title: Heterologous mitochondrial targeting sequences can deliver functional proteins into mitochondria.
abstract: Mitochondrial Targeting Sequences (MTSs) are responsible for trafficking nuclear-encoded proteins into mitochondria. Once entering the mitochondria, the MTS is recognized and cleaved off. Some MTSs are long and undergo two-step processing, as in the case of the human frataxin (FXN) protein (80aa), implicated in Friedreich's ataxia (FA). Therefore, we chose the FXN protein to examine whether nuclear-encoded mitochondrial proteins can efficiently be targeted via a heterologous MTS (hMTS) and deliver a functional protein into mitochondria. We examined three hMTSs; that of citrate synthase (cs), lipoamide deydrogenase (LAD) and C6ORF66 (ORF), as classically MTS sequences, known to be removed by one-step processing, to deliver FXN into mitochondria, in the form of fusion proteins. We demonstrate that using hMTSs for delivering FXN results in the production of 4-5-fold larger amounts of the fusion proteins, and at 4-5-fold higher concentrations. Moreover, hMTSs delivered a functional FXN protein into the mitochondria even more efficiently than the native MTSfxn, as evidenced by the rescue of FA patients' cells from oxidative stress; demonstrating a 18%-54% increase in cell survival; and a 13%-33% increase in ATP levels, as compared to the fusion protein carrying the native MTS. One fusion protein with MTScs increased aconitase activity within patients' cells, by 400-fold. The implications form our studies are of vast importance for both basic and translational research of mitochondrial proteins as any mitochondrial protein can be delivered efficiently by an hMTS. Moreover, effective targeting of functional proteins is important for restoration of mitochondrial function and treatment of related disorders.
authors: Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada (IMRIC), Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem 91120, Israel.
authors: Dana
authors: D
authors: Marcus
authors: Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada (IMRIC), Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem 91120, Israel.
authors: Michal
authors: M
authors: Lichtenstein
authors: Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada (IMRIC), Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem 91120, Israel.
authors: Natali
authors: N
authors: Cohen
authors: Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada (IMRIC), Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem 91120, Israel.
authors: Rita
authors: R
authors: Hadad
authors: Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada (IMRIC), Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem 91120, Israel.
authors: Tal
authors: T
authors: Erlich-Hadad
authors: BioBlast-Pharma Ltd., Israel.
authors: Hagar
authors: H
authors: Greif
authors: Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada (IMRIC), Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem 91120, Israel. Electronic address: hayag@ekmd.huji.ac.il.
authors: Haya
authors: H
authors: Lorberboum-Galski
mesh_terms: D000154:Aconitate Hydratase / Q000378:metabolism; D005621:Friedreich Ataxia / Q000378:metabolism; D006801:Humans; D033862:Iron-Binding Proteins / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism*; D018384:Oxidative Stress; D021381:Protein Transport; D000096923:Frataxin
chemical_list: D000154:Aconitate Hydratase; D033862:Iron-Binding Proteins; D000096923:Frataxin
keywords: Fusion protein; HIV-transactivator of transcription (TAT) peptide; Heterologous mitochondrial targeting sequence (hMTS); Human frataxin (FXN); Mitochondria