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Identification of a monocyte specific pre-interleukin 1 beta convertase activity
M J Kostura1, M J Tocci, G Limjuco
1Department of Biochemical and Molecular Pathology, Merck Sharp & Dohme Research Laboratories, Rahway, NJ 07065.
Summary
A specific protease found in monocytes cleaves inactive Interleukin-1 beta (IL-1 beta) precursor into its active form. This discovery sheds light on the inflammatory response mechanism.
Area of Science:
- Immunology
- Molecular Biology
- Biochemistry
Background:
- Interleukin-1 beta (IL-1 beta) is a key inflammatory cytokine produced by monocytes.
- IL-1 beta is synthesized as an inactive precursor and requires specific cleavage for activation.
- The cellular site and mechanism of IL-1 beta precursor cleavage remain largely unknown.
Purpose of the Study:
- To identify the cellular source and characteristics of the protease responsible for cleaving IL-1 beta precursor.
- To investigate the biochemical properties of the IL-1 beta processing activity.
- To confirm the specificity and biological relevance of the cleavage event.
Main Methods:
- Preparation of cell-free postnuclear extracts from human monocytes, THP.1 cells, lymphocytes, and fibroblasts.
- In vitro transcription and translation of pre-IL-1 beta.
- Assay of cleavage activity using cell extracts and synthesized pre-IL-1 beta.
- Subcellular fractionation to localize the processing activity.
- SDS-PAGE and amino acid sequencing to characterize the cleavage product.
- Site-directed mutagenesis of the IL-1 beta precursor (Asp116 to Ala116).
Main Results:
- Cell-free extracts from human monocytes and THP.1 cells, but not lymphocytes or fibroblasts, cleaved precursor IL-1 beta to mature IL-1 beta.
- The processing activity was localized to the cytosol of monocytes/THP.1 cells.
- The cleavage product exhibited identical mobility on SDS-PAGE and amino acid sequence to authentic IL-1 beta.
- The processed IL-1 beta bound to the IL-1 receptor on fibroblast membranes.
- Mutation of Asp116 to Ala116 abolished precursor cleavage, confirming the cleavage site.
Conclusions:
- A monocyte-specific protease is responsible for processing the inactive IL-1 beta precursor to its active form.
- This protease activity is located in the cytosol of monocytes and monocyte-like cell lines.
- The specific cleavage at Asp116-Ala117 is crucial for IL-1 beta activation and subsequent receptor binding.