme catalyzed the N-hydroxylation of the prototypical substrate 2AF. CYP4B2 preferentially dehydrogenated, rather than hydroxylated, the pneumotoxicant 3-methylindole (3MI) (V(max) = 4.61 versus 0.83 nmol/nmol of P450/min, respectively). To investigate the relevance of covalent heme binding of CYP4 enzymes in CYP4B2-mediated metabolism of 3MI, a site-directed mutant (CYP4B2/A315E) was evaluated. The mutation had little effect on the V(max) of either dehydrogenation or hydroxylation but increased the K(m), which decreased the catalytic efficiency (V/K) for 3MI. The A315E mutation shifted the absorbance maximum of the enzyme from 448 to 451 nm, suggesting that the electron density of the heme was altered. These results demonstrate that CYP4B2 is highly specific for methyl group oxidation of 3MI, without formation of ring-oxidized metabolites, and seems to be predominately responsible for the highly organ-specific toxicity of 3MI in goats.

Cytokine traps: multi-component, high-affinity blockers of cytokine action.

Cytokines can initiate and perpetuate human diseases, and are among the best-validated of therapeutic targets. Cytokines can be blocked by the use of soluble receptors; however, the use of this approach for cytokines such as interleukin (IL)-1, IL-4, IL-6 and IL-13 that use multi-component receptor systems is limited because monomeric soluble receptors generally exhibit low affinity or function as agonists. We describe here a generally applicable method to create very high-affinity blockers called 'cytokine traps' consisting of fusions between the constant region of IgG and the extracellular domains of two distinct cytokine receptor components involved in binding the cytokine. Traps potently block cytokines in vitro and in vivo and represent a substantial advance in creating novel therapeutic candidates for cytokine-driven diseases.