Mimicry of erythropoietin by a nonpeptide molecule.

Erythropoietin (EPO) controls the proliferation and differentiation of erythroid progenitor cells into red blood cells. EPO induces these effects by dimerization of the EPO receptors (EPOR) present on these cells. To discover nonpeptide molecules capable of mimicking the effects of EPO, we identified a small molecule capable of binding to one chain of EPOR and used it to synthesize molecules capable of inducing dimerization of the EPOR. We first identified compound 1 (N-3-[2-(4-biphenyl)-6-chloro-5-methyl]indolyl-acetyl-L-lysine methyl ester) by screening the in-house chemical collection for inhibitors of EPO binding to human EPOR and then prepared compound 5, which contains eight copies of compound 1 held together by a central core. Although both compounds inhibited EPO binding of EPOR, only compound 5 induced dimerization of soluble EPOR. Binding of EPO to its receptor in cells results in activation of many intracellular signaling molecules, including transcription factors like signal transducer and activator of transcription (STAT) proteins, leading to growth and differentiation of these cells. Consistent with its ability to induce dimerization of EPOR in solution, compound 5 exhibited much of the same biological activities as EPO, such as (i) the activation of a STAT-dependent luciferase reporter gene in BAF3 cells expressing human EPOR, (ii) supporting the proliferation of several tumor cell lines expressing the human or mouse EPOR, and (iii) the in vitro differentiation of human progenitor cells into colonies of erythrocytic lineage. These data demonstrate that a nonpeptide molecule is capable of inducing EPOR dimerization and mimicking the biological activities of EPO.

Yeast HOS3 forms a novel trichostatin A-insensitive homodimer with intrinsic histone deacetylase activity.

Histone deacetylases such as human HDAC1 and yeast RPD3 are trichostatin A (TSA)-sensitive enzymes that are members of large, multiprotein complexes. These contain specialized subunits that help target the catalytic protein to histones at the appropriate DNA regulatory element, where the enzyme represses transcription. To date, no deacetylase catalytic subunits have been shown to have intrinsic activity, suggesting that noncatalytic subunits of the deacetylase complex are required for their enzymatic function. In this paper we describe a novel yeast histone deacetylase HOS3 that is relatively insensitive to the histone deacetylase inhibitor TSA, forms a homodimer when expressed ectopically both in yeast and Escherichia coli, and has intrinsic activity when produced in the bacterium. Most HOS3 protein can be found associated with a larger complex in partially purified yeast nuclear extracts, arguing that the HOS3 homodimer may be dissociated from a very large nuclear structure during purification. We also demonstrate, using a combination of mass spectrometry, tandem mass spectrometry, and proteolytic digestion, that recombinant HOS3 has a distinct specificity in vitro for histone H4 sites K5 and K8, H3 sites K14 and K23, H2A site K7, and H2B site K11. We propose that while factors that interact with HOS3 may sequester the catalytic subunit at specific cellular sites, they are not required for HOS3 histone deacetylase activity.

Expression and immunoaffinity purification of human inducible nitric-oxide synthase. Inhibition studies with 2-amino-5,6-dihydro-4H-1,3-thiazine.

Recombinant human inducible nitric-oxide synthase (rH-iNOS) was expressed in the baculovirus system and purified by a novel immunoaffinity column. rH-iNOS and its native counterpart from cytokine-stimulated primary hepatocytes exhibited similar molecular mass of 130 kDa on SDS-polyacrylamide gel electrophoresis, recognition by antipeptide antibodies, specific activities, and IC50 values for inhibitors. The active dimeric form exhibited a specific activity range of 114-260 nmol/min/mg at 37 degrees C and contained 1.15 +/- 0.04 mol of calmodulin/monomer. The enzyme exhibited a Soret lambdamax at 396 nm with a shoulder at 460 nm and contained 0. 28-0.64 mol of heme/monomer. Dithionite reduction under CO yielded an absorbance maximum at 446 nm, indicating a P450-type heme. Imidazole induced a type II difference spectrum, reversible by L-Arg. 2-Amino-5,6-dihydro-4H-1,3-thiazine (ADT) was competitive versus L-Arg (Ki = 22.6 +/- 1.9 nM), reversed the type II difference spectrum induced by imidazole (Kd = 17.7 nM), and altered the CO-ferrous absorbance of rH-iNOS. L-Arg did not perturb the CO-ferrous adduct directly, but it partially reversed the ADT-induced absorbance shift, indicating that both bind similarly to the protein but interact differently with the heme.

Active-site structure analysis of recombinant human inducible nitric oxide synthase using imidazole.

Nitric oxide synthase catalyzes the pyridine nucleotide-dependent oxidation of L-arginine to nitric oxide and L-citrulline. It is a specialized cytochrome P450 monooxygenase that is sensitive to inhibition by imidazole. Steady-state kinetic studies on recombinant human inducible nitric oxide synthase (rH-iNOS) demonstrate that imidazole and 1-phenylimidazole are competitive and reversible inhibitors versus L-arginine. Structure-activity relationship and pH dependence studies on the inhibition suggest that the neutral form of imidazole may be the preferred species and that the only modifications allowed without the loss of inhibition are at the N-1 position of imidazole. Optical spectrophotometric studies of rH-iNOS with imidazole and 1-phenylimidazole yielded type II difference spectra exhibiting Kd values of 63 +/- 2 and 28 +/- 3 microM, respectively. These values were in good agreement with the steady-state Ki of 95 +/- 10 and 38 +/- 4 microM, respectively, and confirms the site of binding is at the sixth axial ligand of the heme. Imidazole (2.2 mM) also perturbed the Kd of L-arginine from 3.03 +/- 0.45 to 209 +/- 10 microM. The observed increase in the Kd for L-arginine is consistent with imidazole being a competitive inhibitor versus L-arginine. The IC50 values of imidazole and 1-phenylimidazole were lower in the absence of exogenous BH4, and both inhibitors also competitively inhibited the BH4-dependent activation of the enzyme. These data taken together suggest that the L-arginine, dioxygen, and the BH4 binding sites are in close proximity in rH-iNOS. Furthermore, these studies demonstrate the usefulness of imidazole compounds as active site probes for recombinant human iNOS.

Use of an antibody against the matrix metalloproteinase-generated aggrecan neoepitope FVDIPEN-COOH to assess the effects of stromelysin in a rabbit model of cartilage degradation.

OBJECTIVE: To define the stromelysin cleavage site in the interglobular domain of rabbit aggrecan, and to determine whether the stromelysin-generated neoepitope can be used as a marker of matrix metalloproteinase (MMP) activity in vivo. METHODS: The carboxy-terminus sequence of the stromelysin-generated hyaluronic acid-binding region (HABR) of rabbit aggrecan was determined by reverse transcription-polymerase chain reaction complementary DNA cloning and DNA sequence analysis, followed by purification and mass spectral protein sequence analysis of the HABR fragment. Active stromelysin was injected into the stifle joints of rabbits, and a stromelysin-generated aggrecan neoepitope was analyzed by Western blotting and localized in situ by indirect immunofluorescence. Proteoglycan fragments in joint fluids were quantified by a dimethylmethylene blue dye-binding assay. RESULTS: Stromelysin cleavage of rabbit aggrecan generated a 55-kd HABR fragment that terminated in the sequence FMDIPEN: An anti-FVDIPEN antibody recognized the FMDIPEN neoepitope in situ in cartilage from stromelysin-injected joints. The appearance of the FMDIPEN neoepitope corresponded to the release of cartilage proteoglycan fragments into the joint fluid, and could be inhibited by pretreatment of the rabbits with a synthetic stromelysin inhibitor. CONCLUSION: These results indicate that the anti-FVDIPEN antibody can be used to assess the role of MMPs in cartilage degradation in vivo.

Interleukin 1 beta (IL-1 beta) processing in murine macrophages requires a structurally conserved homologue of human IL-1 beta converting enzyme.

Murine interleukin 1 beta (IL-1 beta) convertase (mICE) was identified in cytosolic extracts of peritoneal exudate cells (PECs) and macrophage cell lines. mICE cleaves both the human and mouse IL-1 beta precursors (pIL-1 beta) at sites 1 and 2 but fails to cleave a human pIL-1 beta (Asp116 to Ala) mutant at site 2, indicating that Asp is required to the left of the scissile bond. Ac-Tyr-Val-Ala-Asp-amino-4-methyl coumarin, patterned after site 2 of human pIL-1 beta, is a fluorogenic substrate for mICE, while the tetrapeptide aldehyde Ac-Tyr-Val-Ala-Asp-CHO is a potent inhibitor (Ki = 3 nM) that prevents generation and release of mature IL-1 beta by PECs (IC50 = 7 microM). Cloning of a full-length 1.4-kb cDNA shows that mICE is encoded as a 402-aa proenzyme (p45) that can be divided into a prodomain (Met1-Asp122), followed by a p20 subunit (Gly123-Asp296), a connecting peptide (Ser297-Asp314), and a p10 subunit (Gly315-His402). At the amino acid level, p45, p20, and p10 are 62%, 60%, and 81% identical with human IL-1 beta convertase (hICE). The active site Cys284 lies within a completely conserved stretch of 18 residues; however, Ser289 in hICE, which aligns with the catalytic region of serine and viral cysteinyl proteases, is absent from mICE. Expression in Escherichia coli of a truncated cDNA encoding Asn119-His402 generated active enzyme, which was autocatalytically processed at three internal Asp-Xaa bonds to generate a p20 subunit (Asn119-Asp296) complexed with either p11 (Ala309-His402) or p10. Recombinant mICE cleaves murine pIL-1 beta accurately at the Asp117-Val118 bond. The striking similarities of the human and murine enzymes will make it possible to assess the therapeutic potential of hICE inhibitors in murine models of disease.

Purification to homogeneity and the N-terminal sequence of human leukotriene C4 synthase: a homodimeric glutathione S-transferase composed of 18-kDa subunits.

Human leukotriene C4 (LTC4) synthase was purified > 25,000-fold to homogeneity from the monocytic leukemia cell line THP-1. Beginning with taurocholate-solubilized microsomal membranes, LTC4 synthase was chromatographically resolved by (i) anion exchange, (ii) affinity chromatography (through a resin of biotinylated LTC2 immobilized on streptavidin-agarose), and then (iii) gel filtration. The final preparation contained only an 18-kDa polypeptide. The molecular mass of the pure polypeptide was consistent with an 18-kDa polypeptide from THP-1 cell membranes that was specifically photolabeled by an LTC4 photoaffinity probe, 125I-labeled azido-LTC4. On calibrated gel-filtration columns, purified LTC4 synthase activity eluted at a volume corresponding to 39.2 +/- 3.3 kDa (n = 12). The sequence of the N-terminal 35 amino acids was determined and found to be a unique sequence composed predominantly of hydrophobic amino acids and containing a consensus sequence for protein kinase C phosphorylation. We therefore conclude that human LTC4 synthase is a glutathione S-transferase composed of an 18-kDa polypeptide that is enzymatically active as a homodimer and may be phosphoregulated in vivo.

A novel heterodimeric cysteine protease is required for interleukin-1 beta processing in monocytes.

Interleukin-1 beta (IL-1 beta)-converting enzyme cleaves the IL-1 beta precursor to mature IL-1 beta, an important mediator of inflammation. The identification of the enzyme as a unique cysteine protease and the design of potent peptide aldehyde inhibitors are described. Purification and cloning of the complementary DNA indicates that IL-1 beta-converting enzyme is composed of two nonidentical subunits that are derived from a single proenzyme, possibly by autoproteolysis. Selective inhibition of the enzyme in human blood monocytes blocks production of mature IL-1 beta, indicating that it is a potential therapeutic target.