Dominant cell death induction by extramitochondrially targeted apoptosis‐inducing factor

Abstract

The complete AIF cDNA comprising the amino-terminal mitochondrial localization sequence (MLS) and the oxidoreductase domain has been fused in its carboxyl terminus to enhanced green fluorescent protein (GFP), thereby engineering an AIF-GFP fusion protein that is selectively targeted to the mitochondrial intermembrane space. Upon induction of apoptosis, the AIF-GFP protein translocates together with cytochrome c (Cyt-c) to the extramitochondrial compartment. Microinjection of recombinant AIF leads to the release of AIF-GFP and Cyt-c-GFP, indicating that ectopic AIF can favor permeabilization of the outer mitochondrial membrane. These mitochondrial effects of AIF are caspase independent, whereas the Cyt-cmicroinjection induced translocation of AIF-GFP and Cyt-c-GFP is suppressed by the pan-caspase inhibitor Z-VAD.fmk. Upon prolonged culture, transfection-enforced overexpression of AIF results in spontaneous translocation of AIF-GFP from mitochondria, nuclear chromatin condensation, and cell death. These effects are caspase independent and do not rely on the oxidoreductase function of AIF. Spontaneous AIF-GFP translocation and subsequent nuclear apoptosis can be retarded by overexpression of a Bcl-2 protein selectively targeted to mitochondria, but not by a Bcl-2 protein targeted to the endoplasmic reticulum. Overexpression of a mutant AIF protein in which the MLS has been deleted (AIF ⌬ 1-100) results in the primary cytosolic accumulation of AIF. AIF ⌬ 1-100-induced cell death is suppressed by neither Z-VAD.fmk or by Bcl-2. Thus, extramitochondrially targeted AIF is a dominant cell death inducer.-Loeffler, M., Daugas, E., Susin, S. A., Zamzami, N., Métivier, D., Nieminen, A.-L., Brothers, G., Penninger, J. M., Kroemer, G. Dominant cell death induction by extramitochondrially targeted apoptosis-inducing factor. FASEB J. 15, 758 -767 (2001)

Physical interaction of apoptosis-inducing factor with DNA and RNA

“…However, the predominant localization of extra-mitochondrial AIF was the nucleus. This has been extensively documented for human and mouse AIF (Loeffler et al, 2001;Ye et al, 2002;Cande et al, 2002Cande et al, , 2004a, and also applies to the AIF ortholog from yeast (S. cerevisiae). Yeast AIF localized at distinct foci within the nucleus upon induction of apoptosis by chronological aging.…”

“…While the biological significance of the AIF-RNA interaction remains a conundrum, AIF binding to DNA has been amply documented in apoptotic cells (Susin et al, 1999Daugas et al, 2000;Cande et al, 2002Cande et al, , 2004aZhu et al, 2003), and it appears clear that AIF can mediate chromatin condensation. Thus, recombinant AIF induces marked pyknosis when added to isolated nuclei or when microinjected into intact cells Loeffler et al, 2001). Moreover, the injection of AIF-neutralizing antibodies or the knockdown of AIF expression with small interfering RNAs has established that endogenous AIF is rate limiting for caspase-independent chromatin condensation induced by apoptotic stimuli (Cregan et al, 2002;Gallego et al, 2004;Kang et al, 2004;Cheung et al, 2005;Park et al, 2005).…”

“…Thus, mutations that affect the redox activity of AIF compromise mitochondrial metabolism (Urbano et al, 2005), yet do not affect its acute proapoptotic activity (Loeffler et al, 2001). Conversely, mutations that affect the translocation of AIF into the nucleus or its DNA binding do not compromise its capacity to sustain the activity of the respiratory chain and cellular redox homeostasis (Cande et al, 2004b;Urbano et al, 2005).…”

Mitochondria as therapeutic targets for cancer chemotherapy

“…Microinjection of recombinant AIF is sufficient to induce hallmarks of apoptosis (chromatin condensation, rounding up of cells, phosphatidyl serine exposure) . Similarly, transfection of cells with an AIF mutant lacking the mitochondrial localization sequence in the N-terminus, misdirects AIF to the cytosol and to the nucleus, and results in cell killing (Loeffler et al, 2001). Recently, a chimeric protein containing an ErbB-2-specific antibody in the C-terminus and the cytotoxic moiety of AIF in the N-terminus has been shown to direct AIF to ErbB-2-expressing cancer cells, causing their demise (Yu et al, 2006).…”

Methods for the assessment of mitochondrial membrane permeabilization in apoptosis

“…1). Whereas AIF and Cyt c are considered the prototypic IMS proteins released upon MMP (due to microinjection experiments of recombinant molecules in intact cells, which demonstrated that either proteins suffice to induce the nuclear manifestations of apoptosis [61,62]), several other proteins are released from mitochondria upon OM permeabilization [63,64]. These include but are not limited to adenylate kinase [65], Smac/Diablo [63], Omi/HtrA2 [11,12], EndoG [17] as well as several pro-caspases [66].…”