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Identification of caspase 3-mediated cleavage and functional alteration of eukaryotic initiation factor 2alpha in
W E Marissen1, Y Guo, A A Thomas
1Department of Microbiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190, USA.
Abstract:
Induction of apoptosis in a variety of cell types leads to inhibition of protein synthesis. Recently, the cleavage of eukaryotic translation initiation factor 4G (eIF4G) by caspase 3 was described as a possible event contributing to translation inhibition. Here, we report the cleavage of another initiation factor in apoptotic cells, eIF2alpha, that could contribute to regulation of translation during apoptosis. This cleavage event could be completely inhibited by pretreatment of HeLa cells with Z-VAD-fmk. In vitro analysis using purified eIF2 and purified caspases showed cleavage of eIF2alpha by caspase 3, 6, 8, and 10 but not 9. Caspase 3 most efficiently cleaved eIF2alpha and this could be inhibited by addition of Ac-DEVD-CHO in vitro. Comparison of cleavage of phosphorylated versus nonphosphorylated eIF2alpha revealed a modest preference of the caspases for the nonphosphorylated form. When eIF2. 2B complex was used as substrate, only caspase 3 was capable of eIF2alpha cleavage, which was not affected by phosphorylation of the alpha subunit. The eIF2.GDP binary complex was cleaved much less efficiently by caspase 3. Sequence analysis of the cleavage fragment suggested that the cleavage site is located in the C-terminal portion of the protein. Analysis showed that after caspase cleavage, exchange of GDP bound to eIF2 was very rapid and no longer dependent upon eIF2B. Furthermore, in vitro translation experiments indicated that cleavage of eIF2alpha results in functional alteration of the eIF2 complex, which no longer stimulated upstream AUG selection on a mRNA containing a viral internal ribosome entry site and was no longer capable of stimulating overall translation. In conclusion, we describe here the cleavage of a translation initiation factor, eIF2alpha that could contribute to inhibition or alteration of protein synthesis during the late stages of apoptosis.
Insights
During apoptosis, caspases cleave eukaryotic translation initiation factor 2 alpha (eIF2alpha), inhibiting protein synthesis. This cleavage alters eIF2 complex function, contributing to translation regulation in late-stage apoptosis.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Apoptosis, or programmed cell death, involves the inhibition of protein synthesis.
- Cleavage of eukaryotic translation initiation factor 4G (eIF4G) by caspase 3 is a known contributor to translation inhibition during apoptosis.
- The role of other translation initiation factors in this process remains to be fully elucidated.
Purpose of the Study:
- To investigate the potential cleavage of eukaryotic translation initiation factor 2 alpha (eIF2alpha) during apoptosis.
- To determine which caspases are responsible for eIF2alpha cleavage and the functional consequences of this cleavage on protein synthesis.
Main Methods:
- In vitro analysis of purified eIF2 and caspases.
- Inhibition studies using Z-VAD-fmk and Ac-DEVD-CHO.
- Analysis of phosphorylated versus nonphosphorylated eIF2alpha.
- In vitro translation experiments using modified mRNA.
Main Results:
- eIF2alpha is cleaved by caspases 3, 6, 8, and 10, with caspase 3 being the most efficient.
- Cleavage is inhibited by Z-VAD-fmk and Ac-DEVD-CHO.
- Caspase cleavage of eIF2alpha leads to rapid GDP exchange, independent of eIF2B.
- Cleaved eIF2alpha functionally alters the eIF2 complex, impairing translation initiation and overall protein synthesis.
Conclusions:
- eIF2alpha cleavage by caspases is a significant event during apoptosis.
- This cleavage contributes to the inhibition and alteration of protein synthesis in late-stage apoptosis.
- The findings identify a novel mechanism for translational regulation during programmed cell death.