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.

Cysteine proteases related to mammalian interleukin-1 beta-converting enzyme (ICE) and the nematode cell death abnormal ced-3 gene product have been implicated in the effector mechanism of apoptotic cell death. Two novel members of this new family of ICE/CED-3-related proteases, designated ICErel-II and ICErel-III, were cloned from human monocytic cells. Both were highly homologous to human ICE (52% identical) and CED-3 (25% identical) and both contained the absolutely conserved pentapeptide sequence Gln-Ala-Cys-Arg-Asp containing the catalytic cysteine residue. Other structural motifs that were comparable with ICE suggest that ICErel-II and ICErel-III are also synthesized as larger proenzymes which are proteolytically processed to form heterodimeric active enzymes. Pro-interleukin-1 beta processing activity could not be detected in cells transfected with ICErel-II or ICErel-III, but pro-domain-less truncated forms of ICErel-II and ICErel-III were capable of effectively inducing fibroblast apoptosis. ICErel-II and ICErel-III may, therefore, participate in proteolytic events culminating in the apoptotic death of human cells.

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Murine interleukin 1p (IL-10) convertase (mICE) was identified in cytosolic extracts of peritoneal exudate cells (PECs) and macrophage cell lines. mICE cleaves both the human and mouse IL-1lB precursors (pIL-l() at sites 1 and 2 but fails to cleave a human pIL-1p (Asp"" 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(3, is a fluorogenic substrate for mICE, while the tetrapeptide aldehyde Ac-Tyr-Val-AlaAsp-CHO is a potent inhibitor (K, = 3 nM) that prevents generation and release of mature IL-11 by PECs (IC,5 = 7 pM). 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 (Met'-Asp'22), followed by a p20 subunit (Gly"AAsp296), a connecting peptide (Ser97-Asp3l4), and a plO subunit (Gly9'-His402). At the amino acid level, p45, p20, and plO are 62%, 60%, and 81% identical with human IL-13 convertase (hICE). The active site Cys284 lies within a completely conserved stretch of 18 residues; however, Ser2" in hICE, which aligns with the catalytic region of serine and viral cysteinyl proteases, is absent from mICE. Expression in Escherchia coli of a truncated cDNA encoding Asn"'-Hism generated active enzyme, which was autocatalytically processed at three internal Asp-Xaa bonds to generate a p20 subunit (Asn"'-Asp296) complexed with either pll (Ala-0"9-His42) or plO. Recombinant mICE cleaves murine pIL-1P accurately at the Asp"17-Val"'8 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.Interleukin 13 (IL-1p) is synthesized as a 31-kDa precursor polypeptide (pIL-108) that must be proteolytically cleaved to generate the active 17.5-kDa cytokine (1, 2). In stimulated human monocytes, processing requires a specific protease, termed IL-1p converting enzyme (ICE), which cleaves pIL-1,B at site 1 (Asp27-Gly28) and site 2 (Asp"16-Ala"7) to yield 28-and 17.5-kDa products, respectively (3). The recent purification and cloning of human ICE (hICE) (4,5) have revealed that the enzyme is a unique heterodimeric cysteine protease with a highly unusual requirement for Asp to the left of the scissile bond (P1). The active enzyme consists of a 1:1 stoichiometric complex of 19.8-(p20) and 10.2-(plO) kDa subunits, which are derived from a single 45.2-kDa (p45) proenzyme (4). In the proenzyme, Asp-Xaa bonds flank both subunits, as well as an alternatively processed form of p20, termed p24, suggesting that autoproteolysis may be involved in generating the heterodimeric (p20-plO) form of the enzyme (4).Inhibition of hICE prevents both cleavage of the IL-113 precursor and release ofthe mature cytokine from monocytes in lipopolysaccharide (LPS)-stimulated whole blood (4) and thus may represent a therapeutic approach for treating inflammatory diseases. To validate ICE as a therapeutic target, it will be necessary to establish ...