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.
The mechanism of replication of the simian virus 40 (SV40) genome closely resembles that of cellular chromosomes, thereby providing an excellent model system for examining the enzymatic requirements for DNA replication. Only one viral gene product, the large tumour antigen (large-T antigen), is required for viral replication, so the majority of replication enzymes must be cellular. Indeed, a number of enzymatic activities associated with replication and the S phase of the cell cycle are induced upon SV40 infection. Cell-free extracts derived from human cells, when supplemented with immunopurified SV40 large-T antigen support efficient replication of plasmids that contain the SV40 origin of DNA replication. Using this system, a cellular protein of relative molecular mass 36,000 (Mr = 36K) that is required for the elongation stage of SV40 DNA replication in vitro has been purified and identified as a known cell-cycle regulated protein, alternatively called the proliferating cell nuclear antigen (PCNA) or cyclin. It was noticed that, in its physical characteristics, PCNA closely resembles a protein that regulates the activity of calf thymus DNA polymerase-delta. Here we show that PCNA and the polymerase-delta auxiliary protein have similar electrophoretic behaviour and are both recognized by anti-PCNA human autoantibodies. More importantly, both proteins are functionally equivalent; they stimulate SV40 DNA replication in vitro and increase the processivity of calf thymus DNA polymerase-delta. These results implicate a novel animal cell DNA polymerase, DNA polymerase-delta, in the elongation stage of replicative DNA synthesis in vitro.
Cell-free extracts prepared from human 293 cells, supplemented with purified SV40 large-T antigen, support replication of plasmids containing the SV40 origin of DNA replication. A cellular protein (Mr approximately 36,000) that is required for efficient SV40 DNA synthesis in vitro has been purified from these extracts. This protein is recognized by human autoantibodies and is identified as the cell-cycle regulated protein known as proliferating cell nuclear antigen (PCNA) or cyclin.
Interleukin 1 (IL-1) is a lymphokine secreted by monocytes in response to a variety of inflammatory stimuli. IL-1fB the predominant form of IL-1 produced by human monocytes, is synthesized as an inactive precursor of 31 kDa and is cleaved at Asp"'6-Ala"17 to yield a 17.5-kDa extracellular form. The exact cellular site of cleavage and mechanism of secretion is at present unknown. We have prepared cell-free postnuclear extracts from freshly isolated human monocytes as well as THP. (1) provided the first substantive evidence that, in a mouse monocyte cell line, IL-1 was synthesized as a cell-associated precursor that could be chased into an extracellular 17-kDa form. Subsequently, reports emerged which suggested that a 31-kDa form of IL-1i3 was associated with human monocytes (2-5) and that this material was cleaved to release the mature form (2, 4, 5). These studies were corroborated by cDNA sequence data from a number of species which indicated that IL-1 mRNA encodes a larger protein than that identified as mature secreted IL-1 (6-10). As precursor IL-183 (pre-IL-1,j)is unable to bind to IL-1 receptors and is biologically inactive (11), some form of proteolytic processing is apparently required to release active IL-1p8. While the kinetics of IL-1 synthesis and secretion has been analyzed in some detail, little has been uncovered about the mechanism by which IL-1 is synthesized, processed, and secreted. Analysis ofthe predicted amino acid sequence from pre-IL-1,3 cDNA has not revealed the presence of a unique hydrophobic signal sequence domain, common to most secreted proteins (6)(7)(8)(9)(10)12). The N-terminal amino acid of mature monocyte IL-1p from humans has been sequenced by a number of investigators as Ala"17 (6, 13), suggesting that a cleavage site exists between Asp'6 and Ala"7. While the first 116 residues may be considered a signal sequence of sorts, it is not recognized as such by otherwise competent endoplasmic reticulum membranes (G.L., unpublished observation). Young et al. (14) showed that mature pre-IL-1P was not secreted from hamster fibroblasts that were stably transformed with pre-IL-1,i cDNA. Instead, large amounts of the precursor accumulated in the cytoplasm of the cell (14). Lomedico et al. (12) The processing of IL-1f3 has recently been investigated by using purified recombinant precursor as a substrate (5, 17).Hazuda et al. (5) showed that pre-IL-1f3, when added to intact human blood monocytes, was not cleaved or processed in any fashion, arguing against an extracellular site of processing. In another report, a potential pre-IL-1,8 cleavage activity was identified in a pelletable compartment of KG-1 cells, a neutrophil-like cell line. This enzymatic activity was able to generate IL-1 activity of similar size to authentic IL-1 from a partially purified pre-IL-1f3 substrate (17). However, the products were not sequenced and the site ofcleavage was not identified.In this report, we describe an in vitro processing system in which mature 17.
The double-stranded RNA (dsRNA)-dependent protein kinase DAI (also termed dsl and P1) possesses two kinase activities; one is an autophosphorylation activity, and the other phosphorylates initiation factor eIF-2. We purified the enzyme, in a latent form, to near homogeneity from interferon-treated human 293 cells. The purified enzyme consisted of a single polypeptide subunit of -70,000 daltons, retained its dependence on dsRNA for activation, and was sensitive to inhibition by adenovirus VA RNA,. Autophosphorylation required a suitable concentration of dsRNA and was second order with respect to DAI concentration, which suggests an intermolecular mechanism in which one DAI molecule phosphorylates a neighboring molecule. Once autophosphorylated, the enzyme could phosphorylate eIF-2 but seemed unable to phosphorylate other DAI molecules, which implies a change in substrate specificity upon activation. VA RNA, blocked autophosphorylation and activation but permitted the activated enzyme to phosphorylate eIF-2. VA RNA, also blocked the binding of dsRNA to the enzyme. The data are consistent with a model in which activation requires the interaction of two molecules of DAI with dsRNA, followed by intermolecular autophosphorylation of the latent enzyme. VA RNA, would block activation by preventing the interaction between DAI and dsIINA.Phosphorylation of the a subunit of eucaryotic protein synthesis initiation factor eIF-2 (eIF-2oa) prevents catalytic recycling of the protein in polypeptide chain initiation and results in the inhibition of protein synthesis (reviewed in reference 30). Control of translational initiation by this mechanism has been demonstrated, or at least inferred, in a variety of systems, including hemin-deprived rabbit reticulocytes, virus-infected mammalian cells, and cells subjected to heat shock or changes in redox state (see reference 21 for reviews). Although many effectors and environmental conditions influence the level of eIF-2 phosphorylation, only two protein kinases that are capable of catalyzing this reaction have been identified (26): the hemin-controlled regulator HCR, found chiefly in reticulocytes, and the double-stranded RNA (dsRNA)-activated inhibitor (DAI, also known as P1, dsl, and PKdS). DAI, also found in reticulocytes, is present at low levels in most mammalian cells and tissue culture cell lines and at greatly increased levels after interferon treatment (reviewed in references 1, 10, and 19). The enzyme plays a role in establishment of the antiviral state, and several viruses have elaborated defense measures to counter this form of interference in their replication (12,14,15,29,35,36,40,43). Viruses may also exploit the enzyme to achieve selective production of their own proteins (12, 26a). The ubiquity of the enzyme suggests that it may function in normal, uninfected cells (for example, during growth and differentiation events [31]), but this idea remains to be fully substantiated.Exposure of cell extracts containing DAI to dsRNA results in phosphorylation of a protein of -70 ...
Novel potent and selective diarylimidazole inhibitors of p38 MAP (mitogen-activated protein) kinase are described which have activity in both cell-based assays of tumor necrosis factor-alpha (TNF-alpha) release and an animal model of rheumatoid arthritis. The SAR leading to the development of selectivity against c-Raf and JNK2alpha1 kinases is presented, with key features being substitution of the 4-aryl ring with m-trifluoromethyl and substitution of the 5-heteroaryl ring with a 2-amino substituent. Cell-based activity was significantly enhanced by incorporation of a 4-piperidinyl moiety at the 2-position of the imidazole which also enhanced aqueous solubility. In general, oral bioavailability of this class of compounds was found to be poor unless the imidazole was methylated on nitrogen. This work led to identification of 48, a potent (p38 MAP kinase inhibition IC50 0.24 nM) and selective p38 MAP kinase inhibitor which inhibits lipopolysaccharide-stimulated release of TNF-alpha from human blood with an IC50 2.2 nM, shows good oral bioavailability in rat and rhesus monkey, and demonstrates significant improvement in measures of disease progression in a rat adjuvant-induced arthritis model.
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