title: Induction of apoptosis in macrophages by Pseudomonas aeruginosa azurin: tumour-suppressor protein p53 and reactive oxygen species, but not redox activity, as critical elements in cytotoxicity.
abstract: Azurin is a copper-containing protein involved in electron transfer during denitrification. We reported recently that purified azurin demonstrates cytotoxicity to macrophages by forming a complex with the tumour-suppressor protein p53, thereby stabilizing it and enhancing its function as an inducer of proapoptotic activity (Yamada, T., Goto, M., Punj, V., Zaborina, O., Kimbara, K., Das Gupta, T. K., and Chakrabarty, A. M. 2002, Infect Immun70: 7054-7062). It is, however, not known whether the oxidoreductase (redox) activity of azurin or the involvement of copper is important for its cytotoxicity. We have isolated apo-azurin devoid of copper and site-directed mutants that are redox negative because of either replacement of a cysteine residue (Cys-112) involved in co-ordination with copper or mutational replacement of two methionine residues (Met-44 and Met-64) that are present in the hydrophobic patch of azurin and allow interaction of azurin with its redox partner cytochrome c551. We demonstrate that, although the wild type (wt) and the Cys-112 Asp mutant azurin can form complexes with the tumour-suppressor protein p53 and generate high levels of reactive oxygen species (ROS), the redox-negative Met-44LysMet-64Glu mutant azurin is defective in complex formation with p53, generates low levels of ROS and lacks appreciable cytotoxicity towards macrophages. Thus, complex formation with p53 and ROS generation, rather than azurin redox activity, are important in the cytotoxic action of azurin towards macrophages.
authors.affiliation: Department of Microbiology, University of Illinois at Chicago, 835 South Wolcott Avenue, Chicago, Il 60612, USA.
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Goto
authors.forename: Tohru
authors.initials: T
authors.lastname: Yamada
authors.forename: Kazuhide
authors.initials: K
authors.lastname: Kimbara
authors.forename: John
authors.initials: J
authors.lastname: Horner
authors.forename: Martin
authors.initials: M
authors.lastname: Newcomb
authors.forename: T K Das
authors.initials: TK
authors.lastname: Gupta
authors.forename: A M
authors.initials: AM
authors.lastname: Chakrabarty
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001400:Azurin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D002460:Cell Line; D008264:Macrophages / Q000382:microbiology*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011550:Pseudomonas aeruginosa / Q000472:pathogenicity*; D017382:Reactive Oxygen Species / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
chemical_list: D017382:Reactive Oxygen Species; D016159:Tumor Suppressor Protein p53; D001400:Azurin