We have sequenced the structural gene and flanking regions for lanosterol 14 alpha-demethylase (14DM) from Saccharomyces cerevisiae. An open reading frame of 530 codons encodes a 60.7-kDa protein. When this gene is disrupted by integrative transformation, the resulting strain requires ergosterol and, as expected, grows only in the absence of oxygen. The deduced amino acid sequence of 14DM includes a hydrophobic segment near the amino terminus which may be a transmembrane domain. The deduced sequence has been compared with those of eight other eukaryotic P450s, each from a different family within the P450 superfamily. These comparisons indicate that this yeast gene is the first member of a new P450 family, P450LI. The P450, designated P450LIA1, is more closely related to mammalian P450s than to the bacterial P450cam. In fact, both the yeast P450 and several mammalian P450s have equivalent alignment scores when each is compared with the bovine P450scc. Matrix comparisons of the amino acid sequence of this P450 with those of mammalian P450s reveal three conserved regions. The DNA region 5' to the structural 14DM gene includes poly(dA:dT) sequences and a repeating hexamer sequence.

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A wide range of immune and inflammatory responses are influenced by the cytokine IL-1 1 (1). The two agonists IL-1␣ and IL-1␤ can cause both local and systemic inflammatory responses. Prolonged expression of even low levels of IL-1 can have detrimental consequences in chronic inflammatory diseases such as rheumatoid arthritis (2) and inflammatory bowel disease (3). The naturally occurring antagonist IL-1ra plays an important role in physiological regulation of IL-1-induced activities. The three IL-1 ligands, IL-1␣, IL-1␤ and IL-1ra, bind to the two IL-1 receptors (IL-1R), type I and type II (1). Only the type I receptor induces signaling upon binding of IL-1␣ or -␤. The type II receptor appears to act as a negative regulator of activity by effectively competing for binding of ligand and thus regulating IL-1 concentrations (4). The type II receptor released from cells selectively binds IL-1␤ or -␣ over IL-1ra, therefore having little effect on the inhibitory action of the antagonist (5).IL-1 antagonists, including IL-1ra and soluble IL-1R, have been therapeutically effective in animal models of inflammatory disease and are currently in human trials. Recent studies have shown that IL-1ra can reduce some of the symptoms of established disease in model systems, bacterial cell wall-induced arthritis in rats (6), antigen-induced rheumatoid arthritis in rabbits (7), and lung fibrosis in mice (8). A small, active IL-1 antagonist would have several advantages over the recombinant IL-1ra protein including ease of synthesis, the ability to deliver locally in high concentrations, and possible oral delivery. We have screened large libraries of recombinant peptides in search of IL-1 receptor antagonists. The peptide described here, AF12198, is derived from a family of peptide antagonists we recently reported (9). AF12198, Ac-FEWTPGWYQJYALPL-NH 2 , binds specifically to the human type I IL-1R and blocks both in vitro and in vivo IL-1-induced activities. This peptide is the first low molecular weight IL-1 receptor antagonist to show in vivo activity. EXPERIMENTAL PROCEDURESPeptide Synthesis-Routine peptide synthesis was performed on an Advanced Chemtech model ACT350 multiple peptide synthesizer using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry (10), 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate activation, and polystyrene resin derivatization with trifluoroacetic acid-labile Knorr linker. Peptides were cleaved from the resin using a solution of 95:5 trifluoroacetic acid/water and appropriate scavengers for 90 min. The cleavage solution was then filtered through a glass wool plug, concentrated to dryness, and the peptide purified to homogeneity by reverse-phase (C18) high performance liquid chromatography (HPLC) (water/acetonitrile gradient). Peptides prepared in this fashion afforded satisfactory results upon characterization by analytical HPLC, mass spectrometry, and high resolution mass spectrometry.Binding Analysis-Competition binding analysis was as described (9). The soluble extracellular dom...