Toxic proteins released from mitochondria in cell death Xavier Saelens 1 , Nele Festjens 1 , Lieselotte Vande Walle 1 , Maria van Gurp 1 , Geert van Loo 1,2,3 and Peter Vandenabeele* ,1 1 Molecular Signalling and Cell Death Unit, Department for Molecular Biomedical Research, VIB and Ghent University, Fiers-Shell-Van Montagu Building, Ghent B9052, Belgium; 2 EMBL-Monterotondo, Mouse Biology Program, Via Ramarini 32, Monterotondo-Scalo 00016, Italy Aplethoraofapoptoticstimuliconvergeonthemitochon- dria and affect their membrane integrity. As a conse- quence, multiple death-promoting factors residing in the mitochondrial intermembrane space are liberated in the cytosol. Pro- and antiapoptotic Bcl-2 family proteins control the release of these mitochondrial proteins by inducing or preventing permeabilization of the outer mitochondrial membrane. Once released into the cytosol, these mitochondrial proteins activate both caspase-depen- dentand-independentcelldeathpathways.Cytochrome c was the first protein shown to be released from the mitochondria into the cytosol, where it induces apopto- some formation. Other released mitochondrial proteins include apoptosis-inducing factor (AIF) and endonuclease G, both of which contribute to apoptotic nuclear DNA damage in a caspase-independent way. Other examples are Smac/DIABLO (second mitochondria-derived activa- tor of caspase/direct IAP-binding protein with low PI) and the serine protease HtrA2/OMI (high-temperature requirement protein A2), which both promote caspase activation and instigate caspase-independent cytotoxicity. Theprecisemodeofactionandimportanceofcytochrome c in apoptosis in mammalian cells has become clear throughbiochemical,structuralandgeneticstudies.More recently identified factors, for example HtrA2/OMI and Smac/DIABLO,arestillbeingstudiedintensivelyinorder to delineate their functions in apoptosis. A better under- standing of these functions may help to develop new strategies to treat cancer. Oncogene (2004) 23, 2861–2874. doi:10.1038/sj.onc.1207523 Keywords: mitochondria; cytochrome c; Smac/DIA- BLO; endonuclease G; AIF; HtrA2/OMI Introduction Mitochondria are present in almost all eukaryotic species. Present day eukaryotes lacking mitochondria, such as Entamoeba histolytica, may have lost these organelles in the course of their evolutionary deviation from the main eukaryotic lineage (Clark and Roger, 1995). Others, like the anaerobic protozoan parasite Trichomonas vaginalis, comprise hydrogenosomes, sub- cellular compartments where ATP is generated con- comitant with the reduction of protons to molecular hydrogen (Hackstein et al., 1999). Mitochondria are considered remnants of invasive or symbiotic bacteria that predated or inhabited the ‘pre-eukaryotic’ cell (Lang et al., 1999). Shielded from the cytoplasm by an inner and an outer membrane, mitochondria provide the eukaryotic cell with energy in the form of ATP that they produce by a process called oxidative phosphorylation (Tyler, 1992). The cell obtains ATP, which is produced at the matrix side of the inner mitochondrial membrane (IMM), through pores present at the contact sites between the outer and the IMMs. The core components of this transmembrane pore are the voltage-dependent anion channel (VDAC), located in the outer mitochon- drial membrane (OMM) and the adenine nucleotide transporter (ANT), present in the IMM. Besides being guardians of survival, mitochondria also harbour noxious molecules in the intermembrane space (IMS). These death-inducing proteins, although encoded in the nucleus of eukaryotic cells, often have distant relatives in the prokaryotic world (Frade and Michaelidis, 1997; Koonin and Aravind, 2002). The observation that the mitochondrial life-support- ing ATP production is disturbed in cell death had already been reported in the 1950s by studying the consequences of X-ray irradiation of the mouse spleen, and this finding was corroborated in many other examples of cell death (Ashwell, 1952; Kroemer and Reed, 2000). In fact, most proapoptotic signalling pathways, including those induced by DNA damage, growth factor depletion and cytosolic calcium overload, were found to converge on the mitochondria. A wide variety of apoptotic signals activate proapoptotic Bcl-2 members such as Bax, Bak and Bid, resulting in a disturbed balance between pro- and antiapoptotic Bcl-2 family proteins. As a consequence, OMM integrity is lost due to the oligomerization of proapoptotic Bcl-2 members in the OMM (Kuwana et al., 2002) or the formation of a megapore complex composed of VDAC and ANT (Marzo et al., 1998). This results in the permeabilization of the OMM and the release of proteins from the IMS. Some of these proteins have *Correspondence: P Vandenabeele, Technologiepark 927, B-9052 Zwijnaarde (Gent), Belgium; E-mail: peter.vandenabeele@dmbr.ugent.be 3 Current address: EMBL-Monterotondo, Mouse Biology Program, Via Ramarini 32, Monterotondo-Scalo 00016, Italy Oncogene (2004) 23, 2861–2874 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $25.00 www.nature.com/onc