title: Enhanced in-cell folding of reversibly cationized transcription factor using amphipathic peptide.
abstract: The intracellular delivery of functionally active transcription factor proteins is emerging as a promising technique for artificial regulation of cellular functions. However, in addition to the cell membrane, which acts as a barrier to macromolecules, the aggregation-favored properties of structurally flexible transcription factor proteins limit the application of this method. In-cell folding technique can be used to overcome these issues. This technique solubilizes denatured protein by reversible alkyl-disulfide cationization (S-cationization), and simultaneously endows efficient intracellular delivery and folding to the biologically active conformation in the reducing environment of the cytosol. Because cationized protein is internalized into cells by adsorption-mediated endocytosis, endosomal escape is crucial for this technique. In this study, we utilized a sensitive luciferase reporter gene assay to quantitatively evaluate in-cell folding of the artificial transcription factor GAL4-VP16. Although the cationic moiety of S-cationized protein was slightly affected, co-transduction of amphipathic peptide Endo-PORTER dramatically improved in-cell folding efficiency. Live cell imaging of fluorescent-labeled GAL4-VP16 revealed that some of the proteins diffused into the cytosol and nucleus through co-transduction with Endo-PORTER. Real-time monitoring of light output of luciferase revealed the kinetics of in-cell folding, supporting that endosomal-release assisted by Endo-PORTER was stimulated by endosome acidification. Because this method can transduce proteins uniformly and repeatedly into living cells, S-cationized transcription factor proteins are widely applicable for the artificial regulation of cellular functions.
authors.affiliation: Department of Biomedical Engineering, Faculty of Engineering, Okayama University of Science, Okayama 700-0005, Japan.
authors.forename: Midori
authors.initials: M
authors.lastname: Futami
authors.affiliation: Department of Biomedical Engineering, Faculty of Engineering, Okayama University of Science, Okayama 700-0005, Japan.
authors.forename: Tomoki
authors.initials: T
authors.lastname: Nakano
authors.affiliation: Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Kagawa 761-0395, Japan.
authors.forename: Mayu
authors.initials: M
authors.lastname: Yasunaga
authors.affiliation: Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Makihara
authors.affiliation: Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Asama
authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Osaka 563-8577, Japan.
authors.forename: Yoshihisa
authors.initials: Y
authors.lastname: Hagihara
authors.affiliation: Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Kagawa 761-0395, Japan.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Nakajima
authors.affiliation: Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan. Electronic address: futamij@okayama-u.ac.jp.
authors.forename: Junichiro
authors.initials: J
authors.lastname: Futami
mesh_terms: D000595:Amino Acid Sequence; D002412:Cations / Q000737:chemistry / Q000378:metabolism; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism; D011992:Endosomes / Q000737:chemistry / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D011489:Protein Denaturation; D017510:Protein Folding; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014161:Transduction, Genetic
chemical_list: D002412:Cations; C511217:Endo-Porter; C070633:Gal-VP16; D010455:Peptides; D015534:Trans-Activators; D014157:Transcription Factors
keywords: Amphipathic peptide; Cell-permeable peptide; Chemical modification; In-cell folding; Protein transduction