Elsevier

Mitochondrion

Volume 9, Issue 3, June 2009, Pages 196-203
Mitochondrion

Recombinant mitochondrial transcription factor A with N-terminal mitochondrial transduction domain increases respiration and mitochondrial gene expression

https://doi.org/10.1016/j.mito.2009.01.012Get rights and content

Abstract

We developed a scalable procedure to produce human mitochondrial transcription factor A (TFAM) modified with an N-terminal protein transduction domain (PTD) and mitochondrial localization signal (MLS) that allow it to cross membranes and enter mitochondria through its “mitochondrial transduction domain” (MTD = PTD + MLS). Alexa488-labeled MTD–TFAM rapidly entered the mitochondrial compartment of cybrid cells carrying the G11778A LHON mutation. MTD–TFAM reversibly increased respiration and levels of respiratory proteins. In vivo treatment of mice with MTD–TFAM increased motor endurance and complex I-driven respiration in mitochondria from brain and skeletal muscle. MTD–TFAM increases mitochondrial bioenergetics and holds promise for treatment of mitochondrial diseases involving deficiencies of energy production.

Introduction

Mammalian mitochondrial DNA (mtDNA) is a ∼16.6 kilobases circular genome that consists of a regulatory control region (“D-loop”), 13 genes for essential catalytic proteins of the ∼87 proteins in the electron transport chain (ETC), 22 tRNA’s and two ribosomal RNA’s that facilitate translation of the mtDNA-encoded ETC proteins in the mitochondrial matrix (Dimauro and Schon, 2008). The remainder of the ETC proteins and ∼1200–1500 of the other mitochondrial catalytic and structural proteins are imported using multi-protein translocase complexes of the outer (TOM) and inner (TIM) mitochondrial membranes that direct protein precursors formed outside mitochondria to their appropriate location by means of specific N-terminal mitochondrial localization sequences (Kutik et al., 2007). After reaching their final destinations, the localization sequences are cleaved by mitochondrial proteases (Kutik et al., 2007). Because most catalytic ETC proteins coded by mtDNA are hydrophobic, special mitochondrial chaperones are believed to assist in proper folding and insertion into their respective ETC macrocomplexes (Leidhold and Voos, 2007, Szabadkai and Rizzuto, 2007).

Although some of the basics of mtDNA replication and transcription are known, much is either controversial or remains to be discovered (Fernandez-Silva et al., 2003, Brown et al., 2005, Scarpulla, 2008, Shadel, 2008). Abnormalities of mtDNA replication and transcription (such as production of deleted species) or translation (due to mutations in tRNA or coding ETC genes) are responsible for illnesses present in childhood or early adulthood involving high energy, post-mitotic tissues such as brain, retina, heart and skeletal muscle (Wallace, 2005, Dimauro and Schon, 2008). These “mitochondrial” diseases can display variable and overlapping phenotypes, and understanding their genotype–phenotype relationships remains a great challenge (Wallace, 2005, Dimauro and Schon, 2008).

Further insights into understanding mitochondrial genome replication and expression, in addition to development of novel therapies for mitochondrial diseases, would benefit from technology that allows external manipulation of the mitochondrial genome. Mitochondrial transcription factor A (TFAM) is a member of the high-mobility group (HMG) of DNA-binding proteins that participate in mtDNA replication and transcription (Garstka et al., 2003, Ekstrand et al., 2004, Maniura-Weber et al., 2004, Pohjoismaki et al., 2006, Cotney et al., 2007, Kang et al., 2007, Scarpulla, 2008). Genetic deletion of TFAM is embryonic lethal (Wallace, 2002), demonstrating its essential role in mitochondrial function.

Earlier (Khan and Bennett, 2004), we proposed an approach to delivery of mtDNA cargo to the mitochondrial matrix based on the use of recombinant TFAM engineered with an N-terminal protein transduction domain (PTD), followed by a matrix mitochondrial localization sequence (MLS). We refer to the combination of PTD and MLS as “mitochondrial transduction domain” (MTD). We now report the development of a scaled-up technology to produce a recombinant MTD–TFAM possessing similar overall properties but a different primary structure (Fig. 1A). We then asked whether this MTD–TFAM protein by itself could affect mitochondrial function in vitro and in vivo and describe effects of this recombinant MTD–TFAM on respiratory physiology in SH-SY5Y cybrid cells carrying the G11778A LHON mutation and in normal adult mice.

Section snippets

Expression of MTD–TFAM

We included a HA epitope after the 11-Arginine PTD based on reports that the HA epitope facilitates cytosolic escape of transduced protein after macropinocytosis (Wadia et al., 2004). The nucleotide sequence corresponding to PTD-HA-MLS-TFAM (see below) was subcloned into PE-Sumo3 (Life Sensors). The construct was transformed into Tuner (DE3)pLysS cells (Novagen). Recombinant protein was expressed by the transformed bacteria cultured in Overnight Express TB medium (Novagen), an auto-induction

Results

MTD–TFAM (Fig. 1A) was produced initially as a N-terminal 6XHis-SUMO derivative to increase its intracellular solubility and with a rapid induction approach to minimize toxicity. The initial protein extract was treated with benzonase to remove contaminating DNA; 6XHis-SUMO-MTD–TFAM was isolated on a nickel column, eluted and treated with SUMO protease. Subsequent passage through a nickel column isolated the 6XHis-SUMO, and the eluted MTD–TFAM was purified further and shown to bind and retard

Discussion

In this study, we have shown that the naturally occurring TFAM protein which is essential for mtDNA expression and replication can be engineered with a protein transduction domain and mitochondrial localization signal (“MTD–TFAM”) so as to be able to enter rapidly into the mitochondrial compartment of cells. After developing a scalable production procedure for this recombinant protein, we found that incubation for only a few hours with MTD–TFAM, followed by return of cells to regular culture

Disclosures

F.R.P. is an officer in Gencia Corporation and has a personal financial interest in the development of the MTD–TFAM technology described in this paper. None of the other authors has a personal financial interest in Gencia Corp. or the technology described.

Acknowledgements

We would like to thank Russell Swerdlow for access to the LHON cybrid cells and Kate Borland for all the work on confocal ETC immunostaining. We also thank members of the Clayton Laboratory for useful discussions and Shaharyar Khan and Raj Rao for critical reading of the manuscript. This work was supported in part by funds from the National Institute of Health (NS39788, AG023443, Bennett), from the American Parkinson’s Disease Association (Iyer) and the Parkinson’s Disease Foundation (Iyer) and

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