AIF and cyclophilin A cooperate in apoptosis-associated chromatinolysis Ce´line Cande´ 1 , Nicola Vahsen 1 , Ilektra Kouranti 1 , Elise Schmitt 2 , Eric Daugas 1 , Chris Spahr 3 , Jeremy Luban 4,5 , Romano T Kroemer 6 , Fabrizio Giordanetto 6,7 , Carmen Garrido 2 , Josef M Penninger 8 and Guido Kroemer* ,1 1 CNRS-UMR8125, Institut Gustave Roussy, Pavillon de Recherche 1, 39 rue Camille-Desmoulins, F-94805 Villejuif, France; 2 INSERM U-517, Faculty of Medicine and Pharmacy, 7 Boulevard Jeanne d’Arc, 21033 Dijon, France; 3 Amgen Inc., Protein Science, Amgen Center, Thousand Oaks, CA-91320, USA; 4 Department of Microbiology, Columbia University, 701 West 168th Street, Room 1502, New York, NY 10032, USA; 5 Department of Medicine, Columbia University, 701 West 168th Street, Room 1502, New York, NY 10032, USA; 6 Molecular Modeling & Design, Pharmacia, Viale Pasteur 10, 20014 Nerviano (MI), Italy; 7 Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, UK; 8 IMBA, Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Dr Bohrgasse 7, 1030 Vienna, Austria Cyclophilin A (CypA) was determined to interact with apoptosis-inducing factor (AIF) by mass spectroscopy, coimmunoprecipitation, pull-down assays, and molecular modeling. During the initial, caspase-independent stage of chromatin condensation that accompanies apoptosis, AIF and CypA were found to coimmunolocalize in the nucleus. Recombinant AIF and CypA proteins synergized in vitro in the degradation of plasmid DNA, as well as in the capacity to induce DNA loss in purified nuclei. The apoptogenic cooperation between AIF and CypA did not rely on the CypA peptidyl-prolyl cis–trans isomerase activity. In Cyp-expressing cells, AIF overexpression augmented apoptotic chromatinolysis. The AIF-dependent large-scale DNA fragmentation was less pronounced in CypA knockout cells as compared to controls. AIF mutantslackingtheCypA-bindingdomainwereinefficient apoptosis sensitizers in transfection experiments. More- over, AIF failed to sensitize CypA knockout cells to apoptosis induction, and this defect in the AIF response was reversed by reintroduction of the CypA gene into CypA-deficient cells. In summary, AIF and CypA collaborate in chromatinolysis. Oncogene (2004) 23, 1514–1521. doi:10.1038/sj.onc.1207279 Published online 12 January 2004 Keywords: mitochondria; Bcl-2 Introduction The cell-autonomous enzymatic degradation of nuclear DNA (chromatinolysis) is a hallmark of apoptosis (Hengartner, 2000; Wyllie, 284) and presumably con- stitutes an important mechanism designed to avoid the transfer of mutated DNA from tumor cells (which may contain gain-of-function mutations of oncogenes) to surrounding healthy cells (de la Taille et al., 1999; Bergsmedh et al., 2001; Bergsmedh et al., 2002). Much of the oligonucleosomic ‘ladder-type’ DNA degradation is due to the activation of caspase-dependent DNAse (CAD) (Enari et al., 1998; Sakahira et al., 1998). However, caspase-independent mechanisms of apoptotic DNA degradation are well described (Montague et al., 1994; Montague et al., 1997; Dumont et al., 2000; Susin et al., 2000; Li et al., 2001; Widlak et al., 2001; Wyllie and Golstein, 2001; Wang et al., 2002). Apoptosis-inducing factor (AIF) is a flavoprotein with NADH oxidase activity normally contained in the mitochondrial intermembrane space (Susin et al., 1999; Miramar et al., 2001; Klein et al., 2002). Upon apoptosis induction, AIF translocates from mitochondria to the cytosol and to the nucleus (Daugas et al., 2000). During early nuclear condensation, AIF is found to be associated with chromatin (Ye et al., 2002). AIF protein binds to DNA and causes purified nuclei to undergo chromatin condensation, DNA degradation to B50kbp fragments and DNA loss (Susin et al., 1999; Ye et al., 2002). The knock out of the AIF gene indicates that AIF participates in the first wave of caspase-independent morphogenetic cell death, required for cavitation of embryoid bodies in in vitro differentiation cultures (Joza et al., 2001). In addition to acting in the early ontogeny of apoptosis, AIF is a phylogenetically ancient factor (Cande et al., 2002). Thus, AIF homologs undergo a mitochondrio-nuclear translocation during cell death occurring in the slime mold Dictyostelium discoideum (Arnoult et al., 2001) and in the nematode Caenorhab- ditis elegans (Wang et al., 2002). Although the mitochondrial release of AIF is controlled by caspases in some models (Arnoult et al., 2002), it is caspase-independent in several important paradigms of apoptosis induction. Thus, DNA damage can induce the caspase-independent AIF release either through p53 (Cregan et al., 2002), or via PARP- mediated NAD depletion (Yu et al., 2002). AIF is also released in several in vivo models of anoikis (Hisatomi et al., 2001), HIV-1 infection (Ferri et al., 2000), and Received 27 August 2003; revised 2 October 2003; accepted 17 October 2003 *Correspondence: G Kroemer; E-mail: kroemer@igr.fr Oncogene (2004) 23, 1514–1521 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $25.00 www.nature.com/onc