Multiple Translation Factor eIF4G (IFG-1) Isoforms are Required for the Apoptosome-Dependent Activation of Germ Cell Apoptosis

ECU Author/Contributor (non-ECU co-authors, if there are any, appear on document)

Vince Contreras (Creator)

Institution

East Carolina University (ECU )

Web Site: http://www.ecu.edu/lib/

Abstract: Apoptosis is a naturally occurring process during animal development required for the programmed killing and removal of injured cells. Cellular insult induces a switch in translation that allows for the rapid synthesis of proteins that quickly destroy the unhealthy cell and remove it from undamaged tissue. In mammalian cells, this selective modulation of translation has been attributed to modifications of the protein synthesis machinery by apoptotic caspases. Specifically the initiation complex scaffold protein eIF4G becomes cleaved to disrupt cap-dependent translation and induce the IRES-dependent synthesis of apoptogenic mRNAs like Apaf-1. While the biochemistry of these cleavage events has been well characterized, how disruptions in translation mechanism affect apoptotic signaling still remains largely unknown. The nematode Caenorhabditis elegans was used to study how the balance between cap-dependent and -independent translation mechanisms mediate the translation of pro-apoptotic Apaf-1/CED-4 to alter the fate of healthy germ cells. Two major eIF4G (IFG-1) isoforms, derived from a single gene, were found to be broadly expressed throughout all stages of development. IFG-1 p170 and p130 appeared to both associate with ribosomes yet differed in their N-termini. Analysis of mRNA cap complexes showed that only IFG-1 p170 associates with C. elegans eIF4Es (IFEs) and participates in cap-dependent translation. IFG-1 p130, like its human paralog p97, was not retained in bound complexes and likely participates in cap-independent translation. We further showed that disruption of the balance between the C. elegans eIF4G cap-dependent (IFG-1 p170) and cap-independent (IFG-1 p130) isoforms increased apoptosis in developing oocytes. This induction of germ cell apoptosis coincided with the upregulated synthesis of the Apaf-1 homolog, CED-4. In addition, IFG-1 p170, like mammalian eIF4GI, is a substrate for human caspase-3 and its worm homolog, CED-3. Site-directed mutagenesis of the cleavage site indicated that CED-3 processes IFG-1 at a non-canonical caspase motif, TTTD456. This caspase recognition site is located 65 amino acids downstream of the IFG-1 p130 start site indicating that this truncated form may be required to support cap-independent initiation in the germline. Furthermore, genetic analysis involving ced-3/caspase-3 and ced-4/Apaf-1 mutants confirmed that apoptosis induced by loss of IFG-1 p170 is caspase and apoptosome dependent. These findings provide the first demonstration that modal changes in protein synthesis are required to initiate the cell death signal, rather than merely downstream consequences of the apoptotic event.  

Additional Information

Publication

Dissertation

Date: 2010

Keywords

Developmental Biology, Biology, Molecular, Chemistry, Biochemistry

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