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.
Antigen recognition by the T-cell receptor (TCR) initiates events including lymphokine gene transcription, particularly interleukin-2, that lead to T-cell activation. The immunosuppressive drugs, cyclosporin A (CsA) and FK-506, prevent T-cell proliferation by inhibiting a Ca(2+)-dependent event required for induction of interleukin-2 transcription. Complexes of FK-506 or CsA and their respective intracellular binding proteins inhibit the calmodulin-dependent protein phosphatase, calcineurin, in vitro. The pharmacological relevance of this observation to immunosuppression or drug toxicity is undetermined. Calcineurin, although present in lymphocytes, has not been implicated in TCR-mediated activation of lymphokine genes or in transcriptional regulation in general. Here we report that transfection of a calcineurin catalytic subunit increases the 50% inhibitory concentration (IC50) of the immunosuppressants FK-506 and CsA, and that a mutant subunit acts in synergy with phorbol ester alone to activate the interleukin-2 promoter in a drug-sensitive manner. These results implicate calcineurin as a component of the TCR signal transduction pathway by demonstrating its role in the drug-sensitive activation of the interleukin-2 promoter.
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 interleukin-2 (IL-2) promoter consists of several independent T cell receptor (TcR) responsive elements. The induction of promoters dependent on these elements is inhibitable by the immunosuppressants cyclosporin A (CsA) and tacrolimus . Calcineurin, a Ca2+/calmodulin-dependent protein phosphatase, is the FK-506-and CsA-sensitive enzyme required for TcR mediated activation of the LL-2 promoter. We report that a constitutively active form of calcineurin partially substitutes for the Ca2+ co-stimulus required to activate the IL-2 promoter elements IL-2A (which binds the factors OAP and Oct-i) and LL-2E (which binds NF-AT), and completely substitutes for the Ca2+ co-stimulus required to stimulate an NF-xB-dependent element. Calcineurin stimulates the NF-xB element by enhancing inactivation of IxB/MAD3, an inhibitor of NF-xB, thereby increasing the amount of nuclear NF-xB DNA binding activity. These data provide the first demonstration in vivo that activation of a protein phosphatase can inactivate IxB, and suggest one possible explanation for mechanism-based toxicities associated with FK-506 and CsA by demonstrating that these drugs can inhibit the calcineurin-dependent activation of a virtually ubiquitous transcription factor.
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