RACK1 protein interacts with Helicobacter pylori VacA cytotoxin: the yeast two-hybrid approach.
The VacA toxin is the major virulence factor of Helicobacter pylori. The studies on VacA intracellular expression suggest that it interacts with cytosolic proteins and that this interaction contributes significantly to vacuolization. The aim of this study was to identify the host protein(s) that interacts with the VacA protein. We used the fragments of VacA protein fused with GAL4-BD as the baits in the yeast two-hybrid approach. The yeast transformed with plasmids encoding bait proteins were screened with human gastric mucosa cDNA library, encoded C-terminal fusion proteins with GAL4-AD. Three independent His-beta-Gal-positive clones were identified in VacA-b1 screen; they matched two different lengths of cDNA encoding RACK1 protein. The specific activity of beta-galactosidase found in the yeast expressing both VacA-b1 and RACK1 fusion proteins was 12-19 times higher compared to all negative controls used. VacA is capable of binding the RACK1 in vitro as was confirmed by the pull-down assay with GST fusion VacA protein and [(35)S]Met-labeled RACK1 protein fragments.
Hennig
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EE
Department of Gastroenterology, Meidcal Center for Postgraduate Education, Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, 02-781 Warsaw, Poland. hennige@coi.waw.pl
Butruk
E
E
Ostrowski
J
J
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity; D001483:Base Sequence; D003603:Cytotoxins / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity; D004269:DNA, Bacterial / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D005753:Gastric Mucosa / Q000378:metabolism; D016480:Helicobacter pylori / Q000235:genetics / Q000472:pathogenicity*; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000633:toxicity; D012689:Sequence Homology, Nucleic Acid; D020798:Two-Hybrid System Techniques; D014774:Virulence
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D003603:Cytotoxins; D004269:DNA, Bacterial; D018076:DNA, Complementary; D010446:Peptide Fragments; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; C074689:VacA protein, Helicobacter pylori