title: Identification of subtilase cytotoxin (SubAB) receptors whose signaling, in association with SubAB-induced BiP cleavage, is responsible for apoptosis in HeLa cells.
abstract: Subtilase cytotoxin (SubAB), which is produced by certain strains of Shiga-toxigenic Escherichia coli (STEC), causes the 78-kDa glucose-regulated protein (GRP78/BiP) cleavage, followed by induction of endoplasmic reticulum (ER) stress, leading to caspase-dependent apoptosis via mitochondrial membrane damage by Bax/Bak activation. The purpose of the present study was to identify SubAB receptors responsible for HeLa cell death. Four proteins, NG2, α2β1 integrin (ITG), L1 cell adhesion molecule (L1CAM), and hepatocyte growth factor receptor (Met), were identified to be SubAB-binding proteins by immunoprecipitation and purification, followed by liquid chromatography-tandem mass spectrometry analysis. SubAB-induced Bax conformational change, Bax/Bak complex formation, caspase activation, and cell death were decreased in β1 ITG, NG2, and L1CAM small interfering RNA-transfected cells, but unexpectedly, BiP cleavage was still observed. Pretreatment of cells with a function-blocking β1 ITG antibody (monoclonal antibody [MAb] P5D2) enhanced SubAB-induced caspase activation; MAb P5D2 alone had no effect on caspase activation. Furthermore, we found that SubAB induced focal adhesion kinase fragmentation, which was mediated by a proteasome-dependent pathway, and caspase activation was suppressed in the presence of proteasome inhibitor. Thus, β1 ITG serves as a SubAB-binding protein and may interact with SubAB-signaling pathways, leading to cell death. Our results raise the possibility that although BiP cleavage is necessary for SubAB-induced apoptotic cell death, signaling pathways associated with functional SubAB receptors may be required for activation of SubAB-dependent apoptotic pathways.
authors.affiliation: Department of Molecular Infectiology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. yahirok@faculty.chiba-u.jp
authors.forename: Kinnosuke
authors.initials: K
authors.lastname: Yahiro
authors.forename: Mamoru
authors.initials: M
authors.lastname: Satoh
authors.forename: Naoko
authors.initials: N
authors.lastname: Morinaga
authors.forename: Hiroyasu
authors.initials: H
authors.lastname: Tsutsuki
authors.forename: Kohei
authors.initials: K
authors.lastname: Ogura
authors.forename: Sayaka
authors.initials: S
authors.lastname: Nagasawa
authors.forename: Fumio
authors.initials: F
authors.lastname: Nomura
authors.forename: Joel
authors.initials: J
authors.lastname: Moss
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Noda
mesh_terms: D017209:Apoptosis; D000091342:Endoplasmic Reticulum Chaperone BiP; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D020868:Gene Silencing; D006367:HeLa Cells; D006801:Humans; D038982:Integrin alpha2beta1 / Q000378:metabolism; D039842:Neural Cell Adhesion Molecule L1 / Q000378:metabolism; D011485:Protein Binding; D019859:Proto-Oncogene Proteins c-met / Q000378:metabolism; D054323:Shiga-Toxigenic Escherichia coli / Q000378:metabolism*; D013381:Subtilisins / Q000235:genetics / Q000378:metabolism*; D019284:Thapsigargin
chemical_list: D000091342:Endoplasmic Reticulum Chaperone BiP; D029968:Escherichia coli Proteins; C000717247:HSPA5 protein, human; D038982:Integrin alpha2beta1; D039842:Neural Cell Adhesion Molecule L1; D019284:Thapsigargin; D019859:Proto-Oncogene Proteins c-met; D013381:Subtilisins; C527234:subtilase cytotoxin, E coli