title: Intracellular delivery of full length recombinant human mitochondrial L-Sco2 protein into the mitochondria of permanent cell lines and SCO2 deficient patient's primary cells.
abstract: Mutations in human SCO2 gene, encoding the mitochondrial inner membrane Sco2 protein, have been found to be responsible for fatal infantile cardioencephalomyopathy and cytochrome c oxidase (COX) deficiency. One potentially fruitful therapeutic approach for this mitochondrial disorder should be considered the production of human recombinant full length L-Sco2 protein and its deliberate transduction into the mitochondria. Recombinant L-Sco2 protein, fused with TAT, a Protein Transduction Domain (PTD), was produced in bacteria and purified from inclusion bodies (IBs). Following solubilisation with l-arginine, this fusion L-Sco2 protein was transduced in cultured mammalian cells of different origin (U-87 MG, T24, K-562, and patient's primary fibroblasts) and assessed for stability, transduction into mitochondria, processing and impact on recovery of COX activity. Our results indicate that: a) l-Arg solution was effective in solubilising recombinant fusion L-Sco2 protein, derived from IBs; b) fusion L-Sco2 protein was delivered successfully via a time- and concentration-dependent process into the mitochondria of human U-87 MG and T24 cells; c) fusion L-Sco2 protein was also transduced in human K-562 cells, transiently depleted of SCO2 transcripts and thus COX deficient; transduction of this fusion protein led to partial recovery of COX activity in such cells; d) [(35)S]Methionine-labelled fusion L-Sco2 protein, produced in a cell free transcription/translation system and incubated with intact isolated mitochondria derived from K-562 cells, was efficiently processed to yield the corresponding mature Sco2 protein, thus justifying the potential of the transduced fusion L-Sco2 protein to successfully activate COX holoenzyme; and finally, e) recombinant fusion L-Sco2 protein was successfully transduced into the mitochondria of primary fibroblasts derived from SCO2/COX deficient patient and facilitated recovery of COX activity. These findings provide the rationale of delivering recombinant proteins via PTD technology as a model for therapeutic approach of mitochondrial disorders.
authors.affiliation: Laboratory of Pharmacology, Department of Pharmaceutical Sciences, Aristotle University of Thessaloniki, Thessaloniki GR-54124, Macedonia, Greece.
authors.forename: Parthena F
authors.initials: PF
authors.lastname: Foltopoulou
authors.forename: Asterios S
authors.initials: AS
authors.lastname: Tsiftsoglou
authors.forename: Ioannis D
authors.initials: ID
authors.lastname: Bonovolias
authors.forename: Alexandra T
authors.initials: AT
authors.lastname: Ingendoh
authors.forename: Lefkothea C
authors.initials: LC
authors.lastname: Papadopoulou
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002478:Cells, Cultured; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D003576:Electron Transport Complex IV / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D020014:K562 Cells; D008928:Mitochondria / Q000378:metabolism*; D028361:Mitochondrial Diseases / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism*; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D014161:Transduction, Genetic
chemical_list: D002352:Carrier Proteins; D017931:DNA Primers; D024101:Mitochondrial Proteins; D018832:Molecular Chaperones; D011993:Recombinant Fusion Proteins; C400285:SCO2 protein, human; D003576:Electron Transport Complex IV