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Distinct cell death pathways triggered by the adenovirus early region 4 ORF 4 protein
Amélie Robert1, Marie-Joëlle Miron, Claudia Champagne
1Centre de recherche en cancérologie de l'Université Laval, L'Hôtel-Dieu de Québec, CHUQ, Québec G1R 2J6, Canada.
Abstract:
In transformed cells, induction of apoptosis by adenovirus type 2 (Ad2) early region 4 ORF 4 (E4orf4) correlates with accumulation of E4orf4 in the cell membrane-cytoskeleton fraction. However, E4orf4 is largely expressed in nuclear regions before the onset of apoptosis. To determine the relative contribution of nuclear E4orf4 versus membrane-associated E4orf4 to cell death signaling, we engineered green fluorescent fusion proteins to target E4orf4 to specific cell compartments. The targeting of Ad2 E4orf4 to cell membranes through a CAAX-box or a myristylation consensus signal sufficed to mimic the fast Src-dependent apoptotic program induced by wild-type E4orf4. In marked contrast, the nuclear targeting of E4orf4 abolished the early induction of extranuclear apoptosis. However, nuclear E4orf4 still induced a delayed cell death response independent of Src-like activity and of E4orf4 tyrosine phosphorylation. The zVAD.fmk-inhibitable caspases were dispensable for execution of both cell death programs. Nevertheless, both pathways led to caspase activation in some cell types through the mitochondrial pathway. Finally, our data support a critical role for calpains upstream in the death effector pathway triggered by the Src-mediated cytoplasmic death signal. We conclude that Ad2 E4orf4 induces two distinct cell death responses, whose relative contributions to cell killing may be determined by the genetic background.
Insights
Adenovirus type 2 (Ad2) early region 4 ORF 4 (E4orf4) protein triggers distinct cell death pathways. Membrane-associated E4orf4 induces rapid apoptosis, while nuclear E4orf4 causes delayed cell death.
Area of Science:
- Molecular Biology
- Cell Biology
- Virology
Background:
- Adenovirus type 2 (Ad2) early region 4 ORF 4 (E4orf4) protein is implicated in inducing apoptosis in transformed cells.
- E4orf4 localization shifts from nuclear to membrane-cytoskeleton fractions during apoptosis induction.
- The precise role of E4orf4 subcellular localization in initiating cell death signaling remains unclear.
Purpose of the Study:
- To investigate the distinct contributions of nuclear versus membrane-associated E4orf4 to Ad2-induced cell death.
- To elucidate the signaling pathways and molecular players involved in E4orf4-mediated apoptosis.
Main Methods:
- Engineering of green fluorescent fusion proteins to specifically target E4orf4 to cellular compartments (nucleus or membrane).
- Utilizing CAAX-box and myristylation signals for membrane targeting.
- Assessing apoptosis induction, Src-dependent signaling, caspase activation, and calpain involvement.
Main Results:
- Targeting E4orf4 to the cell membrane mimicked wild-type E4orf4-induced rapid, Src-dependent apoptosis.
- Nuclear targeting of E4orf4 abolished early apoptosis but induced a delayed cell death response, independent of Src activity.
- Caspases were dispensable for initial cell death execution but activated later via the mitochondrial pathway in some cell types.
- Calpains were identified as critical upstream effectors in the Src-mediated cytoplasmic death pathway.
Conclusions:
- Ad2 E4orf4 induces at least two distinct cell death pathways: a rapid membrane-initiated Src-dependent pathway and a delayed nuclear-initiated pathway.
- The relative contribution of these pathways to overall cell killing may be influenced by cellular genetic background.
- Understanding these distinct pathways offers insights into viral oncogenesis and potential therapeutic targets.