title: Engineering anthrax toxin variants that exclusively form octamers and their application to targeting tumors.
abstract: Anthrax toxin protective antigen (PA) delivers its effector proteins into the host cell cytosol through formation of an oligomeric pore, which can assume heptameric or octameric states. By screening a highly directed library of PA mutants, we identified variants that complement each other to exclusively form octamers. These PA variants were individually nontoxic and demonstrated toxicity only when combined with their complementary partner. We then engineered requirements for activation by matrix metalloproteases and urokinase plasminogen activator into two of these variants. The resulting therapeutic toxin specifically targeted cells expressing both tumor associated proteases and completely stopped tumor growth in mice when used at a dose far below that which caused toxicity. This scheme for obtaining intercomplementing subunits can be employed with other oligomeric proteins and potentially has wide application.
authors: Laboratory of Parasitic Diseases, NIAID, National Institutes of Health, Bethesda, MD 20892, USA.
authors: Damilola D
authors: DD
authors: Phillips
authors: Rasem J
authors: RJ
authors: Fattah
authors: Devorah
authors: D
authors: Crown
authors: Yi
authors: Y
authors: Zhang
authors: Shihui
authors: S
authors: Liu
authors: Mahtab
authors: M
authors: Moayeri
authors: Elizabeth R
authors: ER
authors: Fischer
authors: Bryan T
authors: BT
authors: Hansen
authors: Rodolfo
authors: R
authors: Ghirlando
authors: Ekaterina M
authors: EM
authors: Nestorovich
authors: Alexander N
authors: AN
authors: Wein
authors: Lacy
authors: L
authors: Simons
authors: Stephen H
authors: SH
authors: Leppla
authors: Clinton E
authors: CE
authors: Leysath
mesh_terms: D000818:Animals; D000942:Antigens, Bacterial / Q000737:chemistry*; D001408:Bacillus anthracis / Q000378:metabolism; D001427:Bacterial Toxins / Q000737:chemistry*; D045744:Cell Line, Tumor; D005260:Female; D015723:Gene Library; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008819:Mice, Nude; D008968:Molecular Conformation; D009154:Mutation; D009369:Neoplasms / Q000188:drug therapy* / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods; D025941:Protein Interaction Mapping / Q000379:methods; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry; D014461:Ultracentrifugation
chemical_list: D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D011506:Proteins; C030325:anthrax toxin
references: Proc Natl Acad Sci U S A. 1982 May;79(10):3162-6
references: 6285339
references: Microbiol Mol Biol Rev. 2004 Sep;68(3):373-402, table of contents
references: 15353562
references: Phys Rev Lett. 2004 Aug 27;93(9):098106
references: 15447147
references: Proc Natl Acad Sci U S A. 2004 Sep 7;101(36):13147-51
references: 15326297
references: Infect Immun. 1999 Apr;67(4):1853-9
references: 10085027
references: J Mol Biol. 1998 Feb 20;276(2):325-30
references: 9512705
references: J Biol Chem. 2001 May 25;276(21):17976-84
references: 11278833
references: Mol Med. 1998 Feb;4(2):87-95
references: 9508786
references: Science. 1996 Dec 13;274(5294):1859-66
references: 8943190
references: J Biol Chem. 1989 Nov 15;264(32):19103-7
references: 2509473
references: PLoS Pathog. 2012;8(3):e1002638
references: 22479187
references: Cancer Res. 2000 Nov 1;60(21):6061-7
references: 11085528
references: Science. 1998 May 1;280(5364):734-7
references: 9563949
references: Protein Expr Purif. 2000 Apr;18(3):293-302
references: 10733882
references: Am J Pathol. 2011 Jun;178(6):2523-35
references: 21641378
references: Nat Struct Mol Biol. 2010 Nov;17(11):1383-90
references: 21037566
references: J Biol Chem. 1994 Aug 12;269(32):20607-12
references: 8051159
references: Biophys J. 2000 Mar;78(3):1606-19
references: 10692345
references: Proc Natl Acad Sci U S A. 2002 May 14;99(10):7045-8
references: 11997437
references: Gene. 1989 Apr 15;77(1):51-9
references: 2744487
references: Protein Expr Purif. 2011 Nov;80(1):80-90
references: 21827967
references: Nat Biotechnol. 2005 Jun;23(6):725-30
references: 15895075
references: Mol Aspects Med. 2009 Dec;30(6):439-55
references: 19638283
references: Annu Rev Biochem. 2007;76:243-65
references: 17335404
references: J Mol Biol. 2012 Jan 6;415(1):159-74
references: 22063095
references: Biochem Biophys Res Commun. 2001 Sep 21;287(2):542-9
references: 11554763
references: Mol Aspects Med. 2009 Dec;30(6):374-85
references: 19607856
references: J Bacteriol. 2001 Mar;183(6):2111-6
references: 11222612
references: Methods Cell Biol. 2008;84:143-79
references: 17964931
references: PLoS One. 2010 Nov 08;5(11):e13888
references: 21079738
references: J Mol Biol. 2009 Sep 25;392(3):614-29
references: 19627991
references: Biochem J. 2000 Dec 15;352 Pt 3:739-45
references: 11104681