Cytoprotective Peptide Humanin Binds and Inhibits Proapoptotic Bcl-2/Bax Family Protein BimEL

Humanin (HN) is a recently identified endogenous peptide that protects cells against cytotoxicity induced by various stimuli. Recently, we showed that HN binds to and inhibits Bax, a proapoptotic Bcl-2 family protein, suggesting a mechanism for HN action. In this study, we identified Bim, a Bcl-2 homology 3-only member of the Bcl-2/Bax family, as an additional HN target protein. Using in vitro protein binding, immunoprecipitation, and coimmunolocalization assays, we demonstrated that HN binds directly to the extra long isoform of Bim (BimEL) but not the long (BimL) or short (BimS) isoforms. HN also protects cells against apoptosis induced by BimEL but not BimL and BimS in gene transfection studies. In contrast, mutants of HN which failed to bind BimEL failed to protect from BimEL-induced cell death. Moreover, HN inhibited BimEL-induced release of SMAC and cytochrome c from mitochondria isolated from bax–/–cells, indicating that HN can suppress BimEL independently of its effect on Bax. Finally, we demonstrate that HN prevents BimEL-induced oligomerization of Bak using isolated mitochondria. Taken together, our results indicate that the inhibition of BimEL may contribute to the antiapoptotic properties of the HN peptide. Humanin (HN) is a recently identified endogenous peptide that protects cells against cytotoxicity induced by various stimuli. Recently, we showed that HN binds to and inhibits Bax, a proapoptotic Bcl-2 family protein, suggesting a mechanism for HN action. In this study, we identified Bim, a Bcl-2 homology 3-only member of the Bcl-2/Bax family, as an additional HN target protein. Using in vitro protein binding, immunoprecipitation, and coimmunolocalization assays, we demonstrated that HN binds directly to the extra long isoform of Bim (BimEL) but not the long (BimL) or short (BimS) isoforms. HN also protects cells against apoptosis induced by BimEL but not BimL and BimS in gene transfection studies. In contrast, mutants of HN which failed to bind BimEL failed to protect from BimEL-induced cell death. Moreover, HN inhibited BimEL-induced release of SMAC and cytochrome c from mitochondria isolated from bax–/–cells, indicating that HN can suppress BimEL independently of its effect on Bax. Finally, we demonstrate that HN prevents BimEL-induced oligomerization of Bak using isolated mitochondria. Taken together, our results indicate that the inhibition of BimEL may contribute to the antiapoptotic properties of the HN peptide. Apoptosis or programmed cell death is a genetically regulated cellular suicide mechanism that plays critical roles in normal development, tissue homeostasis, and elimination of infected or damaged cells (1Jacobson M.D. Weil M. Raff M.C. Cell. 1997; 88: 347-354Abstract Full Text Full Text PDF PubMed Scopus (2383) Google Scholar, 2Hengartner M.O. Nature. 2000; 407: 770-776Crossref PubMed Scopus (6170) Google Scholar). Mitochondria represent crucial organelles for the integration of various cell death stimuli and execution of the cell death program (3Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar, 4Rathmell J.C. Thompson C.B. Cell. 2002; 109: S97-S107Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar). Mitochondria are capable of releasing several apoptogenic proteins into the cytosol, including cytochrome c, AIF, SMAC/Diablo, endonuclease G, and Omi/HtrA2 (5Kroemer G. Reed J.C. Nat. Med. 2000; 6: 513-519Crossref PubMed Scopus (2741) Google Scholar). The integrity of mitochondrial membranes is controlled primarily by a balance between the antagonistic actions of the proapoptotic and antiapoptotic members of the Bcl-2 family. Bcl-2 family proteins comprise three principal subfamilies: (a) antiapoptotic members, such as Bcl-2/Bcl-XL, which possess the Bcl-2 homology (BH) 1The abbreviations used are: BH, Bcl-2 homology; BimS, BimL, and BimEL, Bim short isoform, Bim long isoform, and Bim extra long isoform, respectively; BMH, bismaleimidoxane; BOP, BH3-only protein; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; EGFP, enhanced green fluorescent protein; FITC, fluorescein isothiocyanate; Fmoc, N-(9-fluorenyl)methoxycarbonyl; FPA, fluorescence polarization assay; GFP, green fluorescent protein; GST, glutathione S-transferase; HEK, human embryonic kidney; His6, hexahistidine; HN, Humanin; mAb, monoclonal antibody; MEF, mouse embryonic fibroblast; Ni-NTA, nickel-nitrilotriacetic acid; PBS, phosphate-buffered saline; ZVAD-fmk, benzyloxycarbonyl-Val-Ala-Asp (O-methyl)-fluoromethylketone; Ac-DEVD-AFC, Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin. 1The abbreviations used are: BH, Bcl-2 homology; BimS, BimL, and BimEL, Bim short isoform, Bim long isoform, and Bim extra long isoform, respectively; BMH, bismaleimidoxane; BOP, BH3-only protein; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; EGFP, enhanced green fluorescent protein; FITC, fluorescein isothiocyanate; Fmoc, N-(9-fluorenyl)methoxycarbonyl; FPA, fluorescence polarization assay; GFP, green fluorescent protein; GST, glutathione S-transferase; HEK, human embryonic kidney; His6, hexahistidine; HN, Humanin; mAb, monoclonal antibody; MEF, mouse embryonic fibroblast; Ni-NTA, nickel-nitrilotriacetic acid; PBS, phosphate-buffered saline; ZVAD-fmk, benzyloxycarbonyl-Val-Ala-Asp (O-methyl)-fluoromethylketone; Ac-DEVD-AFC, Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin. domains, BH1, BH2, BH3, and BH4; (b) proapoptotic members, including Bax, Bak, and Bok, which have the BH1, BH2, and BH3 domains; and (c) BH3-only proteins (BOPs), such as Bid, Bim, Bad, Bik, Puma, and Bmf, which generally possess only the BH3 domain (6Adams J.M. Cory S. Science. 1998; 281: 1322-1326Crossref PubMed Scopus (4754) Google Scholar). The BH3 domain mediates interactions among Bcl-2 family proteins, allowing for networks of protein interactions (7Wang X. Genes Dev. 2001; 15: 2922-2933Crossref PubMed Scopus (92) Google Scholar, 8Puthalakath H. Strasser A. Cell Death Differ. 2002; 9: 505-512Crossref PubMed Scopus (620) Google Scholar). Most BOPs function as antagonists of antiapoptotic Bcl-2 family proteins. However, some BOPs operate as both antagonists of the antiapoptotic proteins and as agonists of proapoptotic family members Bax and Bak. These agonists bind via their BH3 domains, inducing oligomerization of Bax and Bak. The pore-forming capability of oligomerized Bax and Bak results in the destabilization of the mitochondrial outer membrane and the subsequent release of the death molecules from the confines of these organelles. Cells derived from bax/bak double knock-out mice are resistant to a wide spectrum of apoptotic stimuli, including BOPs (9Wei M.C. Zong W.X. Cheng E.H. Lindsten T. Panoutsakopoulou V. Ross A.J. Roth K.A. MacGregor G.R. Thompson C.B. Korsmeyer S.J. Science. 2001; 292: 727-730Crossref PubMed Scopus (3297) Google Scholar, 10Zong W.X. Lindsten T. Ross A.J. MacGregor G.R. Thompson C.B. Genes Dev. 2001; 15: 1481-1486Crossref PubMed Scopus (704) Google Scholar), formally demonstrating that Bax and Bak mediate death signals from various BOPs. The BOP Bim was first identified as a Bcl-2-interacting protein (11O'Connor L. Strasser A. O'Reilly L.A. Hausmann G. Adams J.M. Cory S. Huang D.C. EMBO J. 1998; 17: 384-395Crossref PubMed Scopus (943) Google Scholar) but was subsequently shown to bind both antiapoptotic and proapoptotic members of the Bcl-2 family. Bim is expressed in hematopoietic, epithelial, neuronal, and germ cells (12O'Reilly L.A. Cullen L. Visvader J. Lindeman Huang D.C. Strasser A. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), and three short long and extra long BimEL the isoform in using gene knock-out mice have identified the gene as a of apoptosis in the plays a critical in and of as as cell Huang D.C. Adams J.M. Strasser A. Science. PubMed Scopus Google Scholar, L. H. M. Cory S. Adams J.M. Strasser A. Nature. 2002; PubMed Scopus Google Scholar, Strasser A. J. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). The gene is also for death of in to of A. H. Strasser A. J. Med. PubMed Scopus Google Scholar). Bim proteins also apoptosis in and of cells Huang D.C. Adams J.M. Strasser A. Science. PubMed Scopus Google Scholar, Adams Strasser A. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, T. T. H. T. A. T. Cell. 2001; PubMed Scopus Google Scholar, T. T. H. A. A. H. T. T. A. H. H. Strasser A. S. EMBO J. PubMed Scopus Google Scholar), their is regulated both the of protein and by such as S. A. PubMed Scopus Google Scholar, S. S. S. A. Full Text Full Text PDF PubMed Scopus Google Scholar, A. M. S. G. PubMed Scopus Google Scholar). In BimEL and BimL are on the the H. Huang D.C. O'Reilly L.A. Strasser A. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). In contrast, BimS not bind to a that may the of BimS BimEL and BimL in gene apoptosis such as and to release of BimEL and BimL from the in their to mitochondria. Bim is among the BOPs that can function as agonists of Bax and Bak, inducing oligomerization of these proteins in a and apoptogenic in mitochondrial membrane W.X. Lindsten T. Ross A.J. MacGregor G.R. Thompson C.B. Genes Dev. 2001; 15: 1481-1486Crossref PubMed Scopus (704) Google Scholar). of a derived from an T. M. 2001; PubMed Scopus Google Scholar, T. H. M. J. 2001; PubMed Google Scholar) identified an endogenous Humanin which cell death by including and in and cell HN is a short which is the in and some cell H. T. A. S. 2002; PubMed Scopus Google Scholar, S. Reed J.C. Nature. PubMed Scopus Google Scholar). The for of HN peptide in are of HN protein by interactions proteins is among the H. Huang D.C. O'Reilly L.A. Strasser A. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). an to HN is in a gene the mitochondrial in to the of several (11O'Connor L. Strasser A. O'Reilly L.A. Hausmann G. Adams J.M. Cory S. Huang D.C. EMBO J. 1998; 17: 384-395Crossref PubMed Scopus (943) Google Scholar), the of gene from mitochondrial to The mechanism of HN that HN is from cells T. M. 2001; PubMed Scopus Google Scholar, T. PubMed Scopus Google Scholar) and that a cell is by this peptide. our an mechanism of for HN, that this peptide binds to Bax and prevents the of this proapoptotic protein from to cells from that the mitochondrial not for cell death S. Reed J.C. Nature. PubMed Scopus Google Scholar). In contrast, HN not bind Bcl-2 family members, including Bak, Bok, and we the of HN by BimEL as a target of demonstrate that the of HN to BimEL its proapoptotic by BimEL-induced of Bax and Bak and cells from BimEL-induced cell death. the normal in of HN to these results the of proapoptotic Bcl-2/Bax family proteins that the HN peptide is capable of suggesting for this antiapoptotic peptide directly or for of Cell and and cells in and of cells was using or to the and the and BimEL, BimL, and BimS have A. M. S. G. PubMed Scopus Google Scholar). The a also S. Reed J.C. Nature. PubMed Scopus Google Scholar). human Bak was into the and of the of HN additional was by using an expressed HN as a The and into the and of mutants of HN by using the was by The the BimEL protein was a of T. S. 2002; PubMed Scopus Google Scholar). and was as using cell for protein S. Reed J.C. Nature. PubMed Scopus Google Scholar). cells in benzyloxycarbonyl-Val-Ala-Asp to Cells in and of Bim or monoclonal and of protein of in and to and for and and monoclonal various of or proteins HN peptide for in the in phosphate-buffered as S. Reed J.C. Nature. PubMed Scopus Google Scholar). polarization was in using an cells or in the of cells and for and for cells monoclonal for in PBS, and for in cell was using a which was in and as a cells in PBS, and in and and the protein of the using a protein of for protein in of in a in CHAPS, and was by in using are as fluorescence of of protein. using the an Apoptosis cells as both and cells PBS, and in to the by The of apoptotic cells by cells as apoptotic protein BimEL was expressed from in cells in of to a of was and the for Cells and in and by by for and the to The was of and a of in and using and an peptide HN and its on to and its and on The on the and The Bak BH3 peptide was and on the is to the and from In the of HN and its was used for and the of was used for to the was used in to the for the of The and from the by and for for The by the of and a The was and by an Mitochondria isolated from and Mitochondria using a cells for and in and Cells by in a for to and which The for and the mitochondria was Mitochondria in and used Mitochondria human was or various of HN peptide or peptide for The or peptide BimEL protein of mitochondria in a of of for for to and cytochrome c, and by using monoclonal cytochrome c SMAC and monoclonal Bak using the protein was in of or various of HN peptide for in of isolated mitochondria from cells was to a of for was for by the of by using Bax in Bax of this protein, of mitochondria was in and to monoclonal the by using Bax SMAC SMAC in using peptide or protein SMAC protein was as a protein and as A. Reed J.C. J. Full Text Full Text PDF PubMed Scopus Google Scholar). The SMAC was using a peptide to of human which was an to to proteins and S. M. A. T. Reed J.C. J. Google Scholar). The of for their protein was by HN to showed that HN can bind the proapoptotic protein Bax but not several antiapoptotic Bcl-2 family members S. Reed J.C. Nature. PubMed Scopus Google Scholar). to of HN to Bcl-2 family proteins, we to BimEL by both from cell the expressed proteins and by in vitro protein using from cells BimEL and HN an we using and the for by These demonstrated that can both endogenous or BimEL an In contrast, protein not results using HN expressed a not of HN BimEL was demonstrated by using BimEL protein and a HN peptide. various of a of peptide in in fluorescence allowing an of of the of In contrast, protein not bind the as a BimEL the Bim BimL and BimS, by these we the three of Bim, BimEL, BimL, and BimS, as proteins in cells by these cell we using monoclonal and the using to BimEL, BimL, and BimS from by immunoprecipitation, only BimEL the that HN can BimEL in we to the various of Bim in cells or protein. GFP, which was cells and in the was to in the of cells BimEL was to as by In contrast, and the of cells and not the protein not in cells among the various of Bim, the BimEL protein HN in HN BimEL-induced BimL, and BimS can apoptosis in cells (11O'Connor L. Strasser A. O'Reilly L.A. Hausmann G. Adams J.M. Cory S. Huang D.C. EMBO J. 1998; 17: 384-395Crossref PubMed Scopus (943) Google Scholar, Huang D.C. Adams J.M. Strasser A. Science. PubMed Scopus Google Scholar). the of HN BimEL can its to these cells BimEL in or was in cell as a of BimEL induced BimEL, but not BimEL-induced also the of on induced by BimEL, BimL, and BimS to to of shown in three Bim induced in in However, only induced by BimEL but not by BimL or BimS that the of HN to suppress to Bim protein, such that only the isoform of Bim which binds HN is the of these we in which a of HN was for to In this of for of the HN peptide shown to its to cells from apoptosis T. M. 2001; PubMed Scopus Google Scholar, T. H. M. J. 2001; PubMed Google Scholar). Moreover, we showed that the HN to bind Bax and is to protect cells against cell death S. Reed J.C. Nature. PubMed Scopus Google Scholar). the of HN and the of HN to protect cells against apoptosis induced by BimEL the results protein of BimEL induced and apoptosis which in a by of but not a also demonstrating of BimEL-induced and of the and proteins for the of these proteins The protein also showed that not of BimEL, BimEL-induced and Finally, in using was in of these proteins. of HN to BimEL its to suppress apoptosis induced by BimEL to HN interactions Bax S. Reed J.C. Nature. PubMed Scopus Google Scholar). HN BimEL-induced Apoptosis from have for the of mechanism HN as a peptide that protects by a cell and signals T. M. 2001; PubMed Scopus Google Scholar, T. H. M. J. 2001; PubMed Google Scholar, T. H. J. 2002; PubMed Scopus Google Scholar). The mechanism an of HN as an of family proteins S. Reed J.C. Nature. PubMed Scopus Google Scholar). between these we the of HN or a of these by cells X. Reed J.C. J. Full Text Full Text PDF PubMed Scopus Google Scholar). These in to Bcl-2/Bax family target proteins to of cells that BimEL or Bak. Bak was as a on that HN binds Bak apoptosis S. Reed J.C. Nature. PubMed Scopus Google Scholar). to of HN peptide a failed to suppress induced by BimEL or Bak In contrast, HN induced by of BimEL but not Bak. of these to of cells effect on of These results the that HN peptide cells for its a mechanism of family protein such as HN BimEL-induced SMAC and c from showed that HN peptide can the release of apoptogenic proteins from isolated mitochondria induced by Bax protein S. Reed J.C. Nature. PubMed Scopus Google Scholar). BimEL also to release of apoptogenic proteins directly to isolated mitochondria T. S. 2002; PubMed Scopus Google Scholar, H. J. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). the of HN peptide on BimEL-induced release of SMAC and cytochrome c using isolated mitochondria. we various of to its to SMAC release from isolated mitochondria from cells BimEL mitochondria by and the of SMAC in the mitochondria SMAC into the by using as to that the of mitochondria. These showed that SMAC release of of mitochondria In to BimEL, protein not endogenous SMAC release from isolated mitochondria the of these Using of we was for or various of HN peptide to mitochondria HN peptide SMAC and cytochrome c release from isolated mitochondria. the of these results HN we the of using isolated mitochondria. In to HN peptide not BimEL-induced SMAC release In we the of HN peptide to mitochondrial release of SMAC induced by an to resistant to we used a BH3 peptide of Bak, which is to cytochrome c release from isolated mitochondria H. J. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), by antiapoptotic Bcl-2 family proteins from proapoptotic proteins. In to BimEL, SMAC release induced by Bak BH3 peptide was not by HN peptide Taken together, these results the of of BimEL-induced release of apoptogenic proteins from isolated mitochondria. that the HN peptide used is the as the peptide used for cell that this peptide is an of BimEL in vitro cell HN BimEL-induced Bax additional to the mechanism by which HN the proapoptotic of BimEL is of the BOPs that can operate as an of antiapoptotic or as an of proapoptotic Bcl-2/Bax family proteins M. T. J. Cell. 2002; PubMed Scopus Google Scholar). to of antiapoptotic Bcl-2/Bax family proteins, we the of HN on the of BimEL antiapoptotic proteins Bcl-2 and by but we effect not HN is to the of BimEL to antiapoptotic members of the Bcl-2 family. our to an of the of HN on proapoptotic protein Bax, HN the of BimEL to of Bax, as by of this protein from the to Using a monoclonal for the of Bax, we mitochondria BimEL in the or of HN peptide. that shown that Bax is mitochondria in some of cells Bax in the to to mitochondria S. A. S. S. J.C. J. Cell PubMed Scopus Google BimEL induced of Bax to the as shown by the In contrast, BimEL HN peptide of Bax to the also BimEL-induced release of SMAC from mitochondria. using a the of of Bax in the of these results HN the of BimEL to proapoptotic protein Bax. HN BimEL-induced SMAC and c of HN peptide binds both BimEL and Bax S. Reed J.C. Nature. PubMed Scopus Google Scholar), the of BimEL-induced of Bax the of a effect on BimEL or Bax. between these we using mitochondria isolated from (9Wei M.C. Zong W.X. Cheng E.H. Lindsten T. Panoutsakopoulou V. Ross A.J. Roth K.A. MacGregor G.R. Thompson C.B. Korsmeyer S.J. Science. 2001; 292: 727-730Crossref PubMed Scopus (3297) Google Scholar, A. M. L. M. S. Korsmeyer S. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, E.H. M.C. S. Lindsten T. Korsmeyer S.J. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). BimEL induced release of SMAC and cytochrome c from both and mitochondria the of BimEL to bind and the protein Bak independently of Bax E.H. M.C. S. Lindsten T. Korsmeyer S.J. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). The of HN peptide inhibited BimEL-induced release of SMAC and cytochrome c from both and that HN can BimEL independently of Bax, a effect of HN on the BimEL protein. HN BimEL-induced of of apoptogenic proteins from mitochondria is oligomerization of Bax and Bak, which can by of S.J. M.C. M. S. Cell Death Differ. 2000; PubMed Scopus Google Scholar). Bim proteins are among the BOPs capable of inducing Bax and Bak oligomerization in mitochondrial membranes H. J. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, E.H. M.C. S. Lindsten T. Korsmeyer S.J. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). the of HN peptide on oligomerization of Bak, using to of the these BimEL was or HN to by the of BMH, and of the by using In Bak was as a of of mitochondria BimEL induced of Bak, as by the of of Bak in by of BimEL HN peptide in inhibition of Bak oligomerization for we these Bak oligomerization using peptide. HN, which inhibited Bax oligomerization as as the peptide was as as of HN showed that the first of HN are for to Bax S. Reed J.C. Nature. PubMed Scopus Google Scholar). to some of the of the HN peptide to BimEL and we the of of the HN peptide the first or the of this peptide and the or of HN can bind BimEL, we expressed these of HN as proteins in cells BimEL and HN protein. of BimEL using the of and but not of the of the and proteins in the of these proteins as a for the to BimEL we the of and on BimEL-induced release of SMAC from isolated mitochondria peptide in a the of SMAC from mitochondria BimEL protein, Finally, the of to suppress apoptosis induced by of BimEL was using as a for In BimEL induced an in which was by of or but not that the first of the HN peptide are and to bind BimEL and to suppress BimEL in vitro and in we a mechanism of for the endogenous peptide HN, that binds and inhibits proapoptotic protein Bax S. Reed J.C. Nature. PubMed Scopus Google Scholar). In this study, we the of HN by BimEL as a target of HN HN binds BimEL and inhibits its proapoptotic as demonstrated in cells and in using isolated mitochondria HN BimEL-induced oligomerization of Bak and release of apoptogenic proteins cytochrome c and BimEL can inhibited directly by HN, from Bax and Bak and the of BimEL on these proapoptotic members of the Bcl-2 family. we that HN binds and Bax, and in an we also that HN binds to and inhibits X. Reed J.C. J. Full Text Full Text PDF PubMed Scopus Google Scholar). The Bax, Bid, and BimEL proteins have in that are in the in an subsequently to mitochondria Bcl-2/Bax family members and release of apoptogenic proteins. is that HN peptide can target three of these proapoptotic proteins, their In and BimEL are the only BOPs the identified in that function as agonists of Bax and Bak. Bax, Bid, and BimEL that these proteins to bind However, the of BimEL is not is not to HN of is also by (a) the that the first of the HN peptide are to bind and suppress both BimEL and X. Reed J.C. J. Full Text Full Text PDF PubMed Scopus Google and by (b) using that a of HN for Bax, Bid, and in the HN peptide also directly or to binding, by peptide our that in HN are for Bax S. Reed J.C. Nature. PubMed Scopus Google Scholar). that BimEL is the isoform of Bim in of the BimL and BimS proteins (12O'Reilly L.A. Cullen L. Visvader J. Lindeman Huang D.C. Strasser A. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). However, to the that cells of the BimL and BimS proteins HN to from cell death induced BimEL the both BimEL and BimL are on in a the H. Huang D.C. O'Reilly L.A. Strasser A. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). to the of HN on the of the and HN the of these protein or of BimEL from the in vitro demonstrate that HN is capable of the of isolated BimEL, and we a for this peptide of the release of that the HN peptide can operate via cell to signals into the T. M. 2001; PubMed Scopus Google Scholar, T. PubMed Scopus Google Scholar) that to against HN from suggesting that a cell is the target of HN action. In was shown that HN peptide into can from cell death induced by T. H. J. 2002; PubMed Scopus Google Scholar). this was recently that HN can protein M. L. T. S. A. PubMed Scopus Google Scholar), that HN may we the that HN a function as both an and of cell our for an of HN peptide as an of Bcl-2/Bax family proapoptotic proteins. The this for HN from this study, the on X. Reed J.C. J. Full Text Full Text PDF PubMed Scopus Google Scholar), and our on Bax S. Reed J.C. Nature. PubMed Scopus Google Scholar), and (a) of HN apoptosis induced only by members and of the Bcl-2/Bax family that binds and mutants of HN to bind Bcl-2/Bax family (b) of HN peptide to suppress apoptosis a that to (c) of HN expressed as a protein; and of HN peptide to on isolated release of apoptogenic proteins in the of cell membrane The of the HN peptide not The of this peptide to for its as of a this antiapoptotic T. H. M. J. 2001; PubMed Google Scholar) and to Bax and the of the HN peptide. the in HN a in HN to cellular but a of of can In this that the of HN mediates of this peptide T. PubMed Scopus Google Scholar). However, the of the is not to and this is to to the of HN as an of BimEL, Bid, and Bax. HN is from cells and also by the in HN of the of the HN peptide and are to additional into the of this peptide. the in of the endogenous HN peptide to the that HN can bind and Bax, Bid, and BimEL the of this peptide or that for the of cell to the that BimEL, Bid, and Bax possess their to bind the of the HN may for or that three of these proapoptotic proteins. In this several have Bax, Bid, and Bim in cell death Adams Strasser A. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, S. S. J. A. Korsmeyer S.J. S. A. 2001; PubMed Scopus Google Scholar, L.A. J. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, K.A. J. 2002; PubMed Google Scholar, J. 2001; Full Text Full Text PDF PubMed Google Scholar, S. T. A. D.C. J. PubMed Scopus Google Scholar, J. S.J. L. J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, J.M. J. J. 2002; PubMed Google Scholar, A. J. J. 1997; PubMed Google Scholar, G. L. J. 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