Identification and Characterization of AIFsh2, a Mitochondrial Apoptosis-inducing Factor (AIF) Isoform with NADH Oxidase Activity

Santos A Susin,Victor J Yuste,Cécile Delettre,Rana S Moubarak,Nadine Robert,Marlène Bras

doi:10.1074/jbc.m601751200

Santos A Susin, Victor J Yuste + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m601751200

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Abstract

Apoptosis-inducing factor (AIF) is a bifunctional NADH oxidase involved in mitochondrial respiration and caspase-independent apoptosis. Three alternatively spliced mRNA isoforms of AIF have been identified previously: AIF, AIF-exB, and AIFsh. Here, we report the cloning and the biochemical characterization of a new isoform named AIF short 2 (AIFsh2). AIFsh2 transcript includes a previously unknown exon placed between exons 9 and 10 of AIF. The resulting AIFsh2 protein, which localizes in mitochondria, corresponds to the oxidoreductase domain of AIF. In this way, AIFsh2 exhibits similar NADH oxidase activity to AIF and generates reactive oxygen species. Like AIF, AIFsh2 is released from mitochondria to cytosol after an apoptotic insult in a calpain or cathepsin-dependent manner. However, in contrast to AIF, AIFsh2 does not induce nuclear apoptosis. Thus, it seems that the reactive oxygen species produced by the oxidoreductase domain of AIF/AIFsh2 are not important for AIF-dependent nuclear apoptosis. In addition, we demonstrate that the AIFsh2 mRNA is absent in normal brain tissue, whereas it is expressed in neuroblastoma-derived cells, suggesting a different regulation in normal and transformed cells from the brain lineage. Together, our results reveal that AIF yields an original and independent genetic regulation of the two AIF functions. This is an important issue to understand the physiological role of this protein.

Highlights

Apoptosis-inducing factor (AIF) was described as a central mediator of relevant experimental models of cell death such as As2O3-induced cell death in human cervical cancer cells [11], DNA damage-mediated p53 activation [12, 13], Sulindac-induced PCD in colon cancer cells [14], geldanamycin-mediated PCD in human glioma cells [15], staurosporine-induced PCD in neuroblastoma cells [10], caspase-independent apoptosis induced by Survivin in melanoma cells [16], hexaminolevulinate-mediated photodynamic therapy in human leukemia cells [17], or poly(ADP-ribose) polymerase-mediated cell death [18, 19]
Identification of a Novel Exon in the Human AIF Gene, Which Results in Two Novel AIF Isoforms: AIF short 2 (AIFsh2) and AIF short 3 (AIFsh3)—Recently, by 3Ј- and 5Ј-Rapid Amplification of cDNA End (RACE), we have identified a new AIF transcript that results from an alternate transcriptional start site located in intron 9 of AIF [24]
By a similar 3Јand 5Ј-RACE approach, we identified here two new AIF cDNA species in a human kidney cDNA Marathon library: AIF short 2 (AIFsh2) and AIF short 3 (AIFsh3)

Summary

Introduction

Cellular, and molecular biology approaches, we localized the AIFsh2 resulting protein in mitochondria from mouse liver or human HeLa cells. 48 h after the indicated transfection of HeLa cells, AIF, AIFsh, or AIFsh2 mRNA expression was assessed by RT-PCR as described above. The resulting putative AIFsh3 protein (237 amino acids) lacked both the mitochondrial localization sequences and the C-terminal domain of AIF

Results

Conclusion