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.

Active interleukin-1beta-converting enzyme (ICE) is composed of 20- and 10-kDa polypeptides (p20 and p10) derived from the processing of a cytosolic 45-kDa precursor protein (p45). The cleavage and activation of the native p45 ICE precursor have been characterized by use of specific inhibitors and antibodies recognizing various regions of ICE. The processing of p45 in vitro in THP.1 monocytic cell cytoplasmic extracts is inhibited only by protease inhibitors that inhibit ICE and not by inhibitors of other protease classes. The addition of L-742,395, a biotinylated irreversible ICE inhibitor, to these extracts labels only p45 and simultaneously inhibits p45 processing, demonstrating that the p45 has catalytic activity. Following a cleavage of p45 at a site that becomes the COOH terminus of p20, a more active intermediate is formed which migrates on SDS-polyacrylamide gel electrophoresis with an molecular mass of 35 kDa (ED50 of approximately 0.1 microM L-742,395 labeling versus 5 microM for p45). This new more active ICE form serves both as an intermediate enzyme to cleave p45 as well as a substrate for the formation of the final active ICE (ED50 of 1 nM L-742,395 labeling of p20 and for p22, an NH2-terminally extended form of p20). While initial cleavage of p45 can be found at the sites corresponding to both the NH2 termini of p22 and p20, these fragments cannot be labeled by L-742,395 and are hence inactive. p45 is not processed at the site corresponding to the NH2 terminus of the p10. Less than 50% of the p45 is cleaved down to active p20 or p22 ICE as determined by band shift on SDS-polyacrylamide gel electrophoresis of the biotinylated fragments, indicating that the in vitro activation is highly inefficient. The ICE fragmentation occurs by an intermolecular process and is highly dilution sensitive. Cleavage of p45 by exogenous p20/p10 ICE differs from that of the endogenous p45 cleavage activity in that the p20/p10 activity is more salt sensitive, and it produces a different pattern of cleavage fragments, principally 35- and 12-kDa fragments. These results indicate that the nature of the ICE activity changes as p45 is processed down to the p20/p10 form of the enzyme.

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Endopeptidase 24.15, a metalloendopeptidase (EC 3.4.24.15) with an Mr of about 70,000, was purified to homogeneity from rat testes. The enzyme cleaves preferentially bonds on the carboxyl side of hydrophobic amino acids. Secondary enzyme-substrate interactions at sites removed from the scissile bond are indicated by the finding that a hydrophobic or bulky residue in the P3' position greatly contributes to substrate binding and catalytic efficiency. The isolated enzyme is inhibited by metal chelators and by thiols. Loss of enzymic activity after dialysis against EDTA can be restored by low concentrations of Zn2+ and Co2+ ions. The rate of reaction of the Co2+ enzyme with a synthetic substrate was higher than that of the Zn2+ enzyme. These results are consistent with the classification of the enzyme as a metalloendopeptidase. N-Carboxymethyl peptides that fulfil the binding requirements of the substrate recognition site of the enzyme act as potent competitive inhibitors. Biologically active peptides such as luteinizing hormone-releasing hormone, bradykinin and neurotensin are cleaved at sites consistent with the specificity of the enzyme deduced from studies with synthetic peptides. Dynorphin A (1-8)-peptide, beta-neoendorphin, metorphamide, and Metenkephalin-Arg6-Gly7-Leu8 are rapidly converted to the corresponding enkephalins. The testis enzyme is catalytically and immunologically closely related to the previously identified brain enzyme.

Objective-Leukotriene B 4 (LTB 4 ), a product of the 5-lipoxygenase (5-LO) pathway of arachidonic acid metabolism, has been implicated in atherosclerosis. However, the molecular mechanisms for the atherogenic effect of LTB 4 are not well understood. This study is to determine candidate mechanisms. Method and Results-Primary human monocytes were treated with LTB 4 and the supernatant was analyzed for cytokine/chemokine production by an immuno-protein array. This analysis revealed a strong increase of the monocyte chemoattractant protein-1 (MCP-1), a proinflammatory cytokine. See page 1748Recent studies suggest a strong link between LTB 4 pathways with atherosclerosis. For example, human atherogenic plaques produce LTB 4 , 7 and expression of both BLT 1 and BLT 2 increases with the progression of atherosclerotic lesions. 8 Treatment of atherosclerosis-prone mice (apolipoprotein E [ApoE] or low-density lipoprotein receptor [LDLR]-deficient mice) with the BLT 1 -specific antagonist CP-105,696 9,10 markedly decreased lesion size. 11 Interestingly, the anti-atherogenic effects of CP-105,696 were diminished in mice deficient in the chemoattractant monocyte chemotactic protein-1 (MCP-1), 11 indicating a critical role for MCP-1 in mediating the LTB 4 atherogenic signals.MCP-1 is a prototype of the C-C chemokine ␤ subfamily and exhibits the most potent chemotactic activity for monocytes. 12 Overexpression of MCP-1 contributes to the development of atherosclerosis in mouse models. 13 Deficiency of either MCP-1 or its cognate high-affinity receptor C-C chemokine receptor 2 (CCR2) results in a marked decrease in atheromas and fewer monocytes in vascular lesions. 14,15 Additionally, therapeutic gene transfer of a dominantnegative MCP-1 mutant attenuated the development of early atherosclerosis and also limited progression of preexisting atherosclerotic lesions in ApoE-null mice. 16 Despite the critical role played by LTB 4 in atherogenesis, the molecular mechanisms for these activities are poorly understood. In this study, we investigated specifically whether LTB 4 regulates MCP-1 production in primary human monocytes to broaden the mechanistic understanding for LTB 4 -induced atherosclerosis. Our study shows that LTB 4 induced MCP-1 protein by several hundred-fold in primary human monocytes. LTB 4 induced MCP-1 mRNA by 500-fold Original