Related Experiment Videos
Cloning and expression of four novel isoforms of human interleukin-1 beta converting enzyme with different apoptotic
E S Alnemri1, T Fernandes-Alnemri, G Litwack
1Department of Pharmacology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
Abstract:
To understand the mechanism of interleukin-1 beta converting enzyme (ICE) activation in apoptosis, we analyzed the expression of ICE mRNA in two human cell lines by reverse transcription-polymerase chain reaction technique. This resulted in the identification and cloning of four alternatively spliced ICE mRNA isoforms. Although all the alternative splicing events were within the coding sequence of ICE, the four ICE isoforms maintained open reading frames and were designated as ICE beta, gamma, delta, and epsilon. In ICE gamma, most of the propeptide (amino acids 20-112) is deleted, which suggests that it may function as a catalyst for ICE autoprocessing in vivo. In ICE delta, amino acids 288-335, which contain the cleavage sites between the p20 and p10 subunits of ICE, are deleted thus resulting in its inactivation. Intriguingly, in ICE epsilon amino acids 20-335, which encompass most of the propeptide and the p20 subunit, are deleted resulting in the formation of a molecule that is homologous to the p10 subunit. Examination of the ability of these four ICE isoforms to cause apoptosis revealed that only the parental ICE alpha and isoforms beta and gamma, but not isoforms delta and epsilon, can induce apoptosis when overexpressed in Sf9 insect cells. In addition, coexpression of the p20 and p10 but not the p20 and ICE epsilon in Sf9 cells results in apoptosis. Interestingly, expression of ICE epsilon and to a lesser degree ICE delta resulted in extension of the survival of baculovirus-infected cells in a manner similar to expression of BCL2. The ability of ICE epsilon to extend the survival of Sf9 cells suggests that baculovirus-induced apoptosis in these cells is mediated by an ICE-like protease. We show that ICE epsilon can bind to the p20 subunit of ICE and potentially may compete with the p10 subunit to form an inactive ICE complex. Therefore, by acting as a dominant inhibitor of ICE activity, ICE epsilon may regulate ICE activation in vivo.
Insights
Researchers identified four new forms of interleukin-1 beta converting enzyme (ICE) mRNA. Some forms, like ICE epsilon, can inhibit ICE activity, potentially regulating apoptosis and cell survival.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Apoptosis, or programmed cell death, is crucial for development and tissue homeostasis.
- Interleukin-1 beta converting enzyme (ICE) is a key protease regulating apoptosis.
- Understanding ICE activation mechanisms is vital for controlling cell death pathways.
Purpose of the Study:
- To investigate the alternative splicing of ICE mRNA and its impact on ICE function.
- To identify novel ICE isoforms and characterize their roles in apoptosis.
Main Methods:
- Reverse transcription-polymerase chain reaction (RT-PCR) was used to analyze ICE mRNA expression.
- Cloning and overexpression of ICE isoforms in Sf9 insect cells were performed.
- Apoptosis induction and cell survival assays were conducted.
Main Results:
- Four alternatively spliced ICE mRNA isoforms (beta, gamma, delta, epsilon) were identified.
- ICE alpha, beta, and gamma induced apoptosis upon overexpression; delta and epsilon did not.
- ICE epsilon acted as a dominant inhibitor of ICE activity, extending cell survival.
Conclusions:
- Alternative splicing generates diverse ICE isoforms with distinct apoptotic functions.
- ICE epsilon functions as a dominant-negative regulator of ICE, modulating apoptosis.
- These findings provide insights into the complex regulation of ICE-mediated apoptosis.