title: A HA2-Fusion tag limits the endosomal release of its protein cargo despite causing endosomal lysis.
abstract: BACKGROUND
  Protein transduction domains (PTDs) can be fused to a protein to render it cell-permeable. The delivery efficiencies of PTDs are, however, often poor because PTD-protein conjugates cannot escape from endosomes. A potential solution to this problem consists in adding HA2 analogs to the PTD-protein construct as these peptides can cause endosomal lysis upon acidification of the endosomal lumen. To date, however, the utility of HA2-based PTDs has not been clearly established.
  
  
  METHODS
  We investigate the biophysical and cellular properties of the glutamate-rich HA2 analog E5 fused to the model protein TAT-mCherry.
  
  
  RESULTS
  E5-TAT-mCherry causes the release of fluorescent dextrans trapped with the protein inside endosomes. Yet, E5-TAT-mCherry itself is not released in the cytosol of cells, indicating that the protein remained trapped inside endosomes even after endosomal lysis takes place. Cytosolic delivery of the protein could be achieved, however, by insertion of a disulfide bond between E5 and its cargo.
  
  
  CONCLUSIONS
  These results show that E5 causes the retention of its fused protein inside endosomes even after lysis takes place.
  
  
  CONCLUSIONS
  These data establish that HA2 analogs might not be useful PTDs unless cleavable linkers are engineered between PTD and protein cargo.
authors.affiliation: Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843, USA.
authors.forename: Ya-Jung
authors.initials: YJ
authors.lastname: Lee
authors.forename: Gregory
authors.initials: G
authors.lastname: Johnson
authors.forename: Grantham C
authors.initials: GC
authors.lastname: Peltier
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Pellois
mesh_terms: D011992:Endosomes / Q000235:genetics / Q000378:metabolism*; D015707:HN Protein / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D009980:Influenza A virus; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D015707:HN Protein; D011993:Recombinant Fusion Proteins