A Small Molecule Inhibitor of Redox-Regulated Protein Translocation into Mitochondria.
ABSTRACT The mitochondrial disulfide relay system of Mia40 and Erv1/ALR facilitates import of the small translocase of the inner membrane (Tim) proteins and cysteine-rich proteins. A chemical screen identified small molecules that inhibit Erv1 oxidase activity, thereby facilitating dissection of the disulfide relay system in yeast and vertebrate mitochondria. One molecule, mitochondrial protein import blockers from the Carla Koehler laboratory (MitoBloCK-6), attenuated the import of Erv1 substrates into yeast mitochondria and inhibited oxidation of Tim13 and Cmc1 in in vitro reconstitution assays. In addition, MitoBloCK-6 revealed an unexpected role for Erv1 in the carrier import pathway, namely transferring substrates from the translocase of the outer membrane complex onto the small Tim complexes. Cardiac development was impaired in MitoBloCK-6-exposed zebrafish embryos. Finally, MitoBloCK-6 induced apoptosis via cytochrome c release in human embryonic stem cells (hESCs) but not in differentiated cells, suggesting an important role for ALR in hESC homeostasis.
- SourceAvailable from: Lawrence I Grossman
[Show abstract] [Hide abstract]
- "When all four cysteine residues were converted to serines (C–S) no interaction was seen with Mia40 and no localization to mitochondria was seen (Fig. 4C). MitoBloCK-6 has been shown to be a selective inhibitor of the Mia40/Erv1 pathway (Dabir et al., 2013). To evaluate whether MNRR1 is imported via this pathway, 293 cells were co-transfected with a Flag-tagged expression plasmid for MNRR1 and an HA-tagged expression plasmid for Akt. "
ABSTRACT: Our understanding of stress-associated regulatory mechanisms for mitochondria remains incomplete. We now report a new regulator of mitochondrial metabolism, the coiled-coil-helix-coiled-coil-helix domain-containing protein 2 (CHCHD2) which, based on the functionality described here, is renamed MNRR1 (Mitochondria Nuclear Retrograde Regulator 1). It functions in a novel way by acting in two cellular compartments, mitochondria and nucleus. In normally growing cells most MNRR1 is located in mitochondria; during stress most MNRR1 is now located in the nucleus. MNRR1 is imported to the mitochondrial intermembrane space by a Mia40-mediated pathway, where it binds to cytochrome c oxidase (COX). This association is required for full COX activity. Decreased MNRR1 levels produce widespread dysfunction including reduced COX activity, membrane potential, and growth rate, and increased reactive oxygen species and mitochondrial fragmentation. In the nucleus, MNRR1 acts as a transcription factor, one of whose targets is the COX subunit 4 isoform, COX4I2, which is transcriptionally stimulated by hypoxia. This MNRR1-mediated stress response may provide an important survival mechanism for cells under conditions of oxidative or hypoxic stress, both in the acute phase by altering mitochondrial oxygen utilization and in the chronic phase by promoting COX remodeling.Mitochondrion 10/2014; 20. DOI:10.1016/j.mito.2014.10.003 · 3.52 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Disulfide bond formation drives protein import of most proteins of the mitochondrial intermembrane space (IMS). The main components of this disulfide relay machinery are the oxidoreductase Mia40 and the sulfhydryl oxidase Erv1/ALR. Their precise functions have been elucidated in molecular detail for the yeast and human enzymes in vitro and in intact cells. However, we still lack knowledge on how Mia40 and Erv1/ALR impact cellular and organism physiology and whether they have functions beyond their role in disulfide bond formation. Here we summarize the principles of oxidation-dependent protein import mediated by the mitochondrial disulfide relay. We proceed by discussing recently described functions of Mia40 in the hypoxia response and of ALR in influencing mitochondrial morphology and its importance for tissue development and embryogenesis. We also include a discussion of the still mysterious function of Erv1/ALR in liver regeneration.International Journal of Cell Biology 11/2013; 2013:742923. DOI:10.1155/2013/742923
- [Show abstract] [Hide abstract]
ABSTRACT: The tumorigenicity of human pluripotent stem cells (hPSCs) is widely acknowledged as a major obstacle that withholds their application in regenerative medicine. This protocol describes two efficient and robust ways to chemically eliminate the tumor-initiating hPSCs from monolayer culture. The protocol details how to maintain and differentiate hPSCs, how to apply chemical inhibitors to cultures of hPSCs and their differentiated progeny, and how to assess the purity of the resultant cell cultures using in vitro and in vivo assays. It also describes how to rescue the cytotoxic effect. The elimination and the rescue assay can be completed within 3-5 d, the in vitro assessment requires another day, and the in vivo assessment requires up to 12 additional weeks.Nature Protocol 03/2014; 9(3):729-740. DOI:10.1038/nprot.2014.050 · 8.36 Impact Factor