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Interleukin 1 beta (IL-1 beta) processing in murine macrophages requires a structurally conserved homologue of human
S M Molineaux1, F J Casano, A M Rolando
1Department of Molecular Immunology, Merck Research Laboratories, Rahway, NJ 07065.
Abstract:
Murine interleukin 1 beta (IL-1 beta) convertase (mICE) was identified in cytosolic extracts of peritoneal exudate cells (PECs) and macrophage cell lines. mICE cleaves both the human and mouse IL-1 beta precursors (pIL-1 beta) at sites 1 and 2 but fails to cleave a human pIL-1 beta (Asp116 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 beta, is a fluorogenic substrate for mICE, while the tetrapeptide aldehyde Ac-Tyr-Val-Ala-Asp-CHO is a potent inhibitor (Ki = 3 nM) that prevents generation and release of mature IL-1 beta by PECs (IC50 = 7 microM). 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 (Met1-Asp122), followed by a p20 subunit (Gly123-Asp296), a connecting peptide (Ser297-Asp314), and a p10 subunit (Gly315-His402). At the amino acid level, p45, p20, and p10 are 62%, 60%, and 81% identical with human IL-1 beta convertase (hICE). The active site Cys284 lies within a completely conserved stretch of 18 residues; however, Ser289 in hICE, which aligns with the catalytic region of serine and viral cysteinyl proteases, is absent from mICE. Expression in Escherichia coli of a truncated cDNA encoding Asn119-His402 generated active enzyme, which was autocatalytically processed at three internal Asp-Xaa bonds to generate a p20 subunit (Asn119-Asp296) complexed with either p11 (Ala309-His402) or p10. Recombinant mICE cleaves murine pIL-1 beta accurately at the Asp117-Val118 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.
Insights
Murine interleukin 1 beta (IL-1 beta) convertase (mICE) was identified and characterized. This enzyme is crucial for processing IL-1 beta and can be inhibited, offering potential for therapeutic applications in disease models.
Area of Science:
- Biochemistry
- Molecular Biology
- Immunology
Background:
- Interleukin 1 beta (IL-1 beta) is a key inflammatory cytokine.
- The processing of pro-IL-1 beta into its mature, active form is mediated by specific convertases.
- Understanding the murine homolog of human IL-1 beta convertase (hICE) is essential for studying IL-1 beta's role in disease and for developing therapies.
Purpose of the Study:
- To identify and characterize the murine interleukin 1 beta (IL-1 beta) convertase (mICE).
- To elucidate the substrate specificity and catalytic mechanism of mICE.
- To compare mICE with its human counterpart (hICE) and assess the potential for using mICE in disease models.
Main Methods:
- Identification of mICE in cytosolic extracts of peritoneal exudate cells (PECs) and macrophage cell lines.
- Enzymatic assays using fluorogenic substrates and peptide aldehydes to determine substrate specificity and inhibition.
- Cloning and sequencing of the mICE cDNA.
- Expression of recombinant mICE in Escherichia coli and analysis of its processing and activity.
Main Results:
- mICE was identified and shown to cleave murine and human pro-IL-1 beta precursors at specific sites, requiring an aspartic acid residue.
- A fluorogenic substrate and a potent tetrapeptide aldehyde inhibitor (Ki = 3 nM) for mICE were developed.
- The mICE cDNA encodes a 402-amino acid proenzyme (p45) with structural similarities to hICE.
- Recombinant mICE was expressed, autocatalytically processed, and demonstrated accurate cleavage of murine pro-IL-1 beta.
Conclusions:
- Murine IL-1 beta convertase (mICE) shares significant structural and functional similarities with human IL-1 beta convertase (hICE).
- The development of mICE inhibitors provides a tool for studying IL-1 beta in murine models.
- The high degree of similarity between mICE and hICE facilitates the evaluation of hICE inhibitors in preclinical murine disease models.