Mitochondrial DNA Metabolism Research Articles

Modeling and simulating human mitochondrial RNA polymerase nucleotide addition cycle using computational microscope.

Human mitochondrial RNA polymerase (POLRMT) is essential to the transcription of mitochondrial DNA and cell metabolism. This work contributes to an all-atom structural dynamics model for individual steps of the nucleotide addition cycle of POLRMT. We aim to establish a computational model using all-atom molecular dynamics to reveal Watson-Crick base pair regulation of natural nucleotide and nucleotide analog binding and insertion in POLRMT, and a model for the post-catalytic mechanochemical coupling between product release and POLRMT translocation. The models are currently constructed combining the available high-resolution structures of POLRMT in the open and closed conformations with the bound NTP and Mg2+ ions detected in structurally similar T7 bacteriophage RNA polymerase, and by additionally using curve interpolation to place a missing non-template DNA segment. The models have been evaluated for protein subdomain stability and the presence of the appropriate protein conformation, open or closed, via subdomain motion regulation on the active site. Ongoing work will establish a model for the post-translocation mechanochemical regulations that reset for a new nucleotide addition cycle. Since POLRMT is structurally similar to viral RNA polymerases, the studies are particularly important for antiviral drug design, where nucleotide analog drug candidates are expected to be well screened for POLRMT toxicity.

Mitochondrial DNA copy number changes, heteroplasmy, and mutations in plasma-derived exosomes and brain tissue of glioblastoma patients

Glioblastoma is the most common malignant tumor of the central nervous system (CNS) in adults. Glioblastoma cells show increased glucose consumption associated with poor prognosis. Since mitochondria play a crucial role in energy metabolism, mutations and copy number changes of mitochondrial DNA may serve as biomarkers. As the brain is difficult to access, analysis of mitochondria directly from the brain tissue represents a challenge. Exosome analysis is an alternative (still poorly explored) approach to investigate molecular changes in CNS tumors.We analyzed brain tissue DNA and plasma-derived exosomal DNA (exoDNA) of 44 glioblastoma patients and 40 control individuals. Quantitative real-time PCR was performed to determine mtDNA copy numbers and the Kruskal-Wallis and Mann-Whitney U test were used for statistical analysis of data. Subsequently, sequencing libraries were prepared and sequenced on the MiSeq platform to identify mtDNA point mutations.Tissue mtDNA copy number was different among controls and patients in multiple comparisons. A similar tendency was detected in exosomes. Based on NGS analysis, several mtDNA point mutations showed slightly different frequencies between cases and controls, but the clinical relevance of these observations is difficult to assess and likely less than that of overall mtDNA copy number changes. Allele frequencies of variants were used to determine the level of heteroplasmy (found to be higher in exo-mtDNA of control individuals).Despite the suggested potential, the use of such biomarkers for the screening and/or diagnosis of glioblastomas is still limited, thus further studies are needed.

β-Nicotinamide Mononucleotide Supplementation Increases the Nucleotide Pool Through Multiple Pathways, Improving Mitochondrial DNA Metabolism

β-Nicotinamide Mononucleotide Supplementation Increases the Nucleotide Pool Through Multiple Pathways, Improving Mitochondrial DNA Metabolism

Selective Suppression of Endogenous Gene Expression Using RNAi in Drosophila Schneider S2 Cells.

RNA interference (RNAi) is a posttranscriptional gene silencing method that is triggered by double-stranded RNA (dsRNA). RNAi is used to inactivate genes of interest and provides a genetic tool for loss-of-function studies in a variety of organisms.I have used this method to reveal the physiological roles of a number of endogenous proteins involved in mitochondrial DNA metabolism in Schneider cells, including the mitochondrial single-stranded DNA-binding protein. Here, I present experimental schemes of selective suppression of endogenous gene expression using RNAi in Drosophila Schneider S2 cells. With this method, the function of exogenous wild-type or mutant genes can be evaluated.

Mitochondrial Lon-induced mtDNA leakage contributes to PD-L1–mediated immunoescape via STING-IFN signaling and extracellular vesicles

BackgroundMitochondrial Lon is a chaperone and DNA-binding protein that functions in protein quality control and stress response pathways. The level of Lon regulates mitochondrial DNA (mtDNA) metabolism and the production...

The roles of fission yeast exonuclease 5 in nuclear and mitochondrial genome stability

The Exo5 family consists of bi-directional, single-stranded DNA-specific exonucleases that contain an iron-sulfur cluster as a structural motif and have multiple roles in DNA metabolism. S. cerevisiae Exo5 is essential for mitochondrial genome maintenance, while the human ortholog is important for nuclear genome stability and DNA repair. Here, we identify the Exo5 ortholog in Schizosaccharomyes pombe (spExo5). The activity of spExo5 is highly similar to that of the human enzyme. When the single-stranded DNA is coated with single-stranded DNA binding protein RPA, spExo5 become a 5′-specific exonuclease. Exo5Δ mutants are sensitive to various DNA damaging agents, particularly interstrand crosslinking agents. An epistasis analysis places exo5+ in the Fanconi pathway for interstrand crosslink repair. Exo5+ is in a redundant pathway with rad2+, which encodes the flap endonuclease FEN1, for mitochondrial genome maintenance. Deletion of both genes lead to severe depletion of the mitochondrial genome, and defects in respiration, indicating that either spExo5 or spFEN1 is necessary for mitochondrial DNA metabolism.

Mitochondrial Transcription Factor A Regulates Mycobacterium bovis-Induced IFN-β Production by Modulating Mitochondrial DNA Replication in Macrophages.

Mycobacterium bovis persistently survives in macrophages by developing multiple strategies to evade host immune responses, and the early induction of interferon-β (IFN-β) is one of these critical strategies. The mitochondrial transcription factor A (TFAM) plays a vital role in mitochondrial DNA (mtDNA) metabolism and has been suggested to influence IFN-β production in response to viral infection. However, its role in the production of IFN-β by M. bovis has not been elucidated. In the current study, we investigated the role of TFAM in the production of IFN-β in M. bovis-infected macrophages. We found that knockdown of TFAM expression significantly reduced M. bovis-induced IFN-β production, mtDNA copy numbers and cytosolic mtDNA were increased in murine macrophages with M. bovis infection, cytosolic mtDNA contributed to IFN-β production, and TFAM was required for the increase in mtDNA copy numbers induced by M. bovis. We also observed that TFAM affected the intracellular survival of M. bovis. Our results suggest that TFAM plays an essential role in M. bovis-induced IFN-β production by regulating mtDNA copy numbers. This might be a new strategy adopted by M. bovis for its intracellular survival.

Expression of selected mitochondrial genes during in vitro maturation of bovine oocytes related to their meiotic competence

The main goal of this study was to characterize the expression patterns of genes which play a role in mitochondrial DNA biogenesis and metabolism during the maturation of bovine oocytes with different meiotic competence and health. Meiotically more and less competent oocytes were obtained separately either from medium (MF) or small (SF) follicles and categorized according to oocyte morphology into healthy and light-atretic. The four oocyte categories were matured and collected after 0, 3, 7, 16 and 24 h of maturation. Either total RNA or poly(A) RNA were extracted from oocytes and the expression of selected mitochondrial translational factors (TFAM, TFB1M, and TFB2M), MATER, and Luciferase as external standard was assessed using a real-time RT-PCR. The level of TFAM, TFB1M and MATER poly(A) RNA transcripts significantly decreased during maturation in both healthy and light-atretic MF and SF oocytes. On the other hand, the level of TFB2M poly(A) increased during maturation in healthy and light-atretic SF oocytes, in contrast to MF oocytes. The abundance of TFAM total RNA was significantly higher after maturation than that before maturation in all oocyte categories. However, no differences in TFB1M and TFB2M total RNA were found in any oocyte categories. It can be concluded that the gene expression patterns differ in maturing bovine oocytes in dependence on their meiotic competence and health. The TFAM and TFB1M poly(A) RNAs are actively deadenylated at different meiotic stages but TFB2M poly(A) RNA remains elevated in light-atretic less competent oocytes until the completion of meiosis.

Anticancer Cyclometalated Iridium(III) Complexes with Planar Ligands: Mitochondrial DNA Damage and Metabolism Disturbance.

Emerging studies have shown that mitochondrial DNA (mtDNA) is a potential target for cancer therapy. Herein, six cyclometalated Ir(III) complexes Ir1-Ir6 containing a series of extended planar diimine ligands have been designed and assessed for their efficacy as anticancer agents. Ir1-Ir6 show much higher cytotoxicity than cisplatin and they can effectively localize to mitochondria. Among them, complexes Ir3 and Ir4 with dipyrido[3,2- a:2',3'- c]phenazine (dppz) ligands can bind to DNA tightly in vitro, intercalate to mtDNA in situ, and induce mtDNA damage. Ir3- and Ir4-impaired mitochondria exhibit decline ofmitochondrial membrane potential, disability of adenosine triphosphate generation, disruption of mitochondrial energetic and metabolic status, which subsequently cause protective mitophagy, G0/G1 phase cell cycle arrest, and apoptosis. In vivo antitumor evaluations also show that Ir4 can inhibit tumor xenograft growth effectively. Overall, our work proves that targeting the mitochondrial genome may present an effective strategy to develop metal-based anticancer agents to overcome cisplatin resistance.

Homocysteine impairs porcine oocyte quality via deregulation of one-carbon metabolism and hypermethylation of mitochondrial DNA†.

Homocysteine (Hcy) is an intermediate in the one-carbon metabolism that donates methyl groups for methylation processes involved in epigenetic gene regulation. Although poor oocyte quality in polycystic ovarian syndrome (PCOS) patients is associated with elevated Hcy concentration in serum and follicular fluid, whether Hcy directly affects oocyte quality and its mechanisms are poorly understood. Here we show that Hcy treatment impaired oocyte quality and developmental competence, indicated by significantly reduced survival rate, polar body extrusion rate, and cleavage rate. Hcy treatment resulted in mitochondrial dysfunction, with increased production of mitochondrial ROS, reduced mtDNA copy number, and the expression of 7 out of 13 mtDNA-encoded genes and 2 ribosome RNA genes, 12S rRNA and 16S rRNA. Upon Hcy treatment, the expression of one-carbon metabolic enzymes and DNMT1 was enhanced. Interestingly, DNA methyltransferase inhibitor 5'AZA rescued Hcy-induced mitochondrial dysfunction, impaired oocyte quality and developmental competence. Concurrently, expression of one-carbon metabolic enzymes and methylation status of mtDNA coding sequences were also normalized, at least partially, by 5'AZA treatment. Our findings not only extend the understanding about how Hcy induces poor oocyte quality, but also contribute to a novel angle of identifying targets for enhancing the quality of oocyte from PCOS patients.

Clonal expansion of mitochondrial DNA deletions is a private mechanism of aging in long-lived animals.

Disruption of mitochondrial metabolism and loss of mitochondrial DNA (mtDNA) integrity are widely considered as evolutionarily conserved (public) mechanisms of aging (López‐Otín et al., Cell, 153, 2013 and 1194). Human aging is associated with loss in skeletal muscle mass and function (Sarcopenia), contributing significantly to morbidity and mortality. Muscle aging is associated with loss of mtDNA integrity. In humans, clonally expanded mtDNA deletions colocalize with sites of fiber breakage and atrophy in skeletal muscle. mtDNA deletions may therefore play an important, possibly causal role in sarcopenia. The nematode Caenorhabditis elegans also exhibits age‐dependent decline in mitochondrial function and a form of sarcopenia. However, it is unclear if mtDNA deletions play a role in C. elegans aging. Here, we report identification of 266 novel mtDNA deletions in aging nematodes. Analysis of the mtDNA mutation spectrum and quantification of mutation burden indicates that (a) mtDNA deletions in nematode are extremely rare, (b) there is no significant age‐dependent increase in mtDNA deletions, and (c) there is little evidence for clonal expansion driving mtDNA deletion dynamics. Thus, mtDNA deletions are unlikely to drive the age‐dependent functional decline commonly observed in C. elegans. Computational modeling of mtDNA dynamics in C. elegans indicates that the lifespan of short‐lived animals such as C. elegans is likely too short to allow for significant clonal expansion of mtDNA deletions. Together, these findings suggest that clonal expansion of mtDNA deletions is likely a private mechanism of aging predominantly relevant in long‐lived animals such as humans and rhesus monkey and possibly in rodents.

Endonuclease G promotes mitochondrial genome cleavage and replication.

Endonuclease G (EndoG) is a nuclear-encoded endonuclease, mostly localised in mitochondria. In the nucleus EndoG participates in site-specific cleavage during replication stress and genome-wide DNA degradation during apoptosis. However, the impact of EndoG on mitochondrial DNA (mtDNA) metabolism is poorly understood. Here, we investigated whether EndoG is involved in the regulation of mtDNA replication and removal of aberrant copies. We applied the single-cell mitochondrial Transcription and Replication Imaging Protocol (mTRIP) and PCR-based strategies on human cells after knockdown/knockout and re-expression of EndoG. Our analysis revealed that EndoG stimulates both mtDNA replication initiation and mtDNA depletion, the two events being interlinked and dependent on EndoG's nuclease activity. Stimulation of mtDNA replication by EndoG was independent of 7S DNA processing at the replication origin. Importantly, both mtDNA-directed activities of EndoG were promoted by oxidative stress. Inhibition of base excision repair (BER) that repairs oxidative stress-induced DNA damage unveiled a pronounced effect of EndoG on mtDNA removal, reminiscent of recently discovered links between EndoG and BER in the nucleus. Altogether with the downstream effects on mitochondrial transcription, protein expression, redox status and morphology, this study demonstrates that removal of damaged mtDNA by EndoG and compensatory replication play a critical role in mitochondria homeostasis.

Structural insights into mitochondrial EndoG in response to oxidative stress

Endonuclease G (EndoG) is an evolutionarily conserved mitochondrial protein that degrades chromosomal DNA during apoptosis. In mitochondria, EndoG also processes DNA and participates in mitochondrial DNA metabolism. EndoG‐deficient mice have high levels of mitochondria reactive oxygen species (ROS) and develop heart and mental diseases, showing that the EndoG defect is linked to mitochondrial dysfunction and oxidative stress‐linked diseases. However, it remained unclear how EndoG degrades DNA as a homodimer and why EndoG's function is linked to ROS levels in mitochondria. We reported the crystal structures of the C. elegans EndoG homologue, CPS‐6, in complex with single‐stranded DNA at a resolution of 2.3 Å. Two separate DNA strands are bound at the bba‐metal motifs in the homodimer with their nucleobases pointing away from the enzyme, explaining why CPS‐6 degrades DNA without sequence specificity. We also showed that under oxidative stress, homodimeric EndoG becomes oxidized and converts to monomers with a diminished endonuclease activity. High ROS levels are thus linked to mitochondrial dysfunction by repressing EndoG's activity. Modulation of EndoG dimer conformation and endonuclease activity could present an avenue for prevention and treatment of the diseases resulted from oxidative stresses.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Molecular signature pathway of gene protein interaction in human mitochondrial DNA (mtDNA) metabolism linked disease

Abstract Mitochondrial DNA (mtDNA) contains approximately 37 numbers genes which encoded functional proteins that are essential for cellular energy production and regulation of biochemical process within human. Therefore, any kind of mtDNA abnormalities can initiate an energy calamity, ensuing in crucial mitochondrial diseases. It also depends on nuclear genes because its protein products properly help in mtDNA replication and maintenances of dNTP pools within cytosol and mitochondrial matrix part. Mitochondrial DNA dNTP synthesis comes from two broad categories: primarily mitochondrial salvage and cytosolic de novo pathways with the direct help towards some kind of factors and a variety of enzymes. However, defective mtDNA maintenance can lead to severe disease in humans which may have poor curative therapeutic accessible approaches. Existing research supports that a vast number of drugs and inhibitor components have played significant roles in regulating the mtDNA as well as dNTP pool alteration. The review will primarily focus on the protein regulatory pathways of mitochondrial deoxyribonucleotide (mtDNA) metabolism i.e. mtDNA synthesis, maintenances and its degradation with the connection of several mtDNA metabolism linked diseases.

19 - Importing of peroxiredoxins to distinct mitochondrial compartments: possible impacts on physiology and pathology

Peroxiredoxin (Prx) enzymes are considered major systems for reduction of peroxides in mitochondria. Although the import of Prx from cytosolic ribosomes to distinct mitochondrial subcompartments is driven by a cleavable N-terminal presequence, the mechanisms underlying this process are still elusive. Here, we show by cell fractionation/western blot analysis that yeast mitochondrial Prx (Prx1) displays dual localization: matrix and intermembrane space. Prx1 sorting into the intermembrane space likely involves the release of the precursor protein within the lipid bilayer of the inner membrane, followed by cleavage by the inner membrane peptidase complex (IMP). Alternatively, during its translocation into the matrix, Prx1 is sequentially cleaved by mitochondrial processing peptidase (MPP) and then by octapeptidyl aminopeptidase 1 (Oct1). Oct1 removed eight amino acid residues from the N-terminal region of Prx1, but its physiological role is still elusive. Remarkably, the processing of Prx proteins by Oct1 appears to be evolutionarily conserved, since yeast Oct1 could also cleave the human mitochondrial peroxiredoxin Prx3 when this thiol peroxidase was expressed in yeast. In addition, the alignment of the N-terminal amino acid sequences of Prx3 from mammals indicated the putative cleavage sites by MPP and Oct1 proteases. Remarkably, there are orthologues of IMP and Oct1 proteins in human cells and their malfunctioning was associated with diseases. For instance, loss of Oct1 provokes disturbances in iron metabolism and loss of mitochondrial DNA. Currently, we are investigating the import of mammalian Prx enzymes into distinct mitochondrial subcompartments and the involvement of IMP and Oct1orthologues.

ATAD3 gene cluster deletions cause cerebellar dysfunction associated with altered mitochondrial DNA and cholesterol metabolism.

Although mitochondrial disorders are clinically heterogeneous, they frequently involve the central nervous system and are among the most common neurogenetic disorders. Identifying the causal genes has benefited enormously from advances in high-throughput sequencing technologies; however, once the defect is known, researchers face the challenge of deciphering the underlying disease mechanism. Here we characterize large biallelic deletions in the region encoding the ATAD3C, ATAD3B and ATAD3A genes. Although high homology complicates genomic analysis of the ATAD3 defects, they can be identified by targeted analysis of standard single nucleotide polymorphism array and whole exome sequencing data. We report deletions that generate chimeric ATAD3B/ATAD3A fusion genes in individuals from four unrelated families with fatal congenital pontocerebellar hypoplasia, whereas a case with genomic rearrangements affecting the ATAD3C/ATAD3B genes on one allele and ATAD3B/ATAD3A genes on the other displays later-onset encephalopathy with cerebellar atrophy, ataxia and dystonia. Fibroblasts from affected individuals display mitochondrial DNA abnormalities, associated with multiple indicators of altered cholesterol metabolism. Moreover, drug-induced perturbations of cholesterol homeostasis cause mitochondrial DNA disorganization in control cells, while mitochondrial DNA aggregation in the genetic cholesterol trafficking disorder Niemann-Pick type C disease further corroborates the interdependence of mitochondrial DNA organization and cholesterol. These data demonstrate the integration of mitochondria in cellular cholesterol homeostasis, in which ATAD3 plays a critical role. The dual problem of perturbed cholesterol metabolism and mitochondrial dysfunction could be widespread in neurological and neurodegenerative diseases.

Explore molecular mechanism of Chinese herbs with promoting blood circulation and resolving phlegm effects on myocardial ischemia reperfusion injury based on correlation between microRNA and mtDNA

A large number of basic and clinical studies have shown that the Chinese herbs with promoting blood circulation and resolving phlegm effects could prevent and treat myocardial ischemia-reperfusion injury(MIRI) by regulating lipid metabolism. But its mechanism is not yet clear. The studies show that mitochondrial DNA (mtDNA), microRNAs and lipid metabolism participate in the whole process of MIRI and affect the prognosis. mtDNA mutation is the primary factor to cause myocardial ischemia and reperfusion myocardial cell damage. microRNAs aggravate or reduce MIRI injury by down-regulating or up-regulating related genes expression, while miR-33, as a key regulator of cholesterol transport, regulates lipid metabolism through CROT, PGC-1α, AMPK and other genes located in the mitochondria. There are less studies on correlation between miR-33 and mtDNA, microRNAs. Therefore, further studies on the correlation between miR-33 and mtDNA, microRNAs, as well as the discussions on whether the traditional Chinese medicine (TCM) with promoting blood circulation and resolving phlegm effects could target miR-33 to regulate lipid metabolism and inducemt DNA mutations or deletions, would have important significance for the prevention and treatment of MIRI.

Inhibiting Mitochondrial DNA Ligase IIIα Activates Caspase 1-Dependent Apoptosis in Cancer Cells.

Elevated levels of DNA ligase IIIα (LigIIIα) have been identified as a biomarker of an alteration in DNA repair in cancer cells that confers hypersensitivity to a LigIIIα inhibitor, L67, in combination with a poly (ADP-ribose) polymerase inhibitor. Because LigIIIα functions in the nucleus and mitochondria, we examined the effect of L67 on these organelles. Here, we show that, although the DNA ligase inhibitor selectively targets mitochondria, cancer and nonmalignant cells respond differently to disruption of mitochondrial DNA metabolism. Inhibition of mitochondrial LigIIIα in cancer cells resulted in abnormal mitochondrial morphology, reduced levels of mitochondrial DNA, and increased levels of mitochondrially generated reactive oxygen species that caused nuclear DNA damage. In contrast, these effects did not occur in nonmalignant cells. Furthermore, inhibition of mitochondrial LigIIIα activated a caspase 1-dependent apoptotic pathway, which is known to be part of inflammatory responses induced by pathogenic microorganisms in cancer, but not nonmalignant cells. These results demonstrate that the disruption of mitochondrial DNA metabolism elicits different responses in nonmalignant and cancer cells and suggests that the abnormal response in cancer cells may be exploited in the development of novel therapeutic strategies that selectively target cancer cells. Cancer Res; 76(18); 5431-41. ©2016 AACR.

Human DNA ligase III bridges two DNA ends to promote specific intermolecular DNA end joining.

Mammalian DNA ligase III (LigIII) functions in both nuclear and mitochondrial DNA metabolism. In the nucleus, LigIII has functional redundancy with DNA ligase I whereas LigIII is the only mitochondrial DNA ligase and is essential for the survival of cells dependent upon oxidative respiration. The unique LigIII zinc finger (ZnF) domain is not required for catalytic activity but senses DNA strand breaks and stimulates intermolecular ligation of two DNAs by an unknown mechanism. Consistent with this activity, LigIII acts in an alternative pathway of DNA double strand break repair that buttresses canonical non-homologous end joining (NHEJ) and is manifest in NHEJ-defective cancer cells, but how LigIII acts in joining intermolecular DNA ends versus nick ligation is unclear. To investigate how LigIII efficiently joins two DNAs, we developed a real-time, fluorescence-based assay of DNA bridging suitable for high-throughput screening. On a nicked duplex DNA substrate, the results reveal binding competition between the ZnF and the oligonucleotide/oligosaccharide-binding domain, one of three domains constituting the LigIII catalytic core. In contrast, these domains collaborate and are essential for formation of a DNA-bridging intermediate by adenylated LigIII that positions a pair of blunt-ended duplex DNAs for efficient and specific intermolecular ligation.

Mitochondrial dysfunction: a neglected component of skin diseases.

Aberrant mitochondrial structure and function influence tissue homeostasis and thereby contribute to multiple human disorders and ageing. Ten per cent of patients with primary mitochondrial disorders present skin manifestations that can be categorized into hair abnormalities, rashes, pigmentation abnormalities and acrocyanosis. Less attention has been paid to the fact that several disorders of the skin are linked to alterations of mitochondrial energy metabolism. This review article summarizes the contribution of mitochondrial pathology to both common and rare skin diseases. We explore the intriguing observation that a wide array of skin disorders presents with primary or secondary mitochondrial pathology and that a variety of molecular defects can cause dysfunctional mitochondria. Among them are mutations in mitochondrial- and nuclear DNA-encoded subunits and assembly factors of oxidative phosphorylation (OXPHOS) complexes; mutations in intermediate filament proteins involved in linking, moving and shaping of mitochondria; and disorders of mitochondrial DNA metabolism, fatty acid metabolism and heme synthesis. Thus, we assume that mitochondrial involvement is the rule rather than the exception in skin diseases. We conclude the article by discussing how improving mitochondrial function can be beneficial for aged skin and can be used as an adjunct therapy for certain skin disorders. Consideration of mitochondrial energy metabolism in the skin creates a new perspective for both dermatologists and experts in metabolic disease.