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.

Nitric oxide (NO) conveys a variety of messages between cells, including signals for vasorelaxation, neurotransmission, and cytotoxicity. In some endothelial cells and neurons, a constitutive NO synthase is activated transiently by agonists that elevate intracellular calcium concentrations and promote the binding of calmodulin. In contrast, in macrophages, NO synthase activity appears slowly after exposure of the cells to cytokines and bacterial products, is sustained, and functions independently of calcium and calmodulin. A monospecific antibody was used to clone complementary DNA that encoded two isoforms of NO synthase from immunologically activated mouse macrophages. Liquid chromatography-mass spectrometry was used to confirm most of the amino acid sequence. Macrophage NO synthase differs extensively from cerebellar NO synthase. The macrophage enzyme is immunologically induced at the transcriptional level and closely resembles the enzyme in cytokine-treated tumor cells and inflammatory neutrophils.

SummaryThe high-output pathway of nitric oxide production helps protect mice from infection by several pathogens, including Mycobacterium tuberculosis. However, based on studies of cells cultured from blood, it is controversial whether human mononuclear phagocytes can express the corresponding inducible nitric oxide synthase (iNOS; NOS2). The present study examined alveolar macrophages fixed directly after bronchopulmonary lavage. An average of 65% of the macrophages from 11 of 11 patients with untreated, culture-positive pulmonary tuberculosis reacted with an antibody documented herein to be monospecific for human NOS2. In contrast, a mean of 10% ofbronchoalveolar lavage cells were positive from each of five clinically normal subjects. Tuberculosis patients' macrophages displayed diaphorase activity in the same proportion that they stained for NOS2, under assay conditions wherein the diaphorase reaction was strictly dependent on NOS2 expression. Bronchoalveolar lavage specimens also contained NOS2 mRNA. Thus, macrophages in the lungs of people with clinically active Mycobacterium tuberculosis infection often express catalytically competent NOS2.

Insulin elicits a spectrum of biological responses by binding to its cell surface receptor. In a screen for small molecules that activate the human insulin receptor tyrosine kinase, a nonpeptidyl fungal metabolite (L-783,281) was identified that acted as an insulin mimetic in several biochemical and cellular assays. The compound was selective for insulin receptor versus insulin-like growth factor I (IGFI) receptor and other receptor tyrosine kinases. Oral administration of L-783,281 to two mouse models of diabetes resulted in significant lowering in blood glucose levels. These results demonstrate the feasibility of discovering novel insulin receptor activators that may lead to new therapies for diabetes.

SummaryA central issue in nitric oxide (NO) research is to understand how NO can act in some settings as a servoregulator and in others as a cytotoxin. To answer this, we have sought a molecular basis for the differential regulation of the two known types of NO synthase (NOS). Constitutive NOS's in endothelium and neurons are activated by agonist-induced elevation of Ca 2+ and resultant binding of calmodulin (CaM). In contrast, NOS in macrophages does not require added Ca 2+ or CaM, but is regulated instead by transcription. We show here that macrophage NOS contains, as a tightly bound subunit, a molecule with the immunologic reactivity, high performance liquid chromatography retention time, tryptic map, partial amino add sequence, and exact molecular mass of CaM. In contrast to most CaM-dependent enzymes, macrophage NOS binds CaM tightly without a requirement for elevated Ca ~+ . This may explain why NOS that is independent of Ca 2+ and elevated CaM appears to be activated simply by being synthesized.

Cpc2/RACK1 is a highly conserved WD domain protein found in all eucaryotes. Cpc2/RACK1 functions on mammalian signal transduction pathways most notably as an adaptor protein for the ␤II protein kinase C isozyme. In single cell eucaryotes, Cpc2/RACK1 regulates growth, differentiation, and entry into G 0 stationary phase. The exact biochemical function of Cpc2/RACK1 is unknown. Here, we provide evidence that Cpc2 is associated with the ribosome. Using immunoaffinity purification, we isolated ribosomal proteins in association with Cpc2/ RACK1. Polysome and ribosomal subunit analysis using velocity gradient centrifugation of cell lysates demonstrated that Cpc2 co-sediments with the 40 S ribosomal subunit and with polysomes. Conditions known to disrupt ribosome structure alter sedimentation of the ribosome and of Cpc2/RACK1 coordinately. Loss of cpc2 does not dramatically alter the rate of cellular protein synthesis but causes a decrease in the steady state level of numerous proteins, some of which regulate methionine metabolism. Whereas real time PCR analysis demonstrated that transcriptional mechanisms are responsible for down-regulation of some of these proteins, one protein, ribosomal protein L25, is probably regulated at the level of translation.

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.acetylation ͉ mass spectrometry W e previously have identified a yeast (Saccharomyces cerevisiae) histone deacetylase HDA1 (1) that was found to have similarity to four other histone deacetylases, RPD3, HOS1, HOS2, and HOS3, inferred from the yeast genome database (2). Of these enzymes, RPD3 is homologous to a mammalian histone deacetylase subunit, HDAC1 (3), that is recruited in a multiprotein complex (4) to various regulatory DNA sequences, such as Mad-Max binding sites, to repress adjacent genes (5, 6). This involves proteins such as mSIN3 and p48, which interact with repressor proteins and histones, respectively (5-7). Similar mechanisms are involved in repression by the unliganded thyroid hormone receptor (8), retinoblastoma protein (9-11), notch protein (12), and other proteins (13-15). In certain HDAC complexes (e.g., Mi2), ATPases are also implicated in histone deacetylase function (16)(17)(18). Surprisingly, although HDAC1 binds small-molecule deacetylase inhibitors such as trichostatin A (19), apicidin (20), and trapoxin (3), it has not yet been shown that HDAC-like proteins have intrinsic deacetylase activity. This suggests that other proteins in the HDAC complex are required for activity of the catalytic subunit.In yeast, the RPD3 deacetylase is also found in a large, multiprotein complex even after extensive enzyme purification (2,21,22). This trichostatin A (TSA)-sensitive enzyme complex is targeted by SIN3 to UME6-regulated genes in a manner similar to that involving Mad-Max (23). Other yeast histone deacetylases that include HDA1, HOS1, and HOS2 are also found in large comple...

Human leukotriene C4 (LTC4) synthase was purified >25,000-fold to homogeneity from the monocytic leukemia cell line THP-1. Beginning with taurocholatesolubilized microsomal membranes, LTC4 synthase was chromatographically resolved by (s) anion exchange, (ig) affinity chromatography (through a resin of biotinylated LTC2 immobilized on streptavidin-agarose), and then (id) gel ifitration. 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, 12SI-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 ofhydrophobic 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.Leukotrienes (LTs) are potent lipid-derived mediators that are released in response to a variety of immunologic and inflammatory stimuli (1, 2). The enzyme LTC4 synthase catalyzes the first committed step in the biosynthesis of the cysteinyl LTs (LTC4, LTD4, and LTE4) that collectively make up the slow-reacting substance of anaphylaxis and appear to play a pivotal role in the pathogenesis of human bronchial asthma (3). LTC4 synthase is a membrane-bound glutathione S-transferase activity that is distinct from a, ,u, ir, 0, and microsomal glutathione S-transferases and appears to be exclusively committed to the biosynthesis of LTC4 (4). Of the known human glutathione S-transferases, it is the only one that has not yet been purified to homogeneity or cloned, due predominantly to the extreme lability of LTC4 synthase in partially purified forms and the lack of a suitably abundant source of the enzyme. Recently, we have described the induction of high levels of LTC4 synthase activity in the human promonocytic leukemia cell line U937 after differentiation into monocyte/ macrophage-like cells by growth in the presence of dimethyl sulfoxide (Me2SO) (4). From the microsomal membranes of these cells, LTC4 synthase was purified >10,000-fold and an 18-kDa membrane polypeptide was identified by photoaffinity labeling as a strong candidate for being LTC4 synthase or a subunit of the enzyme (5). In this report, we describe the purification to homogeneity of human LTC4 synthase from the monocytic leukemia cell line THP-1 and show that like other known glutathione S-transferases, which are all low molecular mass dimeric or trimeric enzymes, human LTC4 synthase is composed of a single 18-kDa polypeptide that is functionally active as a homodimer. MATERIALS AND METHODSCell Growth and Subcellular Fractionation. U937 and HL-60 cells were grown and differe...