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
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
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DOI：
10.1016/j.bbadis.2010.02.009
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发表时间：
2010-06-01
影响因子：
6.2
通讯作者：
Papadopoulou, Lefkothea C.
中科院分区：
文献类型：
--
作者：
Foltopoulou, Parthena F.;Tsiftsoglou, Asterios S.;Papadopoulou, Lefkothea C.
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, 124, 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 124 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) [S-35]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. (C) 2010 Elsevier B.V. All rights reserved.
