title: Subtilase cytotoxin induces apoptosis in HeLa cells by mitochondrial permeabilization via activation of Bax/Bak, independent of C/EBF-homologue protein (CHOP), Ire1alpha or JNK signaling.
abstract: Subtilase cytotoxin (SubAB) is an AB(5) cytotoxin produced by some strains of Shiga-toxigenic Escherichia coli. The A subunit is a subtilase-like serine protease and cleaves an endoplasmic reticulum (ER) chaperone, BiP, leading to transient inhibition of protein synthesis and cell cycle arrest at G(1) phase, and inducing caspase-dependent apoptosis via mitochondrial membrane damage in Vero cells. Here we investigated the mechanism of mitochondrial permeabilization in HeLa cells. SubAB-induced cytochrome c release into cytosol did not depend on mitochondrial permeability transition pore (PTP), since cyclosporine A did not suppress cytochrome c release. SubAB did not change the expression of anti-apoptotic Bcl-2 or Bcl-XL and pro-apoptotic Bax or Bak, but triggered Bax and Bak conformational changes and association of Bax with Bak. Silencing using siRNA of both bax and bak genes, but not bax, bak, or bim alone, resulted in reduction of cytochrome c release, caspase-3 activation, DNA ladder formation and cytotoxicity, indicating that Bax and Bak were involved in apoptosis. SubAB activated ER transmembrane transducers, Ire1alpha, and cJun N-terminal kinase (JNK), and induced C/EBF-homologue protein (CHOP). To investigate whether these signals were involved in cytochrome c release by Bax activation, we silenced ire1alpha, jnk or chop; however, silencing did not decrease SubAB-induced cytochrome c release, suggesting that these signals were not necessary for SubAB-induced mitochondrial permeabilization by Bax activation.
authors.affiliation: Department of Molecular Infectiology, Graduate School of Medicine, Chiba University, Chuo-ku, Chiba, Japan. yahirok@faculty.chiba-u.jp
authors.forename: Kinnosuke
authors.initials: K
authors.lastname: Yahiro
authors.forename: Naoko
authors.initials: N
authors.lastname: Morinaga
authors.forename: Joel
authors.initials: J
authors.lastname: Moss
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Noda
mesh_terms: D017209:Apoptosis; D053148:Caspase 3 / Q000378:metabolism; D045304:Cytochromes c / Q000378:metabolism; D003593:Cytoplasm / Q000737:chemistry; D004722:Endoribonucleases / Q000378:metabolism; D004926:Escherichia coli / Q000472:pathogenicity*; D029968:Escherichia coli Proteins / Q000633:toxicity*; D020868:Gene Silencing; D006367:HeLa Cells; D006801:Humans; D048670:MAP Kinase Kinase 4 / Q000378:metabolism; D051336:Mitochondrial Membranes / Q000187:drug effects*; D010539:Permeability / Q000187:drug effects*; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D015398:Signal Transduction; D013381:Subtilisins / Q000633:toxicity*; D051742:Transcription Factor CHOP / Q000378:metabolism; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051028:bcl-2-Associated X Protein / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*
chemical_list: C494772:BAK1 protein, human; C494826:BAX protein, human; C497249:DDIT3 protein, human; D029968:Escherichia coli Proteins; D034741:RNA, Small Interfering; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; D051742:Transcription Factor CHOP; D045304:Cytochromes c; C528229:ERN1 protein, human; D017346:Protein Serine-Threonine Kinases; D048670:MAP Kinase Kinase 4; D004722:Endoribonucleases; D013381:Subtilisins; C527234:subtilase cytotoxin, E coli; D053148:Caspase 3