Abstract
AbstractDiseases involving mitochondrial defects usually manifest themselves in high-energy, post-mitotic tissues such as brain, retina, skeletal and cardiac muscle and frequently cause deficiencies in mitochondrial bioenergetics. We have developed a scalable procedure to produce recombinant 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). in vitro studies in a classic mitochondrial disease cell model demonstrated that Alexa488-labeled MTD-TFAM rapidly entered the mitochondrial compartment. MTD-TFAM treatment of these cell lines reversibly increased oxygen consumption (respiration) rates 3-fold, levels of respiratory proteins and mitochondrial gene expression. in vivo results demonstrated that respiration increased to lesser degrees in mitochondria from tissues of mice injected with MTD-TFAM. MTD-TFAM can alter mitochondrial bioenergetics and holds promise for treatment of mitochondrial diseases involving deficiencies of energy production.
Highlights
Some of the basics of mitochondrial DNA (mtDNA) replication and transcription are known, much is either controversial or remains to be discovered 5, 10-12
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[3,4,5, 13,14,15,16]
We report here the development of a technology to produce recombinant TFAM engineered with an N-terminal protein transduction domain (PTD), followed by a matrix mitochondrial localization sequence (MLS)
Summary
Some of the basics of mtDNA replication and transcription are known, much is either controversial or remains to be discovered 5, 10-12. Three consecutive independent experiments were carried out over several months in which LHON cybrid cells at the same initial passage numbers were treated with MTD-TFAM or buffer control (CTL). We observed that exposure to MTD-TFAM caused a time-dependent, reversible increase in basal respiration rates that reached a maximal ~3-fold increase over control samples at around 2 weeks (Figure 2A).
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