bims-cytox1 Biomed News
on Cytochrome oxidase subunit 1
Issue of 2020‒03‒22
seven papers selected by
Gavin McStay
Staffordshire University


  1. Methods Cell Biol. 2020 ;pii: S0091-679X(19)30137-2. [Epub ahead of print]155 121-156
    Frazier AE, Vincent AE, Turnbull DM, Thorburn DR, Taylor RW.
      Measurement of the individual enzymes involved in mitochondrial oxidative phosphorylation (OXPHOS) forms a key part of diagnostic investigations in patients with suspected mitochondrial disease, and can provide crucial information on mitochondrial OXPHOS function in a variety of cells and tissues that are applicable to many research investigations. In this chapter, we present methods for analysis of mitochondrial respiratory chain enzymes in cells and tissues based on assays performed in two geographically separate diagnostic referral centers, as part of clinical diagnostic investigations. Techniques for sample preparation from cells and tissues, and spectrophotometric assays for measurement of the activities of OXPHOS complexes I-V, the combined activity of complexes II+III, and the mitochondrial matrix enzyme citrate synthase, are provided. The activities of mitochondrial respiratory chain enzymes are often expressed relative to citrate synthase activity, since these ratios may be more robust in accounting for variability that may arise due to tissue quality, handling and storage, cell growth conditions, or any mitochondrial proliferation that may be present in tissues from patients with mitochondrial disease. Considerations for adaption of these techniques to other cells, tissues, and organisms are presented, as well as comparisons to alternate methods for analysis of respiratory chain function. In this context, a quantitative immunofluorescence protocol is also provided that is suitable for measurement of the amount of multiple respiratory chain complexes in small diagnostic tissue samples. The analysis and interpretation of OXPHOS enzyme activities are then placed in the context of mitochondrial disease tissue pathology and diagnosis.
    Keywords:  Citrate synthase; Enzymology; Mitochondria; Mitochondrial disease; Oxidative phosphorylation; Quantitative immunofluorescence; Respiratory chain
    DOI:  https://doi.org/10.1016/bs.mcb.2019.11.007
  2. Methods Cell Biol. 2020 ;pii: S0091-679X(19)30136-0. [Epub ahead of print]155 45-79
    Priesnitz C, Pfanner N, Becker T.
      Mitochondria are deeply integrated into crucial functions of eukaryotic cells, including ATP production via oxidative phosphorylation, biosynthesis of iron-sulfur clusters, amino acids, lipids and heme, signaling pathways, and programmed cell death. The import of about 1000 different proteins that are produced as precursors on cytosolic ribosomes is essential for mitochondrial functions and biogenesis. The translocase of the outer mitochondrial membrane (TOM) forms the entry gate for the vast majority of mitochondrial proteins. Research of the last years has uncovered a complicated network of protein translocases and pathways that sort proteins into the mitochondrial subcompartments: outer and inner membranes, intermembrane space, and matrix. The in vitro import of a large number of different precursor proteins into mitochondria has been a pivotal experimental assay to identify these protein-sorting routes. This experimental set-up enables studies on the kinetics of protein transport into isolated mitochondria, on the processing of precursor proteins, and on their assembly into functional protein machineries. In vitro protein import assays are widely used and are indispensable for research on mitochondrial protein biogenesis.
    Keywords:  Blue native electrophoresis; Mitochondria; Protein assembly; Protein import; Protein sorting; TIM23 complex; TOM complex
    DOI:  https://doi.org/10.1016/bs.mcb.2019.11.006
  3. J Intern Med. 2020 Mar 18.
    Wei W, Chinnery PF.
      The first draft human mitochondrial DNA (mtDNA) sequence was published in 1981, paving the way for two decades of discovery linking mtDNA variation with human disease. Severe pathogenic mutations cause sporadic and inherited rare disorders that often involve the nervous system. However, some mutations cause mild organ-specific phenotypes that have a reduced clinical penetrance, and polymorphic variation of mtDNA is associated with an altered risk of developing several late-onset common human diseases including Parkinson's disease. mtDNA mutations also accumulate during human life and are enriched in affected organs in a number of age-related diseases. Thus, mtDNA contributes to a wide range of human pathologies. For many decades, it has generally been accepted that mtDNA is inherited exclusively down the maternal line in humans. Although recent evidence has challenged this dogma, whole-genome sequencing has identified nuclear-encoded mitochondrial sequences (NUMTs) that can give the false impression of paternally inherited mtDNA. This provides a more likely explanation for recent reports of 'bi-parental inheritance', where the paternal alleles are actually transmitted through the nuclear genome. The presence of both mutated and wild-type variant alleles within the same individual (heteroplasmy) and rapid shifts in allele frequency can lead to offspring with variable severity of disease. In addition, there is emerging evidence that selection can act for and against specific mtDNA variants within the developing germ line, and possibly within developing tissues. Thus, understanding how mtDNA is inherited has far-reaching implications across medicine. There is emerging evidence that this highly dynamic system is amenable to therapeutic manipulation, raising the possibility that we can harness new understanding to prevent and treat rare and common human diseases where mtDNA mutations play a key role.
    Keywords:  human mitochondrial DNA; mitochondrial DNA mutation; mitochondrial bottleneck; mitochondrial disorders; mitochondrial inheritance
    DOI:  https://doi.org/10.1111/joim.13047
  4. Mol Biol Cell. 2020 Mar 18. mbcE19120677
    Matta SK, Kumar A, D' Silva P.
      Mgr2, a newly identified subunit of the TIM23 complex function as a gatekeeper of presequence translocase, thereby maintains quality control of inner membrane preproteins sorting. However, precise recruitment of the Mgr2 subunit to the core channel and how it influences the assembly of the TIM23 complex during lateral sorting of preproteins are poorly understood. Present findings provide insights into a direct association of Mgr2 with the channel-forming Tim23 subunit. Furthermore, the mutational analysis uncovers that the TM1 region of Mgr2 critically required for association with Tim23 and Tim21. On the other hand, the TM2 region of Mgr2 involved in bridging respiratory complexes to the TIM23 complex via Tim21. Importantly, both the TM regions of Mgr2 are essential for lateral sorting of preprotein into the inner membrane as well as maintaining mitochondrial morphology. Together, our findings provide mechanistic insights into the role of Mgr2 in regulating the dynamicity of the TIM23 complex assembly required for preprotein import and coupling of respiratory pathways.
    DOI:  https://doi.org/10.1091/mbc.E19-12-0677
  5. Methods Cell Biol. 2020 ;pii: S0091-679X(19)30155-4. [Epub ahead of print]155 415-439
    Bacman SR, Nissanka N, Moraes CT.
      The study of the mitochondrial DNA (mtDNA) has been hampered by the lack of methods to genetically manipulate the mitochondrial genome in living animal cells. This limitation has been partially alleviated by the ability to transfer mitochondria (and their mtDNAs) from one cell into another, as long as they are from the same species. This is done by isolating mtDNA-containing cytoplasts and fusing these to cells lacking mtDNA. This transmitochondrial cytoplasmic hybrid (cybrid) technology has helped the field understand the mechanism of several pathogenic mutations. In this chapter, we describe procedures to obtain transmitochondrial cybrids.
    Keywords:  Mitochondrial diseases; Rho zero cells; Transmitochondrial cybrids; mtDNA
    DOI:  https://doi.org/10.1016/bs.mcb.2019.11.025
  6. Nucleic Acids Res. 2020 Mar 17. pii: gkaa148. [Epub ahead of print]
    Summer S, Smirnova A, Gabriele A, Toth U, Fasemore AM, Förstner KU, Kuhn L, Chicher J, Hammann P, Mitulović G, Entelis N, Tarassov I, Rossmanith W, Smirnov A.
      Ribosome biogenesis requires numerous trans-acting factors, some of which are deeply conserved. In Bacteria, the endoribonuclease YbeY is believed to be involved in 16S rRNA 3'-end processing and its loss was associated with ribosomal abnormalities. In Eukarya, YBEY appears to generally localize to mitochondria (or chloroplasts). Here we show that the deletion of human YBEY results in a severe respiratory deficiency and morphologically abnormal mitochondria as an apparent consequence of impaired mitochondrial translation. Reduced stability of 12S rRNA and the deficiency of several proteins of the small ribosomal subunit in YBEY knockout cells pointed towards a defect in mitochondrial ribosome biogenesis. The specific interaction of mitoribosomal protein uS11m with YBEY suggests that the latter helps to properly incorporate uS11m into the nascent small subunit in its late assembly stage. This scenario shows similarities with final stages of cytosolic ribosome biogenesis, and may represent a late checkpoint before the mitoribosome engages in translation.
    DOI:  https://doi.org/10.1093/nar/gkaa148
  7. J Intern Med. 2020 Mar 16.
    Zekonyte U, Bacman SR, Moraes CT.
      Mutations in the mitochondrial genome are the cause of many debilitating neuromuscular disorders. Currently, there is no cure or treatment for these diseases, and symptom management is the only relief doctors can provide. Although supplements and vitamins are commonly used in treatment, they provide little benefit to the patient and are only palliative. This is why gene therapy is a promising research topic to potentially treat and in theory, even cure diseases caused by mutations in the mitochondrial DNA (mtDNA). Mammalian cells contain approximately a thousand copies of mtDNA, which can lead to a phenomenon called heteroplasmy, where both wild-type and mutant mtDNA molecules co-exist within the cell. Disease only manifests once the percent of mutant mtDNA reaches a high threshold (>80%), which causes mitochondrial dysfunction and reduced ATP production. This is a useful feature to take advantage of for gene therapy applications, as not every mutant copy of mtDNA needs to be eliminated, but only enough to shift the heteroplasmic ratio below the disease threshold. Several DNA editing enzymes have been used to shift heteroplasmy in cell culture and mice. This review provides an overview of these enzymes, and discusses roadblocks of applying these to gene therapy in humans.
    DOI:  https://doi.org/10.1111/joim.13055