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

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. 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. Apoptosis-inducing factor (AIF) 6The abbreviations used are: AIF, apoptosis inducing factor; AIFsh2, apoptosis inducing factor short 2; AIFsh3, apoptosis inducing factor short 3; CHX, cycloheximide; GFP, green fluorescent protein; NBT, nitro blue tetrazolium; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; mAIFsh2, mouse apoptosis inducing factor short 2; MLS, mitochondrial localization sequence; RACE, rapid amplification of cDNA ends; RT, reverse transcription; TNF, tumor necrosis factor; RNAi, RNA interference; siRNA, small interfering RNA; Z, benzyloxycarbonyl; fmk, fluoromethyl ketone; ERK, extracellular signal-regulated kinase; STS, staurosporine. is a flavoprotein, with significant homology to bacterial and plant oxidoreductases, located in the mitochondrial intermembrane space (1Susin S.A. Lorenzo H.K. Zamzami N. Marzo I. Snow B.E. Brothers G.M. Mangion J. Jacotot E. Costantini P. Loeffler M. Larochette N. Goodlett D.R. Aebersold R. Siderovski D.P. Penninger J.M. Kroemer G. Nature. 1999; 397: 441-446Crossref PubMed Scopus (3452) Google Scholar, 2Lorenzo H.K. Susin S.A. Penninger J. Kroemer G. Cell Death Differ. 1999; 6: 516-524Crossref PubMed Scopus (425) Google Scholar, 3Lorenzo H.K. Susin S.A. FEBS Lett. 2004; 557: 14-20Crossref PubMed Scopus (156) Google Scholar). Under physiological conditions, AIF is a NADH oxidase that plays a role in oxidative phosphorylation (4Miramar M.D. Costantini P. Ravagnan L. Saraiva L.M. Haouzi D. Brothers G. Penninger J.M. Peleato M.L. Kroemer G. Susin S.A. J. Biol. Chem. 2001; 276: 16391-16398Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar, 5Vahsen N. Cande C. Briere J.J. Benit P. Joza N. Larochette N. Mastroberardino P.G. Pequignot M.O. Casares N. Lazar V. Feraud O. Debili N. Wissing S. Engelhardt S. Madeo F. Piacentini M. Penninger J.M. Schagger H. Rustin P. Kroemer G. EMBO J. 2004; 23: 4679-4689Crossref PubMed Scopus (521) Google Scholar, 6Joza N. Oudit G.Y. Brown D. Benit P. Kassiri Z. Vahsen N. Benoit L. Patel M.M. Nowikovsky K. Vassault A. Backx P.H. Wada T. Kroemer G. Rustin P. Penninger J.M. Mol. Cell. Biol. 2005; 25: 10261-10272Crossref PubMed Scopus (174) Google Scholar). Moreover, AIF plays a major role in cell death (7Joza N. Susin S.A. Daugas E. Stanford W.L. Cho S.K. Li C.Y. Sasaki T. Elia A.J. Cheng H.Y. Ravagnan L. Ferri K.F. Zamzami N. Wakeham A. Hakem R. Yoshida H. Kong Y.Y. Mak T.W. Zuniga-Pflucker J.C. Kroemer G. Penninger J.M. Nature. 2001; 410: 549-554Crossref PubMed Scopus (1153) Google Scholar). Indeed, after a cellular insult, AIF is cleaved by calpains and/or cathepsins (8Polster B.M. Basanez G. Etxebarria A. Hardwick J.M. Nicholls D.G. J. Biol. Chem. 2005; 280: 6447-6454Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar, 9Yuste C. M. P. N. J. Susin S.A. Cell Death Differ. 2005; PubMed Scopus Google and from mitochondria to cytosol and it I. C. M. Susin S.A. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google and in a caspase-independent (1Susin S.A. Lorenzo H.K. Zamzami N. Marzo I. Snow B.E. Brothers G.M. Mangion J. Jacotot E. Costantini P. Loeffler M. Larochette N. Goodlett D.R. Aebersold R. Siderovski D.P. Penninger J.M. Kroemer G. Nature. 1999; 397: 441-446Crossref PubMed Scopus (3452) Google Scholar). This of AIF seems in of cell In AIF a of of cell death cell death in cells S. Cho H.Y. 2004; PubMed Scopus Google A. F. Kroemer G. J. Cell Biol. PubMed Scopus Google Scholar, C. F. Cell 2004; PubMed Scopus Google in cells 2005; PubMed Scopus Google in cells M. N. C. D. J. Cell. 2004; PubMed Scopus Google in cells I. C. M. Susin S.A. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google caspase-independent apoptosis by in cells T. D. 2004; 23: PubMed Scopus Google in cells S. Z. E. J. K. J.M. 2005; PubMed Scopus Google or cell death H. C. PubMed Scopus Google Scholar, H. J. C. J. 2004; PubMed Scopus Google Scholar). In addition, of the AIF seems to in the of cell S. L. Kroemer G. P. P. 2004; 23: PubMed Scopus Google and E. A. S. Cande C. A. Kroemer F. M. Penninger J.M. A. Kroemer G. C. Google Scholar). the in cells, AIF apoptosis and the transformed of tumor cells A. P.H. J.C. K. S. A. EMBO J. 2005; PubMed Scopus Google Scholar). Thus, AIF is a bifunctional with a activity in mitochondria, and a to the AIF exons and and in and E. D. Ravagnan L. Loeffler M. Susin S.A. Zamzami N. Kroemer G. FEBS Lett. PubMed Scopus Google Scholar). AIF is expressed a of This is and in mitochondria by localization located in the in mitochondria, the AIF is and the a of (1Susin S.A. Lorenzo H.K. Zamzami N. Marzo I. Snow B.E. Brothers G.M. Mangion J. Jacotot E. Costantini P. Loeffler M. Larochette N. Goodlett D.R. Aebersold R. Siderovski D.P. Penninger J.M. Kroemer G. Nature. 1999; 397: 441-446Crossref PubMed Scopus (3452) Google Scholar). This and the domain H.K. Susin S.A. Penninger J. Kroemer G. Cell Death Differ. 1999; 6: 516-524Crossref PubMed Scopus (425) Google Scholar). The two the oxidoreductase of AIF, which an activity to the (4Miramar M.D. Costantini P. Ravagnan L. Saraiva L.M. Haouzi D. Brothers G. Penninger J.M. Peleato M.L. Kroemer G. Susin S.A. J. Biol. Chem. 2001; 276: 16391-16398Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar). The of AIF seems to the domain C. M. J.C. S. J. Susin S.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of AIF, AIF-exB, after the of AIF M. Daugas E. Susin S.A. Zamzami N. D. Brothers G. Penninger J.M. Kroemer G. J. 2001; PubMed Scopus Google Scholar). This an exon of the original exon the of this alternatively spliced is not it is that the exon does not mitochondrial and M. Daugas E. Susin S.A. Zamzami N. D. Brothers G. Penninger J.M. Kroemer G. J. 2001; PubMed Scopus Google Scholar). our identified a AIF C. M. J.C. S. J. Susin S.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). This isoform results from an located 9 of AIF. a the AIF oxidoreductase which is a protein, the and C. M. J.C. S. J. Susin S.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). it seems that the oxidoreductase of AIF is not for the of cell However, the of reactive oxygen the oxidoreductase of AIF an role in caspase-independent apoptosis (4Miramar M.D. Costantini P. Ravagnan L. Saraiva L.M. Haouzi D. Brothers G. Penninger J.M. Peleato M.L. Kroemer G. Susin S.A. J. Biol. Chem. 2001; 276: 16391-16398Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar, A. P.H. J.C. K. S. A. EMBO J. 2005; PubMed Scopus Google Scholar, S. P. E. S. Engelhardt T. A. A. F. M. J. Cande C. Kroemer G. Madeo F. J. Cell Biol. 2004; PubMed Scopus Google Scholar). In the we report the of a exon in AIF, which generates two new AIF mRNA in and and in mouse and we the AIFsh2 resulting in mitochondria from mouse or In to the protein. In addition, we the of AIFsh2 and biochemical and that this AIF which is absent in normal brain tissue, NADH oxidase nuclear with our results C. M. J.C. S. J. Susin S.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google our the of AIF and to understand the and death AIF-dependent functions. RNA from 10 previously C. M. J.C. S. J. Susin S.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google with a exon by with and reverse of cDNA and the cDNA from to the the the of amplification the and reverse The with the 2 and the the the and the and RNA from from from cell and mouse the reverse of RNA with of reverse and of a reverse reverse and AIFsh2 isoforms the and and and AIFsh2 isoform and In a of the from AIF and the and and AIFsh2 the and The used for the for 2 for GAPDH, for AIF, and for AIFsh2, and of for for and for 2 and a for 10 by in a for AIF, AIFsh2, and by amplification of the cDNA and or are to or used Cell and Cell Death cells in with 2 and Cell with cells a of 2 in to the after or AIFsh2 and by cells with AIF AIFsh2 or a AIF, and AIFsh2 a we used an after the of cells, AIF, or AIFsh2 mRNA by In we used by of cells for with 2 and Cell and of AIF/AIFsh2 from cells or mouse previously C. M. P. N. J. Susin S.A. Cell Death Differ. 2005; PubMed Scopus Google and in a 10 and by the of mitochondria with or for with the the or the calpain of The for and to a mitochondrial or released from mitochondria to a In the with 2 or with STS, or in 10 2 and and for and the the by the the localization of and cells with and with for and and green in a and AIF, and AIFsh2 produced from a from a and in and of AIFsh2 and and AIFsh2 with the by (4Miramar M.D. Costantini P. Ravagnan L. Saraiva L.M. Haouzi D. Brothers G. Penninger J.M. Peleato M.L. Kroemer G. Susin S.A. J. Biol. Chem. 2001; 276: 16391-16398Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar). In of nitro blue the E. L.M. J. 1999; PubMed Scopus Google Scholar). a The in the with 2 NADH to and the with after the of the blue oxidase activity of AIFsh2 previously (4Miramar M.D. Costantini P. Ravagnan L. Saraiva L.M. Haouzi D. Brothers G. Penninger J.M. Peleato M.L. Kroemer G. Susin S.A. J. Biol. Chem. 2001; 276: 16391-16398Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar). oxidase activity in a of in The by the of AIFsh2 and by in and in the of AIFsh2 with oxygen (4Miramar M.D. Costantini P. Ravagnan L. Saraiva L.M. Haouzi D. Brothers G. Penninger J.M. Peleato M.L. Kroemer G. Susin S.A. J. Biol. Chem. 2001; 276: 16391-16398Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar). of mitochondria or of or cell to a and a which and with an an or an and with a with In with a an AIF for we used the the of from cells by Susin S.A. Zamzami N. M. T. P. A. Daugas E. M. Kroemer G. J. PubMed Scopus Google Scholar). In conditions, in the of AIF, or AIFsh2 for with and by with a with by S.A. Zamzami N. M. T. P. A. Daugas E. M. Kroemer G. J. PubMed Scopus Google Scholar). of a in the AIF in AIF AIFsh2 and by and we have identified a new AIF transcript that results from an located in 9 of AIF C. M. J.C. S. J. Susin S.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). This new transcript for a new C. M. J.C. S. J. Susin S.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). a similar and we identified two new AIF cDNA species in a cDNA AIF short 2 and AIF short AIFsh2 and from the previously AIF AIF, AIF-exB, and (1Susin S.A. Lorenzo H.K. Zamzami N. Marzo I. Snow B.E. Brothers G.M. Mangion J. Jacotot E. Costantini P. Loeffler M. Larochette N. Goodlett D.R. Aebersold R. Siderovski D.P. Penninger J.M. Kroemer G. Nature. 1999; 397: 441-446Crossref PubMed Scopus (3452) Google Scholar, C. M. J.C. S. J. Susin S.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. Daugas E. Susin S.A. Zamzami N. D. Brothers G. Penninger J.M. Kroemer G. J. 2001; PubMed Scopus Google by of a new exon of exon and This new exon located in 9 of AIF, between exons 9 and 10 of AIF, of the of exon that it a for R. P. PubMed Scopus Google In AIFsh2, exon for two and which are by a The resulting not the AIF domain and The transcript exon AIF exon 2 Here, the of exon 2 a in the that generates a a the for located in exon of AIF The resulting the mitochondrial localization and the domain of AIF and for a mouse of AIFsh2 and in of the mouse AIF to an of exon a between exon and 9 to an mRNA transcript AIFsh2, the for exon This exon and which by a the domain of mouse AIF The resulting similar to the mouse our a new for AIF. Indeed, this includes a new exon and different in AIF, AIF-exB, AIFsh2, and In the mRNA is AIFsh2 in and the of the AIFsh2 and mRNA resulting In this way, that AIF is in mitochondria (1Susin S.A. Lorenzo H.K. Zamzami N. Marzo I. Snow B.E. Brothers G.M. Mangion J. Jacotot E. Costantini P. Loeffler M. Larochette N. Goodlett D.R. Aebersold R. Siderovski D.P. Penninger J.M. Kroemer G. Nature. 1999; 397: 441-446Crossref PubMed Scopus (3452) Google we AIFsh2 and/or in this Thus, we mitochondria from and a The of AIF, and ERK, of the or the mitochondrial of our AIFsh2 we used two with an in the from mouse or This to AIF and a of The of this with the of AIFsh2, suggesting that AIFsh2 and in In this way, of mitochondrial an a of AIF not reveal the the of AIFsh2 Indeed, the domain of AIF absent in AIFsh2, with this we AIF to AIFsh2 The of mitochondrial AIFsh2 with the of the resulting from AIFsh2 the of the mitochondrial AIFsh2 a we and cells and in in contrast to the protein, and a after which with the mitochondrial AIF, AIFsh2 is important to that the of AIFsh2 in mitochondria is in with the of the two previously for AIF in the AIFsh2 mRNA (1Susin S.A. Lorenzo H.K. Zamzami N. Marzo I. Snow B.E. Brothers G.M. Mangion J. Jacotot E. Costantini P. Loeffler M. Larochette N. Goodlett D.R. Aebersold R. Siderovski D.P. Penninger J.M. Kroemer G. Nature. 1999; 397: 441-446Crossref PubMed Scopus (3452) Google Scholar). cellular and to the in or mitochondrial from cells not This in Indeed, this to a in cells, which absent in from cells Thus, it seems in this cell the mRNA is absent or not mRNA is expressed in and used cell a In this the mRNA amplification used a and used to the mRNA a mRNA in and cell similar used to the mRNA in a of In this mRNA a in and results that the transcript different in normal and transformed In cells, the mRNA is of AIFsh2 mRNA in and and of AIFsh2 in a with a located exon we to in This a mRNA transcript in and this transcript to absent in brain AIFsh2 we our by a used RNA from a of and and two are to the and AIFsh2 and to the of two the amplification used an that AIFsh2 mRNA expressed in brain the a different between and AIFsh2 In of the to in the of AIFsh2 that of AIFsh. similar of the two AIF isoforms in a transcript in and of AIFsh2 by a similar in cell AIFsh2 transcript in cell with a in and cells In AIFsh2 expressed in that the AIFsh2 mRNA is absent from brain and it is in neuroblastoma-derived cell and seems to a different regulation in normal and tumor from the brain lineage. In it in normal and AIFsh2 are expressed in tumor that AIFsh2 and are this we used an Indeed, AIFsh2 and are it to with the of mRNA the of the In this way, the in we this a similar in to AIF, or AIFsh2 Thus, seems to AIF, and AIFsh2 which AIFsh2 physiological conditions, AIF is a mitochondrial NADH oxidase that plays a role in oxidative phosphorylation (4Miramar M.D. Costantini P. Ravagnan L. Saraiva L.M. Haouzi D. Brothers G. Penninger J.M. Peleato M.L. Kroemer G. Susin S.A. J. Biol. Chem. 2001; 276: 16391-16398Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar, 5Vahsen N. Cande C. Briere J.J. Benit P. Joza N. Larochette N. Mastroberardino P.G. Pequignot M.O. Casares N. Lazar V. Feraud O. Debili N. Wissing S. Engelhardt S. Madeo F. Piacentini M. Penninger J.M. Schagger H. Rustin P. Kroemer G. EMBO J. 2004; 23: 4679-4689Crossref PubMed Scopus (521) Google Scholar). In the oxidoreductase of AIF, which is in AIFsh2, an activity to the (4Miramar M.D. Costantini P. Ravagnan L. Saraiva L.M. Haouzi D. Brothers G. Penninger J.M. Peleato M.L. Kroemer G. Susin S.A. J. Biol. Chem. 2001; 276: 16391-16398Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar, 5Vahsen N. Cande C. Briere J.J. Benit P. Joza N. Larochette N. Mastroberardino P.G. Pequignot M.O. Casares N. Lazar V. Feraud O. Debili N. Wissing S. Engelhardt S. Madeo F. Piacentini M. Penninger J.M. Schagger H. Rustin P. Kroemer G. EMBO J. 2004; 23: 4679-4689Crossref PubMed Scopus (521) Google Scholar, 6Joza N. Oudit G.Y. Brown D. Benit P. Kassiri Z. Vahsen N. Benoit L. Patel M.M. Nowikovsky K. Vassault A. Backx P.H. Wada T. Kroemer G. Rustin P. Penninger J.M. Mol. Cell. Biol. 2005; 25: 10261-10272Crossref PubMed Scopus (174) Google Scholar). Thus, we the AIFsh2 to AIFsh2 the NADH oxidase activity AIF. which the AIF oxidoreductase used a AIF, and AIFsh2 in E. and the by in the of AIFsh2 the of an flavoprotein, with and and a are similar to the with AIF. In Thus, AIF and AIFsh2 in not this and NADH oxidase activity by in whereas the NADH activity This AIF, AIFsh2 exhibits an oxidoreductase the of the AIFsh2 and that AIFsh2 NADH and oxidase in the of AIFsh2 by the of The for NADH and the used the 9 and the are similar to for AIF (4Miramar M.D. Costantini P. Ravagnan L. Saraiva L.M. Haouzi D. Brothers G. Penninger J.M. Peleato M.L. Kroemer G. Susin S.A. J. Biol. Chem. 2001; 276: 16391-16398Abstract Full Text Full Text PDF PubMed Scopus (344) Google that AIFsh2 major for NADH or AIFsh2 the of This to the of to In this way, the of by 10 of the not In our that AIFsh2 exhibits a similar NADH oxidase activity to AIF (4Miramar M.D. Costantini P. Ravagnan L. Saraiva L.M. Haouzi D. Brothers G. Penninger J.M. Peleato M.L. Kroemer G. Susin S.A. J. Biol. Chem. 2001; 276: 16391-16398Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar). In AIFsh2 from to in a a cellular insult, AIF is released from mitochondria and to cytosol and it a caspase-independent (1Susin S.A. Lorenzo H.K. Zamzami N. Marzo I. Snow B.E. Brothers G.M. Mangion J. Jacotot E. Costantini P. Loeffler M. Larochette N. Goodlett D.R. Aebersold R. Siderovski D.P. Penninger J.M. Kroemer G. Nature. 1999; 397: 441-446Crossref PubMed Scopus (3452) Google Scholar, A. F. Kroemer G. J. Cell Biol. PubMed Scopus Google Scholar, H. J. C. J. 2004; PubMed Scopus Google Scholar, S.A. Zamzami N. M. T. P. A. Daugas E. M. Kroemer G. J. PubMed Scopus Google Scholar, S.A. Zamzami N. M. Daugas E. S. F. J.C. Kroemer G. J. PubMed Scopus Google Scholar, S.A. Daugas E. Ravagnan L. K. Zamzami N. Loeffler M. Costantini P. Ferri K.F. T. Brothers G. Mak T.W. Penninger J. Kroemer G. J. PubMed Scopus Google Scholar). is to released from mitochondria AIF is cleaved by of the mouse AIF C. M. P. N. J. Susin S.A. Cell Death Differ. 2005; PubMed Scopus Google Scholar). This is by or cathepsins or (8Polster B.M. Basanez G. Etxebarria A. Hardwick J.M. Nicholls D.G. J. Biol. Chem. 2005; 280: 6447-6454Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar, 9Yuste C. M. P. N. J. Susin S.A. Cell Death Differ. 2005; PubMed Scopus Google Scholar). AIFsh2 the of AIF, which includes the Thus, the of cell death regulation and M. Susin S.A. 2005; PubMed Scopus Google we that AIFsh2 cleaved in a or cathepsin-dependent this we used two independent AIF and AIFsh2 are released from mitochondria, it to and in from mitochondria with an in inducing mitochondrial AIF C. M. P. N. J. Susin S.A. Cell Death Differ. 2005; PubMed Scopus Google and AIFsh2 in this we AIFsh2 released from mitochondria to cytosol after the STS, the the the of the the death TNF, and the In this to after of the of cell by a not the we that it is to in from mitochondria Thus, AIF, AIFsh2 is cleaved and released from the intermembrane space of AIFsh2 and by the the or the calpain it seems that AIF, AIFsh2 is cleaved and released from mitochondria in a calpain or a cathepsin-dependent the of AIFsh2 in cytosol from STS, or cells, AIF, AIFsh2 is released from the mitochondria to cytosol after an apoptotic insult of AIFsh2 previously that the domain of AIF the to induce nuclear apoptosis C. M. J.C. S. J. Susin S.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). However, is an role of the oxidoreductase of AIF, which is by AIFsh2, in nuclear cell a in in which the AIFsh2 to we this S.A. Zamzami N. M. Daugas E. S. F. J.C. Kroemer G. J. PubMed Scopus Google Scholar). AIF and used a nuclear by fluorescent and S.A. Zamzami N. Larochette N. Marzo I. C. T. M. Kroemer G. Cell PubMed Scopus Google Scholar). AIF and and of In AIFsh2 results of NADH or to activity Moreover, AIFsh2 to or the of in not that AIFsh2 not to the nuclear (1Susin S.A. Lorenzo H.K. Zamzami N. Marzo I. Snow B.E. Brothers G.M. Mangion J. Jacotot E. Costantini P. Loeffler M. Larochette N. Goodlett D.R. Aebersold R. Siderovski D.P. Penninger J.M. Kroemer G. Nature. 1999; 397: 441-446Crossref PubMed Scopus (3452) Google Scholar, C. M. J.C. S. J. Susin S.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). results that the reactive oxygen species produced by AIF/AIFsh2 not induce nuclear apoptosis. The of the new exon a of the AIF are for AIF, AIF-exB, AIFsh2, and The two isoforms in the of exon 2 or exon M. Daugas E. Susin S.A. Zamzami N. D. Brothers G. Penninger J.M. Kroemer G. J. 2001; PubMed Scopus Google and are to the mitochondrial intermembrane a of AIF, which results from an located 9 of AIF C. M. J.C. S. J. Susin S.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In the we identified exon an exon located between exons 9 and 10 of AIF. This exon to two new AIF cDNA AIFsh2 and to the in However, AIFsh2 mRNA transcript yields a mitochondrial of AIF by which the two and the oxidoreductase domain of AIF. In this AIFsh2 is to and NADH oxidase In AIF, AIFsh2 to and AIFsh2 in a AIFsh2 the domain of AIF and activity In the domain of AIF and oxidoreductase activity and and C. M. J.C. S. J. Susin S.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). results that AIF is a bifunctional with and Indeed, to bifunctional FEBS Lett. PubMed Scopus Google Scholar, Z. M.O. H. J. PubMed Scopus Google AIF yields an independent genetic regulation of two different functions. This is an important issue to understand the physiological role of AIF. AIFsh2 mitochondrial the of the domain and oxidoreductase activity 1999; PubMed Scopus Google Scholar, H. Biol. 1999; PubMed Scopus Google AIFsh2 involved in a of and regulation of the cellular Moreover, AIFsh2 similar to AIF, we that two have similar in In this the different of AIF and AIFsh2 in a of and cell that the two are This by an is important to that our new to the role of AIF, and AIFsh2 in cell death and in oxidative Indeed, from it to of AIF and two caspase-independent death Moreover, it to for the a cellular in which AIF, and AIFsh2 isoforms are In it is that AIFsh2 the of AIF in normal tumor The different AIFsh2 in brain this AIF activity a in cell the which an in AIF. This mouse normal for and in of in Nature. PubMed Scopus Google Scholar). The of of AIF in and oxidative in this mouse to a of AIFsh2 in to understand AIF/AIFsh2 it been that a domain of AIF and are for the of of suggesting that AIF is involved in an C. Vahsen N. D. H. K. C. J. Kroemer G. J. Cell 2004; PubMed Scopus Google Scholar). Moreover, AIF oxidative phosphorylation by in and in a for AIF N. Cande C. Briere J.J. Benit P. Joza N. Larochette N. Mastroberardino P.G. Pequignot M.O. Casares N. Lazar V. Feraud O. Debili N. Wissing S. Engelhardt S. Madeo F. Piacentini M. Penninger J.M. Schagger H. Rustin P. Kroemer G. EMBO J. 2004; 23: 4679-4689Crossref PubMed Scopus (521) Google Scholar, 6Joza N. Oudit G.Y. Brown D. Benit P. Kassiri Z. Vahsen N. Benoit L. Patel M.M. Nowikovsky K. Vassault A. Backx P.H. Wada T. Kroemer G. Rustin P. Penninger J.M. Mol. Cell. Biol. 2005; 25: 10261-10272Crossref PubMed Scopus (174) Google Scholar). AIF the transformed of cells NADH oxidase by that A. P.H. J.C. K. S. A. EMBO J. 2005; PubMed Scopus Google Scholar). the between the activity of AIF, oxidative and cell Moreover, in the genetic of the AIF transcript to AIFsh2 Thus, it is to AIF, AIFsh2 a role in and have a in the oxidative that the in mitochondrial AIF and a similar in Thus, it is in apoptotic conditions, in oxidative phosphorylation not to AIF mitochondrial The AIFsh2 to the in oxidative phosphorylation in apoptotic a mouse AIF or AIFsh2 in the of the mitochondrial of two In and the between AIFsh2 and AIF AIFsh2 different AIFsh2 mRNA is not expressed in AIFsh2 the domain of AIF. AIFsh2 the activity of AIF. that the domain of AIF is and to induce AIF-dependent nuclear apoptosis. Indeed, by the AIFsh2, we have that the of the domain in AIF are not for nuclear Thus, AIF does not induce and the of the reactive oxygen with this of the protein. In our the of AIF. The and characterization of a AIF apoptotic a that in the of the role of AIF in mitochondria by in the of the bifunctional role of AIF in and and for and of the and for