{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T21:16:48Z","timestamp":1788470208220,"version":"build-2803163510"},"reference-count":42,"publisher":"American Chemical Society (ACS)","issue":"44","license":[{"start":{"date-parts":[[2015,10,15]],"date-time":"2015-10-15T00:00:00Z","timestamp":1444867200000},"content-version":"vor","delay-in-days":365,"URL":"https:\/\/pubs.acs.org\/page\/policy\/authorchoice_termsofuse.html"}],"funder":[{"DOI":"10.13039\/100000060","name":"National Institute of Allergy and Infectious Diseases","doi-asserted-by":"publisher","award":["AI022021"],"award-info":[{"award-number":["AI022021"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2014,11,11]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>The protective antigen (PA) moiety of anthrax toxin forms oligomeric pores in the endosomal membrane, which translocate the effector proteins of the toxin to the cytosol. Effector proteins bind to oligomeric PA via their respective N-terminal domains and undergo N- to C-terminal translocation through the pore. Earlier we reported that a tract of basic amino acids fused to the N-terminus of an unrelated effector protein (the catalytic domain diphtheria toxin, DTA) potentiated that protein to undergo weak PA-dependent translocation. In this study, we varied the location of the tract (N-terminal or C-terminal) and the length of a poly-Lys tract fused to DTA and examined the effects of these variations on PA-dependent translocation into cells and across planar bilayers in vitro. Entry into cells was most efficient with \u223c12 Lys residues (K12) fused to the N-terminus but also occurred, albeit 10\u2013100-fold less efficiently, with a C-terminal tract of the same length. Similarly, K12 tracts at either terminus occluded PA pores in planar bilayers, and occlusion was more efficient with the N-terminal tag. We used biotin-labeled K12 constructs in conjunction with streptavidin to show that a biotinyl-K12 tag at either terminus is transiently exposed to the trans compartment of planar bilayers at 20 mV; this partial translocation in vitro was more efficient with an N-terminal tag than a C-terminal tag. Significantly, our studies with polycationic tracts fused to the N- and C-termini of DTA suggest that PA-mediated translocation can occur not only in the N to C direction but also in the C to N direction.<\/jats:p>","DOI":"10.1021\/bi500985v","type":"journal-article","created":{"date-parts":[[2014,10,15]],"date-time":"2014-10-15T14:21:47Z","timestamp":1413382907000},"page":"6934-6940","source":"Crossref","is-referenced-by-count":7,"title":["Polylysine-Mediated Translocation of the Diphtheria\nToxin Catalytic Domain through the Anthrax Protective Antigen Pore"],"prefix":"10.1021","volume":"53","author":[{"given":"Onkar","family":"Sharma","sequence":"first","affiliation":[{"name":"Harvard Medical School , , 77 Avenue Louis Pasteur , , ,","place":["Boston, Massachusetts, United States, 02115"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"R. John","family":"Collier","sequence":"additional","affiliation":[{"name":"Harvard Medical School , , 77 Avenue Louis Pasteur , , ,","place":["Boston, Massachusetts, United States, 02115"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2014,10,15]]},"reference":[{"key":"2026090316260373800_cit1","doi-asserted-by":"crossref","first-page":"3162","DOI":"10.1073\/pnas.79.10.3162","article-title":"Anthrax\ntoxin edema factor: A bacterial adenylate cyclase that increases cyclic\nAMP concentrations of eukaryotic cells","volume":"79","author":"Leppla","year":"1982","journal-title":"Proc.\nNatl. Acad. Sci. U.S.A."},{"key":"2026090316260373800_cit2","doi-asserted-by":"crossref","first-page":"396","DOI":"10.1038\/415396a","article-title":"Structural\nbasis for the activation of anthrax adenylyl cyclase exotoxin by calmodulin","volume":"415","author":"Drum","year":"2002","journal-title":"Nature"},{"key":"2026090316260373800_cit3","doi-asserted-by":"crossref","first-page":"647","DOI":"10.1111\/j.1365-2958.1993.tb00936.x","article-title":"pH-dependent permeabilization of the plasma membrane\nof mammalian cells by anthrax protective antigen","volume":"10","author":"Milne","year":"1993","journal-title":"Mol. Microbiol."},{"key":"2026090316260373800_cit4","doi-asserted-by":"crossref","first-page":"20607","DOI":"10.1016\/S0021-9258(17)32036-7","article-title":"Anthrax\nprotective antigen forms oligomers during intoxication of mammalian\ncells","volume":"269","author":"Milne","year":"1994","journal-title":"J. Biol. Chem."},{"key":"2026090316260373800_cit5","doi-asserted-by":"crossref","first-page":"833","DOI":"10.1038\/385833a0","article-title":"Crystal\nstructure of the anthrax toxin protective antigen","volume":"385","author":"Petosa","year":"1997","journal-title":"Nature"},{"key":"2026090316260373800_cit6","doi-asserted-by":"crossref","first-page":"243","DOI":"10.1146\/annurev.biochem.75.103004.142728","article-title":"Anthrax toxin: Receptor binding, internalization, pore\nformation, and translocation","volume":"76","author":"Young","year":"2007","journal-title":"Annu. Rev. Biochem."},{"key":"2026090316260373800_cit7","doi-asserted-by":"crossref","first-page":"225","DOI":"10.1038\/n35101999","article-title":"Identification\nof the cellular receptor for anthrax toxin","volume":"414","author":"Bradley","year":"2001","journal-title":"Nature"},{"key":"2026090316260373800_cit8","doi-asserted-by":"crossref","first-page":"5170","DOI":"10.1073\/pnas.0431098100","article-title":"Human capillary\nmorphogenesis protein 2 functions as an anthrax toxin receptor","volume":"100","author":"Scobie","year":"2003","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"2026090316260373800_cit9","doi-asserted-by":"crossref","first-page":"905","DOI":"10.1038\/nature02763","article-title":"Crystal\nstructure of a complex between anthrax toxin and its host cell receptor","volume":"430","author":"Santelli","year":"2004","journal-title":"Nature"},{"key":"2026090316260373800_cit10","doi-asserted-by":"crossref","first-page":"13147","DOI":"10.1073\/pnas.0405405101","article-title":"Structure\nof heptameric protective antigen bound to an anthrax toxin receptor:\nA role for receptor in pH-dependent pore formation","volume":"101","author":"Lacy","year":"2004","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"2026090316260373800_cit11","doi-asserted-by":"crossref","first-page":"23349","DOI":"10.1074\/jbc.M401292200","article-title":"Binding stoichiometry and kinetics of the interaction\nof a human anthrax toxin receptor, CMG2, with protective antigen","volume":"279","author":"Wigelsworth","year":"2004","journal-title":"J. Biol. Chem."},{"key":"2026090316260373800_cit12","doi-asserted-by":"crossref","first-page":"106","DOI":"10.1016\/j.mib.2004.12.005","article-title":"Interactions between anthrax toxin receptors and protective\nantigen","volume":"8","author":"Scobie","year":"2005","journal-title":"Curr. Opin. Microbiol."},{"key":"2026090316260373800_cit13","doi-asserted-by":"crossref","first-page":"16396","DOI":"10.1016\/S0021-9258(18)42016-9","article-title":"Human furin\nis a calcium-dependent serine endoprotease that recognizes the sequence\nArg-X-X-Arg and efficiently cleaves anthrax toxin protective antigen","volume":"267","author":"Molloy","year":"1992","journal-title":"J. Biol. Chem."},{"key":"2026090316260373800_cit14","doi-asserted-by":"crossref","first-page":"10277","DOI":"10.1073\/pnas.89.21.10277","article-title":"Anthrax toxin protective\nantigen is activated by a cell surface protease with the sequence\nspecificity and catalytic properties of furin","volume":"89","author":"Klimpel","year":"1992","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"2026090316260373800_cit15","doi-asserted-by":"crossref","first-page":"614","DOI":"10.1016\/j.jmb.2009.07.037","article-title":"The protective antigen component of anthrax toxin forms\nfunctional octameric complexes","volume":"392","author":"Kintzer","year":"2009","journal-title":"J. Mol. Biol."},{"key":"2026090316260373800_cit16","doi-asserted-by":"crossref","first-page":"7049","DOI":"10.1073\/pnas.062160399","article-title":"Mapping the lethal\nfactor and edema factor binding sites on oligomeric anthrax protective\nantigen","volume":"99","author":"Cunningham","year":"2002","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"2026090316260373800_cit17","doi-asserted-by":"crossref","first-page":"258","DOI":"10.1016\/j.bbrc.2004.07.105","article-title":"Anthrax toxin complexes:\nHeptameric protective antigen can bind lethal factor and edema factor\nsimultaneously","volume":"322","author":"Pimental","year":"2004","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"2026090316260373800_cit18","doi-asserted-by":"crossref","first-page":"1079","DOI":"10.1021\/bi015860m","article-title":"Stoichiometry of anthrax toxin complexes","volume":"41","author":"Mogridge","year":"2002","journal-title":"Biochemistry"},{"key":"2026090316260373800_cit19","doi-asserted-by":"crossref","first-page":"281","DOI":"10.1078\/0171-9335-00373","article-title":"Effects of\ndynamin inactivation on pathways of anthrax toxin uptake","volume":"83","author":"Boll","year":"2004","journal-title":"Eur. J. 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Microbiol."},{"key":"2026090316260373800_cit23","doi-asserted-by":"crossref","first-page":"15737","DOI":"10.1021\/bi981436i","article-title":"Characterization\nof membrane translocation by anthrax protective antigen","volume":"37","author":"Wesche","year":"1998","journal-title":"Biochemistry"},{"key":"2026090316260373800_cit24","doi-asserted-by":"crossref","first-page":"1059","DOI":"10.1074\/jbc.M609869200","article-title":"Insertion of anthrax\nprotective antigen into liposomal membranes: Effects of a receptor","volume":"282","author":"Sun","year":"2007","journal-title":"J. Biol. Chem."},{"key":"2026090316260373800_cit25","doi-asserted-by":"crossref","first-page":"13278","DOI":"10.1073\/pnas.0505865102","article-title":"Receptor-specific requirements for anthrax toxin delivery\ninto cells","volume":"102","author":"Rainey","year":"2005","journal-title":"Proc. Natl. Acad. Sci. 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Oncol."},{"key":"2026090316260373800_cit30","doi-asserted-by":"crossref","first-page":"4955","DOI":"10.1128\/iai.62.11.4955-4961.1994","article-title":"Fusions of anthrax toxin lethal factor with shiga toxin\nand diphtheria toxin enzymatic domains are toxic to mammalian cells","volume":"62","author":"Arora","year":"1994","journal-title":"Infect. Immun."},{"key":"2026090316260373800_cit31","doi-asserted-by":"crossref","first-page":"12531","DOI":"10.1073\/pnas.93.22.12531","article-title":"Anthrax toxin-mediated delivery of\na cytotoxic T-cell epitope in vivo","volume":"93","author":"Ballard","year":"1996","journal-title":"Proc. Natl.\nAcad. Sci. U.S.A."},{"key":"2026090316260373800_cit32","doi-asserted-by":"crossref","first-page":"1383","DOI":"10.1038\/nsmb.1923","article-title":"Structural basis\nfor the unfolding of anthrax lethal factor by protective antigen oligomers","volume":"17","author":"Feld","year":"2010","journal-title":"Nat. Struct. Mol. Biol."},{"key":"2026090316260373800_cit33","doi-asserted-by":"crossref","first-page":"16756","DOI":"10.1073\/pnas.0405754101","article-title":"Evidence that translocation of anthrax\ntoxin\u2019s lethal factor is initiated by entry of its N terminus\ninto the protective antigen channel","volume":"101","author":"Zhang","year":"2004","journal-title":"Proc. Natl.\nAcad. Sci. U.S.A."},{"key":"2026090316260373800_cit34","doi-asserted-by":"crossref","first-page":"777","DOI":"10.1126\/science.1113380","article-title":"A phenylalanine clamp catalyzes protein\ntranslocation through the anthrax toxin pore","volume":"309","author":"Krantz","year":"2005","journal-title":"Science"},{"key":"2026090316260373800_cit35","doi-asserted-by":"crossref","first-page":"8437","DOI":"10.1073\/pnas.93.16.8437","article-title":"Fused polycationic\npeptide mediates delivery of diphtheria toxin A chain to the cytosol\nin the presence of anthrax protective antigen","volume":"93","author":"Blanke","year":"1996","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"2026090316260373800_cit36","doi-asserted-by":"crossref","first-page":"e46964","DOI":"10.1371\/journal.pone.0046964","article-title":"Role of N-terminal His6-Tags in binding\nand efficient translocation of polypeptides into cells using anthrax\nprotective antigen (PA)","volume":"7","author":"Beitzinger","year":"2012","journal-title":"PLoS One"},{"key":"2026090316260373800_cit37","doi-asserted-by":"crossref","first-page":"4346","DOI":"10.1073\/pnas.0800701105","article-title":"Phenylalanine-427\nof anthrax protective antigen functions in both pore formation and\nprotein translocation","volume":"105","author":"Sun","year":"2008","journal-title":"Proc. Natl. Acad. Sci.\nU.S.A."},{"key":"2026090316260373800_cit38","doi-asserted-by":"crossref","first-page":"8130","DOI":"10.1074\/jbc.M109.093195","article-title":"Effects of introducing a single charged\nresidue into the phenylalanine clamp of multimeric anthrax protective\nantigen","volume":"285","author":"Janowiak","year":"2010","journal-title":"J. Biol. Chem."},{"key":"2026090316260373800_cit39","doi-asserted-by":"crossref","first-page":"3842","DOI":"10.1529\/biophysj.104.050864","article-title":"Protein translocation\nthrough anthrax toxin channels formed in planar lipid bilayers","volume":"87","author":"Zhang","year":"2004","journal-title":"Biophys. J."},{"key":"2026090316260373800_cit40","doi-asserted-by":"crossref","first-page":"29","DOI":"10.1016\/0300-483X(94)90153-8","article-title":"The channel\nformed in planar lipid bilayers by the protective antigen component\nof anthrax toxin","volume":"87","author":"Finkelstein","year":"1994","journal-title":"Toxicology"},{"key":"2026090316260373800_cit41","doi-asserted-by":"crossref","first-page":"1868","DOI":"10.1073\/pnas.1018965108","article-title":"Interactions of anthrax lethal factor\nwith protective antigen defined by site-directed spin labeling","volume":"108","author":"Jennings-Antipov","year":"2011","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"2026090316260373800_cit42","doi-asserted-by":"crossref","first-page":"1390","DOI":"10.1111\/j.1365-2958.2011.07770.x","article-title":"GRP78(BiP) facilitates the cytosolic\ndelivery of anthrax lethal factor (LF) in vivo and functions as an\nunfoldase in vitro","volume":"81","author":"Tamayo","year":"2011","journal-title":"Mol. Microbiol."}],"container-title":["Biochemistry"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/bi500985v","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/bichaw\/article-pdf\/53\/44\/6934\/15695783\/bi500985v.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/bichaw\/article-pdf\/53\/44\/6934\/15695783\/bi500985v.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T20:26:17Z","timestamp":1788467177000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/bichaw\/article\/53\/44\/6934\/1476891\/Polylysine-Mediated-Translocation-of-the"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2014,10,15]]},"references-count":42,"journal-issue":{"issue":"44","published-online":{"date-parts":[[2014,10,15]]},"published-print":{"date-parts":[[2014,11,11]]}},"URL":"https:\/\/doi.org\/10.1021\/bi500985v","relation":{},"ISSN":["0006-2960","1520-4995"],"issn-type":[{"value":"0006-2960","type":"print"},{"value":"1520-4995","type":"electronic"}],"subject":[],"published":{"date-parts":[[2014,10,15]]}}}