{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,12]],"date-time":"2025-10-12T02:16:10Z","timestamp":1760235370941,"version":"build-2065373602"},"reference-count":35,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2021,8,18]],"date-time":"2021-08-18T00:00:00Z","timestamp":1629244800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001711","name":"Schweizerischer Nationalfonds zur F\u00f6rderung der Wissenschaftlichen Forschung","doi-asserted-by":"publisher","award":["310030_192689"],"award-info":[{"award-number":["310030_192689"]}],"id":[{"id":"10.13039\/501100001711","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceutics"],"abstract":"<jats:p>Anthrax toxin has evolved to translocate its toxic cargo proteins to the cytosol of cells carrying its cognate receptor. Cargo molecules need to unfold to penetrate the narrow pore formed by its membrane-spanning subunit, protective antigen (PA). Various alternative cargo molecules have previously been tested, with some showing only limited translocation efficiency, and it may be assumed that these were too stable to be unfolded before passing through the anthrax pore. In this study, we systematically and quantitatively analyzed the correlation between the translocation of various designed ankyrin repeat proteins (DARPins) and their different sizes and thermodynamic stabilities. To measure cytosolic uptake, we used biotinylation of the cargo by cytosolic BirA, and we measured cargo equilibrium stability via denaturant-induced unfolding, monitored by circular dichroism (CD). Most of the tested DARPin cargoes, including target-binding ones, were translocated to the cytosol. Those DARPins, which remained trapped in the endosome, were confirmed by CD to show a high equilibrium stability. We could pinpoint a stability threshold up to which cargo DARPins still get translocated to the cytosol. These experiments have outlined the requirements for translocatable binding proteins, relevant stability measurements to assess translocatable candidates, and guidelines to further engineer this property if needed.<\/jats:p>","DOI":"10.3390\/pharmaceutics13081285","type":"journal-article","created":{"date-parts":[[2021,8,18]],"date-time":"2021-08-18T01:54:06Z","timestamp":1629251646000},"page":"1285","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Thermodynamic Stability Is a Strong Predictor for the Delivery of DARPins to the Cytosol via Anthrax Toxin"],"prefix":"10.3390","volume":"13","author":[{"given":"Lukas","family":"Becker","sequence":"first","affiliation":[{"name":"Department of Biochemistry, University of Zurich, Winterthurerstr. 190, 8057 Zurich, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jasleen","family":"Singh Badwal","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, University of Zurich, Winterthurerstr. 190, 8057 Zurich, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7927-5442","authenticated-orcid":false,"given":"Fabian","family":"Brandl","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, University of Zurich, Winterthurerstr. 190, 8057 Zurich, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2463-277X","authenticated-orcid":false,"given":"Wouter P. R.","family":"Verdurmen","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Radboud Institute for Molecular Life Sciences (RIMLS), Radboud University Medical Center, Geert Grooteplein 28, 6525 GA Nijmegen, The Netherlands"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4191-5306","authenticated-orcid":false,"given":"Andreas","family":"Pl\u00fcckthun","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, University of Zurich, Winterthurerstr. 190, 8057 Zurich, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2021,8,18]]},"reference":[{"key":"ref_1","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 functional octameric complexes","volume":"392","author":"Kintzer","year":"2009","journal-title":"J. Mol. Biol."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Friebe, S., van der Goot, F., and B\u00fcrgi, J. (2016). The ins and outs of anthrax toxin. Toxins, 8.","DOI":"10.3390\/toxins8030069"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"606","DOI":"10.1002\/pro.2052","article-title":"Ratcheting up protein translocation with anthrax toxin","volume":"21","author":"Feld","year":"2012","journal-title":"Protein Sci."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"817","DOI":"10.1097\/CJI.0b013e3181ad4d5d","article-title":"Affitoxin\u2014A novel recombinant, HER2-specific, anticancer agent for targeted therapy of HER2-positive tumors","volume":"32","author":"Zielinski","year":"2009","journal-title":"J. Immunother."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Mechaly, A., McCluskey, A.J., and Collier, R.J. (2012). Changing the receptor specificity of anthrax toxin. mBio, 3.","DOI":"10.1128\/mBio.00088-12"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"13","DOI":"10.1016\/j.jconrel.2014.12.019","article-title":"Efficient cell-specific uptake of binding proteins into the cytoplasm through engineered modular transport systems","volume":"200","author":"Verdurmen","year":"2015","journal-title":"J. Control. Release"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"440","DOI":"10.1016\/j.molonc.2012.12.003","article-title":"Targeting HER2-positive cancer cells with receptor-redirected anthrax protective antigen","volume":"7","author":"McCluskey","year":"2013","journal-title":"Mol. Oncol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"87","DOI":"10.1007\/BF03401732","article-title":"Internalization of a Bacillus anthracis protective antigen-c-myc fusion protein mediated by cell surface anti-c-myc antibodies","volume":"4","author":"Varughese","year":"1998","journal-title":"Mol. Med."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Becker, L., Verdurmen, W.P.R., and Pl\u00fcckthun, A. (2020). Reengineering anthrax toxin protective antigen for improved receptor-specific protein delivery. BMC Biol., 18.","DOI":"10.1186\/s12915-020-00827-y"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1490","DOI":"10.1021\/acschembio.6b00169","article-title":"Delivery of non-native cargo into mammalian cells using anthrax lethal toxin","volume":"11","author":"Rabideau","year":"2016","journal-title":"ACS Chem. Biol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"15737","DOI":"10.1021\/bi981436i","article-title":"Characterization of membrane translocation by anthrax protective antigen","volume":"37","author":"Wesche","year":"1998","journal-title":"Biochemistry"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"5263","DOI":"10.1038\/s41467-019-13253-8","article-title":"Towards the application of Tc toxins as a universal protein translocation system","volume":"10","author":"Roderer","year":"2019","journal-title":"Nat. Commun."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"21555","DOI":"10.1073\/pnas.0905880106","article-title":"Lethal factor unfolding is the most force-dependent step of anthrax toxin translocation","volume":"106","author":"Thoren","year":"2009","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"16756","DOI":"10.1073\/pnas.0405754101","article-title":"Evidence that translocation of anthrax toxin\u2019s lethal factor is initiated by entry of its N terminus into the protective antigen channel","volume":"101","author":"Zhang","year":"2004","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"2294","DOI":"10.1002\/anie.201006460","article-title":"Chemical dissection of protein translocation through the anthrax toxin pore","volume":"50","author":"Pentelute","year":"2011","journal-title":"Angew. Chem. Int. Ed. Engl."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"11944","DOI":"10.1038\/srep11944","article-title":"Translocation of non-canonical polypeptides into cells using protective antigen","volume":"5","author":"Rabideau","year":"2015","journal-title":"Sci. Rep."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"545","DOI":"10.1038\/nature14247","article-title":"Atomic structure of anthrax protective antigen pore elucidates toxin translocation","volume":"521","author":"Jiang","year":"2015","journal-title":"Nature"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1383","DOI":"10.1038\/nsmb.1923","article-title":"Structural basis for the unfolding of anthrax lethal factor by protective antigen oligomers","volume":"17","author":"Feld","year":"2010","journal-title":"Nat. Struct. Mol. Biol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"13","DOI":"10.1016\/j.bcp.2017.04.009","article-title":"Repurposing bacterial toxins for intracellular delivery of therapeutic proteins","volume":"142","author":"Beilhartz","year":"2017","journal-title":"Biochem. Pharmacol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"223","DOI":"10.1007\/978-1-4939-6857-2_14","article-title":"A Biotin ligase-based assay for the quantification of the cytosolic delivery of therapeutic proteins","volume":"Volume 1575","author":"Tiller","year":"2017","journal-title":"Synthetic Antibodies"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"575","DOI":"10.1038\/nbt962","article-title":"High-affinity binders selected from designed ankyrin repeat protein libraries","volume":"22","author":"Binz","year":"2004","journal-title":"Nat. Biotechnol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1700","DOI":"10.1073\/pnas.0337680100","article-title":"Designed to be stable: Crystal structure of a consensus ankyrin repeat protein","volume":"100","author":"Kohl","year":"2003","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1211","DOI":"10.1016\/j.jmb.2008.07.085","article-title":"Efficient selection of DARPins with sub-nanomolar affinities using SRP phage display","volume":"382","author":"Steiner","year":"2008","journal-title":"J. Mol. Biol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1252","DOI":"10.1242\/bio.201410041","article-title":"Protein interference applications in cellular and developmental biology using DARPins that recognize GFP and mCherry","volume":"3","author":"Brauchle","year":"2014","journal-title":"Biol. Open"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1356","DOI":"10.1021\/cb3001167","article-title":"Designed Ankyrin Repeat Proteins (DARPins) as novel isoform-specific intracellular inhibitors of c-Jun N-terminal kinases","volume":"7","author":"Parizek","year":"2012","journal-title":"ACS Chem. Biol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"837","DOI":"10.1016\/j.jmb.2007.09.042","article-title":"Characterization and further stabilization of designed ankyrin repeat proteins by combining molecular dynamics simulations and experiments","volume":"375","author":"Interlandi","year":"2008","journal-title":"J. Mol. Biol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"3334","DOI":"10.1016\/S0021-9258(18)53698-X","article-title":"Residues 1-254 of anthrax toxin lethal factor are sufficient to cause cellular uptake of fused polypeptides","volume":"268","author":"Arora","year":"1993","journal-title":"J. Biol. Chem."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"241","DOI":"10.1016\/j.jmb.2007.11.046","article-title":"Folding and unfolding mechanism of highly stable full-consensus ankyrin repeat proteins","volume":"376","author":"Wetzel","year":"2008","journal-title":"J. Mol. Biol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"691","DOI":"10.1016\/j.jmb.2013.10.026","article-title":"From DARPins to LoopDARPins: Novel LoopDARPin design allows the selection of low picomolar binders in a single round of ribosome display","volume":"426","author":"Schilling","year":"2014","journal-title":"J. Mol. Biol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"280","DOI":"10.1002\/prot.20930","article-title":"Crystal structure of a consensus-designed ankyrin repeat protein: Implications for stability","volume":"65","author":"Binz","year":"2006","journal-title":"Proteins"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"2458","DOI":"10.1002\/cbic.201402290","article-title":"Delivery of antibody mimics into mammalian cells via anthrax toxin protective antigen","volume":"15","author":"Liao","year":"2014","journal-title":"ChemBioChem"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"356","DOI":"10.1016\/j.jmb.2007.04.039","article-title":"Stable intermediate states and high energy barriers in the unfolding of GFP","volume":"370","author":"Huang","year":"2007","journal-title":"J. Mol. Biol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"739","DOI":"10.1016\/j.jmb.2004.09.067","article-title":"Acid-induced unfolding of the amino-terminal domains of the lethal and edema factors of anthrax toxin","volume":"344","author":"Krantz","year":"2004","journal-title":"J. Mol. Biol."},{"key":"ref_34","first-page":"68","article-title":"Influence of chemical denaturants on the activity, fold and zinc status of anthrax lethal factor","volume":"1","author":"Lo","year":"2015","journal-title":"Biochem. Biophys. Rep."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"568","DOI":"10.1006\/bbrc.2001.5018","article-title":"Effect of pH on stability of anthrax lethal factor: Correlation between denaturation and activity","volume":"284","author":"Gupta","year":"2001","journal-title":"Biochem. Biophys. Res. Commun."}],"container-title":["Pharmaceutics"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4923\/13\/8\/1285\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T06:46:08Z","timestamp":1760165168000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4923\/13\/8\/1285"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,8,18]]},"references-count":35,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2021,8]]}},"alternative-id":["pharmaceutics13081285"],"URL":"https:\/\/doi.org\/10.3390\/pharmaceutics13081285","relation":{},"ISSN":["1999-4923"],"issn-type":[{"type":"electronic","value":"1999-4923"}],"subject":[],"published":{"date-parts":[[2021,8,18]]}}}