bims-midmar 2021-09-26 papers

bims-midmar

Biomed News

on Mitochondrial DNA maintenance and replication

Issue of 2021–09–26
thirteen papers selected by
Flavia Söllner, Ludwig-Maximilians University

Controlling the topology of mammalian mitochondrial DNA.
Ablation of mitochondrial DNA results in widespread remodeling of the mitochondrial complexome.
Physical and Functional Interaction of Mitochondrial Single-Stranded DNA-Binding Protein and the Catalytic Subunit of DNA Polymerase Gamma.
Clinico-Genetic Spectrum of POLG1 Mutation Carriers from India.
Assessment of Mitochondrial Protein Topology and Membrane Insertion.
Measuring the Activity of DNA Repair Enzymes in Isolated Mitochondria.
Mitochondrial tRNA-Derived Fragments and Their Contribution to Gene Expression Regulation.
Rescuing mitochondria in traumatic brain injury and intracerebral hemorrhages - A potential therapeutic approach.
Mass Spectrometry-Based Quantitative Cysteine Redox Proteome Profiling of Isolated Mitochondria Using Differential iodoTMT Labeling.
Quantification of Mitochondrial RNA Editing Efficiency Using Sanger Sequencing Data.
Assessment of Mitochondrial Protein Composition and Purity by Mass Spectroscopy.
The 3D organisation of mitochondria in primate photoreceptors.
MitoTALENs: A Method for Targeted Gene Disruption in Plant Mitochondrial Genomes.

Open Biol. 2021 Sep;11(9): 210168

Controlling the topology of mammalian mitochondrial DNA.

Katja E Menger, Alejandro Rodríguez-Luis, James Chapman, Thomas J Nicholls.

  The genome of mitochondria, called mtDNA, is a small circular DNA molecule present at thousands of copies per human cell. MtDNA is packaged into nucleoprotein complexes called nucleoids, and the density of mtDNA packaging affects mitochondrial gene expression. Genetic processes such as transcription, DNA replication and DNA packaging alter DNA topology, and these topological problems are solved by a family of enzymes called topoisomerases. Within mitochondria, topoisomerases are involved firstly in the regulation of mtDNA supercoiling and secondly in disentangling interlinked mtDNA molecules following mtDNA replication. The loss of mitochondrial topoisomerase activity leads to defects in mitochondrial function, and variants in the dual-localized type IA topoisomerase TOP3A have also been reported to cause human mitochondrial disease. We review the current knowledge on processes that alter mtDNA topology, how mtDNA topology is modulated by the action of topoisomerases, and the consequences of altered mtDNA topology for mitochondrial function and human health.

Keywords:  DNA topology; mitochondria; mitochondrial DNA; mitochondrial disease; topoisomerases

DOI:  https://doi.org/10.1098/rsob.210168
EMBO J. 2021 Sep 20. e108648

Ablation of mitochondrial DNA results in widespread remodeling of the mitochondrial complexome.

Sergio Guerrero-Castillo, Joeri van Strien, Ulrich Brandt, Susanne Arnold.

  So-called ρ0 cells lack mitochondrial DNA and are therefore incapable of aerobic ATP synthesis. How cells adapt to survive ablation of oxidative phosphorylation remains poorly understood. Complexome profiling analysis of ρ0 cells covered 1,002 mitochondrial proteins and revealed changes in abundance and organization of numerous multiprotein complexes including previously not described assemblies. Beyond multiple subassemblies of complexes that would normally contain components encoded by mitochondrial DNA, we observed widespread reorganization of the complexome. This included distinct changes in the expression pattern of adenine nucleotide carrier isoforms, other mitochondrial transporters, and components of the protein import machinery. Remarkably, ablation of mitochondrial DNA hardly affected the complexes organizing cristae junctions indicating that the altered cristae morphology in ρ0 mitochondria predominantly resulted from the loss of complex V dimers required to impose narrow curvatures to the inner membrane. Our data provide a comprehensive resource for in-depth analysis of remodeling of the mitochondrial complexome in response to respiratory deficiency.

Keywords:  OXPHOS; complexome profiling; mitochondria; mtDNA; rho0 cells

DOI:  https://doi.org/10.15252/embj.2021108648
Front Genet. 2021 ;12 721864

Physical and Functional Interaction of Mitochondrial Single-Stranded DNA-Binding Protein and the Catalytic Subunit of DNA Polymerase Gamma.

Grzegorz L Ciesielski, Shalom Kim, Carolina de Bovi Pontes, Laurie S Kaguni.

  The maintenance of the mitochondrial genome depends on a suite of nucleus-encoded proteins, among which the catalytic subunit of the mitochondrial replicative DNA polymerase, Pol γα, plays a pivotal role. Mutations in the Pol γα-encoding gene, POLG, are a major cause of human mitochondrial disorders. Here we present a study of direct and functional interactions of Pol γα with the mitochondrial single-stranded DNA-binding protein (mtSSB). mtSSB coordinates the activity of the enzymes at the DNA replication fork. However, the mechanism of this functional relationship is elusive, and no direct interactions between the replicative factors have been identified to date. This contrasts strikingly with the extensive interactomes of SSB proteins identified in other homologous replication systems. Here we show for the first time that mtSSB binds Pol γα directly, in a DNA-independent manner. This interaction is strengthened in the absence of the loop 2.3 structure in mtSSB, and is abolished upon preincubation with Pol γβ. Together, our findings suggest that the interaction between mtSSB and polymerase gamma holoenzyme (Pol γ) involves a balance between attractive and repulsive affinities, which have distinct effects on DNA synthesis and exonucleolysis.

Keywords:  DNA polymerase gamma; intermolecular interactions; mitochondrial DNA replication; mitochondrial biogenesis; mitochondrial single-stranded DNA-binding protein

DOI:  https://doi.org/10.3389/fgene.2021.721864
J Mol Neurosci. 2021 Sep 24.

Clinico-Genetic Spectrum of POLG1 Mutation Carriers from India.

Josef Finsterer.



Keywords:  Heterogeneity; Mitochondrial disorder; POLG1; Phenotype; mtDNA

DOI:  https://doi.org/10.1007/s12031-021-01905-8
Methods Mol Biol. 2022 ;2363 165-181

Assessment of Mitochondrial Protein Topology and Membrane Insertion.

Kerstin Schäfer, Carina Engstler, Korbinian Dischinger, Chris Carrie.

  Analyzing the membrane integrity and topology of a mitochondrial protein is essential for truly understanding its function. In this chapter, we demonstrate how to analyze mitochondrial membrane proteins using both an immunological-based assay and an in vivo self-assembling GFP approach. First, immunological approaches to investigate the solubility or membrane association of a protein within mitochondria are described. With this method, we demonstrate how the topology of soluble domains of a membrane-integrated protein can be determined. Using protein-specific antibodies, the localization of the soluble domains of a protein are analyzed by a proteolytic-cleavage approach using proteinase K in mitochondria, outer membrane-ruptured mitochondria, and solubilized mitochondrial membranes. In a second approach, we determine the topology of plant mitochondrial proteins using an in vivo self-assembling GFP localization approach.

Keywords:  Carbonate extraction; In vivo GFP localization; Membrane integration; Membrane protein topology; Membrane solubilization; Mitochondrial membrane proteins; Mitoplasts; Osmotic swelling; Proteinase K digestion; Self-assembling GFP

DOI:  https://doi.org/10.1007/978-1-0716-1653-6_13
Methods Mol Biol. 2022 ;2363 321-334

Measuring the Activity of DNA Repair Enzymes in Isolated Mitochondria.

Beatriz Ferrando, Ian Max Møller, Tinna Stevnsner.

  Nuclear, mitochondrial and plastidic DNA is constantly exposed to conditions, such as ultraviolet radiation or reactive oxygen species, which will induce chemical modifications to the nucleotides. Unless repaired, these modifications can lead to mutations, so the nucleus, mitochondria and plastids each contains a number of DNA repair systems. We here describe assays for measuring the enzyme activities associated with the base-excision repair pathway in potato tuber mitochondria. As the name implies, this pathway involves removing a modified base and replacing it with an undamaged base. Activity of each of the enzymes involved, DNA glycosylase, apurinic/apyrimidinic endonuclease, DNA polymerase and DNA ligase can be measured by incubating a mitochondrial extract with a specifically designed oligonucleotide. After incubation, the reaction mixture is separated on a polyacrylamide gel, and the amounts of specific products formed is estimated by autoradiography, which makes it possible to calculate the enzymatic activity.

Keywords:  Base excision repair; DNA repair; Plant mitochondria; mtDNA

DOI:  https://doi.org/10.1007/978-1-0716-1653-6_21
Front Physiol. 2021 ;12 729452

Mitochondrial tRNA-Derived Fragments and Their Contribution to Gene Expression Regulation.

Athanasios-Nasir Shaukat, Eleni G Kaliatsi, Vassiliki Stamatopoulou, Constantinos Stathopoulos.

  Mutations in human mitochondrial tRNAs (mt-tRNAs) are responsible for several and sometimes severe clinical phenotypes, classified among mitochondrial diseases. In addition, post-transcriptional modifications of mt-tRNAs in correlation with several stress signals can affect their stability similarly to what has been described for their nuclear-encoded counterparts. Many of the perturbations related to either point mutations or aberrant modifications of mt-tRNAs can lead to specific cleavage and the production of mitochondrial tRNA-derived fragments (mt-tRFs). Although mt-tRFs have been detected in several studies, the exact biogenesis steps and biological role remain, to a great extent, unexplored. Several mt-tRFs are produced because of the excessive oxidative stress which predominantly affects mitochondrial DNA integrity. In addition, mt-tRFs have been detected in various diseases with possible detrimental consequences, but also their production may represent a response mechanism to external stimuli, including infections from pathogens. Finally, specific point mutations on mt-tRNAs have been reported to impact the pool of the produced mt-tRFs and there is growing evidence suggesting that mt-tRFs can be exported and act in the cytoplasm. In this review, we summarize current knowledge on mitochondrial tRNA-deriving fragments and their possible contribution to gene expression regulation.

Keywords:  mitochondria; mitochondrial tRNA-derived fragments; mitochondrial tRNAs; ncRNAs; tRNA-derived fragments

DOI:  https://doi.org/10.3389/fphys.2021.729452
Neurochem Int. 2021 Sep 21. pii: S0197-0186(21)00238-2. [Epub ahead of print] 105192

Rescuing mitochondria in traumatic brain injury and intracerebral hemorrhages - A potential therapeutic approach.

Meenakshi Ahluwalia, Manish Kumar, Pankaj Ahluwalia, Scott Rahimi, John R Vender, Raghavan P Raju, David C Hess, Babak Baban, Fernando L Vale, Krishnan M Dhandapani, Kumar Vaibhav.

  Mitochondria are dynamic organelles responsible for cellular energy production. Besides, regulating energy homeostasis, mitochondria are responsible for calcium homeostasis, signal transmission, and the fate of cellular survival in case of injury and pathologies. Accumulating reports have suggested multiple roles of mitochondria in neuropathologies, neurodegeneration, and immune activation under physiological and pathological conditions. Mitochondrial dysfunction, which occurs at the initial phase of brain injury, involves oxidative stress, inflammation, deficits in mitochondrial bioenergetics, biogenesis, transport, and autophagy. Thus, development of targeted therapeutics to protect mitochondria may improve functional outcomes following traumatic brain injury (TBI) and intracerebral hemorrhages (ICH). In this review, we summarize mitochondrial dysfunction related to TBI and ICH, including the mechanisms involved, and discuss therapeutic approaches with special emphasis on past and current clinical trials.

Keywords:  Brain injury; Immune activation; Mitochondrial bioenergetics; Mitochondrial biogenesis; Mitophagy; Therapeutic approach

DOI:  https://doi.org/10.1016/j.neuint.2021.105192
Methods Mol Biol. 2022 ;2363 215-234

Mass Spectrometry-Based Quantitative Cysteine Redox Proteome Profiling of Isolated Mitochondria Using Differential iodoTMT Labeling.

Jonas Giese, Jürgen Eirich, Frederik Post, Markus Schwarzländer, Iris Finkemeier.

  Mitochondria are central hubs of redox biochemistry in the cell. An important role of mitochondrial carbon metabolism is to oxidize respiratory substrates and to pass the electrons down the mitochondrial electron transport chain to reduce oxygen and to drive oxidative phosphorylation. During respiration, reactive oxygen species are produced as a side reaction, some of which in turn oxidize cysteine thiols in proteins. Hence, the redox status of cysteine-containing mitochondrial proteins has to be controlled by the mitochondrial glutathione and thioredoxin systems, which draw electrons from metabolically derived NADPH. The redox status of mitochondrial cysteines can undergo fast transitions depending on the metabolic status of the cell, as for instance at early seed germination. Here, we describe a state-of-the-art method to quantify redox state of protein cysteines in isolated Arabidopsis seedling mitochondria of controlled metabolic and respiratory state by MS2-based redox proteomics using the isobaric thiol labeling reagent Iodoacetyl Tandem Mass Tag™ (iodoTMT). The procedure is also applicable to isolated mitochondria of other plant and nonplant systems.

Keywords:  Cysteine; Iodoacetyl Tandem Mass Tag; Isobaric labeling; LC-MS/MS; MaxQuant; Mitochondria; Redox; Thiols

DOI:  https://doi.org/10.1007/978-1-0716-1653-6_16
Methods Mol Biol. 2022 ;2363 263-278

Quantification of Mitochondrial RNA Editing Efficiency Using Sanger Sequencing Data.

Mizuki Takenaka.

  C-to-U RNA editing in mitochondria and plastids is widespread in almost all terrestrial plants, where it mainly changes codons to encode conserved amino acids in organelle mRNAs. In flowering plants, the number of RNA editing sites reaches 400-600 in mitochondria and about 40 in plastids, respectively. To date, more than 100 factors involved in RNA editing have been identified. Since target cytidines of each factor are often distributed across multiple transcripts, comprehensive monitoring of all RNA editing sites is necessary for their characterization. Comparing the signals of C and T in the Sanger sequencing chromatogram of RT-PCR products is the most frequently employed method for quantification of RNA editing efficiency, although several methods based on next-generation sequencing have been developed. I here describe a quick and easy method for quantification of RNA editing efficiency at several hundred sites using the Sanger sequencing chromatogram data.

Keywords:  C-to-U conversion; Mitochondria; RNA editing; RT-PCR; Sanger sequencing

DOI:  https://doi.org/10.1007/978-1-0716-1653-6_18
Methods Mol Biol. 2022 ;2363 121-152

Assessment of Mitochondrial Protein Composition and Purity by Mass Spectroscopy.

Anqiang Tang, Yimin Wang, Nicolas L Taylor.

  While the routine mitochondrial extraction and isolation protocols have not fundamentally changed since the introduction of density gradients, the approaches we use to examine the proteome have. The initial characterisation of mitochondrial proteomes was carried out using two-dimensional gel electrophoresis in 2001 and gel spot mass spectrometry have now largely been superseded as the throughput and sensitivity of commercial mass spectrometers increases. Whist many of these early studies established the components of the mitochondrial proteome, as gels were replaced by gel free approaches the numbers of confirmed components rapidly increased. In this chapter we present gel-based approaches for the separation and concentration of mitochondrial proteins for their characterization by mass spectrometry. We also describe two gel-free approaches which can be used to quantity the degree of contamination arising during the isolation of mitochondria. These approaches are equally suitable for studies comparing one treatment to another.

Keywords:  Gel fractionation; Gel purification; Mass spectrometry; Mitochondria; Proteomics; Selected reaction monitoring (SRM) mass spectrometry

DOI:  https://doi.org/10.1007/978-1-0716-1653-6_11
Sci Rep. 2021 Sep 22. 11(1): 18863

The 3D organisation of mitochondria in primate photoreceptors.

Matthew J Hayes, Dhani Tracey-White, Jaimie Hoh Kam, Michael B Powner, Glen Jeffery.

Vertebrate photoreceptors contain large numbers of closely-packed mitochondria which sustain the high metabolic demands of these cells. These mitochondria populations are dynamic and undergo fusion and fission events. This activity serves to maintain the population in a healthy state. In the event of mitochondrial damage, sub-domains, or indeed whole mitochondria, can be degraded and population homeostasis achieved. If this process is overwhelmed cell death may result. Death of photoreceptors contributes to loss of vision in aging individuals and is associated with many eye diseases. In this study we used serial block face scanning electron microscopy of adult Macaca fascicularis retinae to examine the 3D structure of mitochondria in rod and cone photoreceptors. We show that healthy-looking photoreceptors contain mitochondria exhibiting a range of shapes which are associated with different regions of the cell. In some photoreceptors we observe mitochondrial swelling and other changes often associated with cellular stress. In rods and cones that appear stressed we identify elongated domains of mitochondria with densely-packed normal cristae associated with photoreceptor ciliary rootlet bundles. We observe mitochondrial fission and mitochondrion fragments localised to these domains. Swollen mitochondria with few intact cristae are located towards the periphery of the photoreceptor inner-segment in rods, whilst they are found throughout the cell in cones. Swollen mitochondria exhibit sites on the mitochondrial inner membrane which have undergone complex invagination resulting in membranous, electron-dense aggregates. Membrane contact occurs between the mitochondrion and the photoreceptor plasma membrane in the vicinity of these aggregates, and a series of subsequent membrane fusions results in expulsion of the mitochondrial aggregate from the photoreceptor. These events are primarily associated with rods. The potential fate of this purged material and consequences of its clearance by retinal pigment epithelia are discussed.

DOI: https://doi.org/10.1038/s41598-021-98409-7
Methods Mol Biol. 2022 ;2363 335-340

MitoTALENs: A Method for Targeted Gene Disruption in Plant Mitochondrial Genomes.

Shin-Ichi Arimura.

  Modification of plant mitochondrial genomes is still a difficult task, especially in multicellular plants. Transcription activator-like effector nucleases with a mitochondrial localization signal (mitoTALENs) can cut out a desired sequence from the mitochondrial genome in plants. Although vector construction of mitoTALENs is complicated, the modification efficiency is high enough to achieve homoplasmy of multicopy mitochondrial genomes. Here I describe how to design mitoTALENs to select a target, construct a vector, and select the mitochondrial transformants.

Keywords:  Gene targeting; Genome editing; Organelle genome; Organelle transformation; Plant mitochondrial genome; TALEN

DOI:  https://doi.org/10.1007/978-1-0716-1653-6_22