Showing papers on "Mitochondrial DNA replication published in 2020"

A cnidarian parasite of salmon (Myxozoa: Henneguya) lacks a mitochondrial genome

Abstract: Although aerobic respiration is a hallmark of eukaryotes, a few unicellular lineages, growing in hypoxic environments, have secondarily lost this ability. In the absence of oxygen, the mitochondria of these organisms have lost all or parts of their genomes and evolved into mitochondria-related organelles (MROs). There has been debate regarding the presence of MROs in animals. Using deep sequencing approaches, we discovered that a member of the Cnidaria, the myxozoan Henneguya salminicola, has no mitochondrial genome, and thus has lost the ability to perform aerobic cellular respiration. This indicates that these core eukaryotic features are not ubiquitous among animals. Our analyses suggest that H. salminicola lost not only its mitochondrial genome but also nearly all nuclear genes involved in transcription and replication of the mitochondrial genome. In contrast, we identified many genes that encode proteins involved in other mitochondrial pathways and determined that genes involved in aerobic respiration or mitochondrial DNA replication were either absent or present only as pseudogenes. As a control, we used the same sequencing and annotation methods to show that a closely related myxozoan, Myxobolus squamalis, has a mitochondrial genome. The molecular results are supported by fluorescence micrographs, which show the presence of mitochondrial DNA in M. squamalis, but not in H. salminicola. Our discovery confirms that adaptation to an anaerobic environment is not unique to single-celled eukaryotes, but has also evolved in a multicellular, parasitic animal. Hence, H. salminicola provides an opportunity for understanding the evolutionary transition from an aerobic to an exclusive anaerobic metabolism.

Ultrasensitive deletion detection links mitochondrial DNA replication, disease, and aging

Abstract: Acquired human mitochondrial genome (mtDNA) deletions are symptoms and drivers of focal mitochondrial respiratory deficiency, a pathological hallmark of aging and late-onset mitochondrial disease. To decipher connections between these processes, we create LostArc, an ultrasensitive method for quantifying deletions in circular mtDNA molecules. LostArc reveals 35 million deletions (~ 470,000 unique spans) in skeletal muscle from 22 individuals with and 19 individuals without pathogenic variants in POLG. This nuclear gene encodes the catalytic subunit of replicative mitochondrial DNA polymerase γ. Ablation, the deleted mtDNA fraction, suffices to explain skeletal muscle phenotypes of aging and POLG-derived disease. Unsupervised bioinformatic analyses reveal distinct age- and disease-correlated deletion patterns. These patterns implicate replication by DNA polymerase γ as the deletion driver and suggest little purifying selection against mtDNA deletions by mitophagy in postmitotic muscle fibers. Observed deletion patterns are best modeled as mtDNA deletions initiated by replication fork stalling during strand displacement mtDNA synthesis.

Altered Mitochondrial Dynamics in Motor Neuron Disease: An Emerging Perspective.

Abstract: Mitochondria plays privotal role in diverse pathways that regulate cellular function and survival, and have emerged as a prime focus in aging and age-associated motor neuron diseases (MNDs), such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Accumulating evidence suggests that many amyloidogenic proteins, including MND-associated RNA/DNA-binding proteins fused in sarcoma (FUS) and TAR DNA binding protein (TDP)-43, are strongly linked to mitochondrial dysfunction. Animal model and patient studies have highlighted changes in mitochondrial structure, plasticity, replication/copy number, mitochondrial DNA instability, and altered membrane potential in several subsets of MNDs, and these observations are consistent with the evidence of increased excitotoxicity, induction of reactive oxygen species, and activation of intrinsic apoptotic pathways. Studies in MND rodent models also indicate that mitochondrial abnormalities begin prior to the clinical and pathological onset of the disease, suggesting a causal role of mitochondrial dysfunction. Our recent studies, which demonstrated the involvement of specific defects in DNA break-ligation mediated by DNA ligase 3 (LIG3) in FUS-associated ALS, raised a key question of its potential implication in mitochondrial DNA transactions because LIG3 is essential for both mitochondrial DNA replication and repair. This question, as well as how wild-type and mutant MND-associated factors affect mitochondria, remain to be elucidated. These new investigation avenues into the mechanistic role of mitochondrial dysfunction in MNDs are critical to identify therapeutic targets to alleviate mitochondrial toxicity and its consequences. In this article, we critically review recent advances in our understanding of mitochondrial dysfunction in diverse subgroups of MNDs and discuss challenges and future directions.

Mammalian mitochondrial DNA replication and mechanisms of deletion formation.

Abstract: Mammalian mitochondria contain multiple copies of a circular, double-stranded DNA genome (mtDNA) that codes for subunits of the oxidative phosphorylation machinery. Mutations in mtDNA cause a number of rare, human disorders and are also associated with more common conditions, such as neurodegeneration and biological aging. In this review, we discuss our current understanding of mtDNA replication in mammalian cells and how this process is regulated. We also discuss how deletions can be formed during mtDNA replication.

Mitochondrial genetic variation is enriched in G-quadruplex regions that stall DNA synthesis in vitro.

Abstract: As the powerhouses of the eukaryotic cell, mitochondria must maintain their genomes which encode proteins essential for energy production. Mitochondria are characterized by guanine-rich DNA sequences that spontaneously form unusual three-dimensional structures known as G-quadruplexes (G4). G4 structures can be problematic for the essential processes of DNA replication and transcription because they deter normal progression of the enzymatic-driven processes. In this study, we addressed the hypothesis that mitochondrial G4 is a source of mutagenesis leading to base-pair substitutions. Our computational analysis of 2757 individual genomes from two Italian population cohorts (SardiNIA and InCHIANTI) revealed a statistically significant enrichment of mitochondrial mutations within sequences corresponding to stable G4 DNA structures. Guided by the computational analysis results, we designed biochemical reconstitution experiments and demonstrated that DNA synthesis by two known mitochondrial DNA polymerases (Pol γ, PrimPol) in vitro was strongly blocked by representative stable G4 mitochondrial DNA structures, which could be overcome in a specific manner by the ATP-dependent G4-resolving helicase Pif1. However, error-prone DNA synthesis by PrimPol using the G4 template sequence persisted even in the presence of Pif1. Altogether, our results suggest that genetic variation is enriched in G-quadruplex regions that impede mitochondrial DNA replication.

A-Kinase Anchoring Protein 1: Emerging Roles in Regulating Mitochondrial Form and Function in Health and Disease.

Abstract: Best known as the powerhouse of the cell, mitochondria have many other important functions such as buffering intracellular calcium and reactive oxygen species levels, initiating apoptosis and supporting cell proliferation and survival. Mitochondria are also dynamic organelles that are constantly undergoing fission and fusion to meet specific functional needs. These processes and functions are regulated by intracellular signaling at the mitochondria. A-kinase anchoring protein 1 (AKAP1) is a scaffold protein that recruits protein kinase A (PKA), other signaling proteins, as well as RNA to the outer mitochondrial membrane. Hence, AKAP1 can be considered a mitochondrial signaling hub. In this review, we discuss what is currently known about AKAP1's function in health and diseases. We focus on the recent literature on AKAP1's roles in metabolic homeostasis, cancer and cardiovascular and neurodegenerative diseases. In healthy tissues, AKAP1 has been shown to be important for driving mitochondrial respiration during exercise and for mitochondrial DNA replication and quality control. Several recent in vivo studies using AKAP1 knockout mice have elucidated the role of AKAP1 in supporting cardiovascular, lung and neuronal cell survival in the stressful post-ischemic environment. In addition, we discuss the unique involvement of AKAP1 in cancer tumor growth, metastasis and resistance to chemotherapy. Collectively, the data indicate that AKAP1 promotes cell survival throug regulating mitochondrial form and function. Lastly, we discuss the potential of targeting of AKAP1 for therapy of various disorders.

Mitochondrial Bioenergetics at the Onset of Drug Resistance in Hematological Malignancies: An Overview.

Abstract: The combined derangements in mitochondria network, function and dynamics can affect metabolism and ATP production, redox homeostasis and apoptosis triggering, contributing to cancer development in many different complex ways. In hematological malignancies, there is a strong relationship between cellular metabolism, mitochondrial bioenergetics, interconnections with supportive microenvironment and drug resistance. Lymphoma and chronic lymphocytic leukemia cells, e.g., adapt to intrinsic oxidative stress by increasing mitochondrial biogenesis. In other hematological disorders such as myeloma, on the contrary, bioenergetics changes, associated to increased mitochondrial fitness, derive from the adaptive response to drug-induced stress. In the bone marrow niche, a reverse Warburg effect has been recently described, consisting in metabolic changes occurring in stromal cells in the attempt to metabolically support adjacent cancer cells. Moreover, a physiological dynamic, based on mitochondria transfer, between tumor cells and their supporting stromal microenvironment has been described to sustain oxidative stress associated to proteostasis maintenance in multiple myeloma and leukemia. Increased mitochondrial biogenesis of tumor cells associated to acquisition of new mitochondria transferred by mesenchymal stromal cells results in augmented ATP production through increased oxidative phosphorylation (OX-PHOS), higher drug resistance, and resurgence after treatment. Accordingly, targeting mitochondrial biogenesis, electron transfer, mitochondrial DNA replication, or mitochondrial fatty acid transport increases therapy efficacy. In this review, we summarize selected examples of the mitochondrial derangements in hematological malignancies, which provide metabolic adaptation and apoptosis resistance, also supported by the crosstalk with tumor microenvironment. This field promises a rational design to improve target-therapy including the metabolic phenotype.

Blood-derived mitochondrial DNA copy number is associated with gene expression across multiple tissues and is predictive for incident neurodegenerative disease

Abstract: BackgroundMitochondrial DNA copy number (mtDNA-CN) can be used as a proxy for mitochondrial function and is associated with a number of aging-related diseases. However, it is unclear how mtDNA-CN measured in blood can reflect risk for diseases that primarily manifest in other tissues. Using the Genotype-Tissue Expression Project, we interrogated the relationships between mtDNA-CN measured in whole blood and gene expression from whole blood as well as 47 additional tissues. ResultsWe evaluated associations between blood-derived mtDNA-CN and gene expression in whole blood for 418 individuals, correcting for known confounders and surrogate variables derived from RNA-sequencing. Using a permutation-derived cutoff (p<2.70e-6), mtDNA-CN was significantly associated with expression for 721 genes in whole blood, including nuclear genes that are required for mitochondrial DNA replication. Significantly enriched pathways included splicing (p=1.03e-8) and ubiquitin-mediated proteolysis (p=2.4e-10). Genes with target sequences for the mitochondrial transcription factor NRF1 were also enriched (p=1.76e-35). In non-blood tissues, there were more significantly associated genes than expected in 30 out of 47 tested tissues, suggesting that global gene expression in those tissues is correlated with mtDNA-CN. Pathways that were associated in multiple tissues included RNA-binding, catalysis, and neurodegenerative disease. We evaluated the association between mtDNA-CN and incident neurodegenerative disease in an independent dataset, the UK Biobank, using a Cox proportional-hazards model. Higher mtDNA-CN was significantly associated with lower risk for incident neurodegenerative disease (HR=0.73, 95% CI= 0.66;0.90). ConclusionsThe observation that mtDNA-CN measured in whole blood is associated with gene expression in other tissues suggests that blood-derived mtDNA-CN can reflect metabolic health across multiple tissues. Key pathways in maintaining cellular homeostasis, including splicing, RNA binding, and catalytic genes were significantly associated with mtDNA-CN, reinforcing the importance of mitochondria in aging-related disease. As a specific example, genes involved in neurodegenerative disease were significantly enriched in multiple tissues. This finding, validated in a large independent cohort study showing an inverse association between mtDNA-CN and neurodegenerative disease, solidifies the link between blood-derived mtDNA-CN, altered gene expression in both blood and non-blood tissues, and aging-related disease.

Mitochondrial Inheritance in Phytopathogenic Fungi—Everything Is Known, or Is It?

Abstract: Mitochondria are important organelles in eukaryotes that provide energy for cellular processes. Their function is highly conserved and depends on the expression of nuclear encoded genes and genes encoded in the organellar genome. Mitochondrial DNA replication is independent of the replication control of nuclear DNA and as such, mitochondria may behave as selfish elements, so they need to be controlled, maintained and reliably inherited to progeny. Phytopathogenic fungi meet with special environmental challenges within the plant host that might depend on and influence mitochondrial functions and services. We find that this topic is basically unexplored in the literature, so this review largely depends on work published in other systems. In trying to answer elemental questions on mitochondrial functioning, we aim to introduce the aspect of mitochondrial functions and services to the study of plant-microbe-interactions and stimulate phytopathologists to consider research on this important organelle in their future projects.

Potential causes and consequences of rapid mitochondrial genome evolution in thermoacidophilic Galdieria (Rhodophyta)

Abstract: The Cyanidiophyceae is an early-diverged red algal class that thrives in extreme conditions around acidic hot springs. Although this lineage has been highlighted as a model for understanding the biology of extremophilic eukaryotes, little is known about the molecular evolution of their mitochondrial genomes (mitogenomes). To fill this knowledge gap, we sequenced five mitogenomes from representative clades of Cyanidiophyceae and identified two major groups, here referred to as Galdieria-type (G-type) and Cyanidium-type (C-type). G-type mitogenomes exhibit the following three features: (i) reduction in genome size and gene inventory, (ii) evolution of unique protein properties including charge, hydropathy, stability, amino acid composition, and protein size, and (iii) distinctive GC-content and skewness of nucleotides. Based on GC-skew-associated characteristics, we postulate that unidirectional DNA replication may have resulted in the rapid evolution of G-type mitogenomes. The high divergence of G-type mitogenomes was likely driven by natural selection in the multiple extreme environments that Galdieria species inhabit combined with their highly flexible heterotrophic metabolism. We speculate that the interplay between mitogenome divergence and adaptation may help explain the dominance of Galdieria species in diverse extreme habitats.

Microbiome in Healthy Women Between Two Districts With Different Air Quality Index.

Abstract: Although the diversity and abundance of skin microbiome are mainly determined by intrinsic factors, including gender, age, anatomical site, and ethnicity, we question whether facial microbiome could be affected by long-term exposure to airborne pollution. Using 16S ribosomal RNA (rRNA) gene amplicon sequencing, we analyzed the facial bacterial microbiome of healthy and young Chinese women (25-35 years old) between two districts with different air quality indices (AQIs) in Zhejiang Province. The overall microbiome structure was obviously different between these two districts. It revealed an increase in both the abundance and diversity of facial bacterial microbiome in Hangzhou (HZ) with higher AQI compared with those in Yunhe (YH) with lower AQI. Linear discriminant analysis (LDA) and Lefse analysis identified a total of 45 genera showing significant overrepresentation in the HZ group. Furthermore, PICRUSt analysis showed that functional pathways associated with metabolism of saturated fatty acid were relatively more predominant in the HZ group, whereas those with DNA repair or mitochondrial DNA replication were more predominant in the YH group. Our present data can provide useful information for further researches on the composition and function of the skin microbiome related to air pollution factors as well as for the development of therapeutic agents targeting the microbes and their metabolites to resist damages of airborne pollutants.

Novel POLG mutation in a patient with early-onset parkinsonism, progressive external ophthalmoplegia and optic atrophy.

Abstract: Introduction: Mitochondrial DNA polymerase gamma (pol γ) encoded by POLG plays an indispensable role in the process of mitochondrial DNA replication and repair. The mutation of POLG can result in m...

Rapid progressive ALS in a patient with a DNAJC7 loss-of-function mutation.

Abstract: In S. cerevisiae, the Pif1 helicase functions to impact both nuclear and mitochondrial DNA replication and repair processes. Pif1 is a 5’-3’ helicase, which preferentially unwinds RNA-DNA hybrids and resolves G-quadruplex structures. Further, regulation of Pif1 by phosphorylation negatively impacts its interaction with telomerase during double strand break repair. Here, we report that in addition to phosphorylation, Pif1 is also modified by lysine acetylation, which influences both its cellular and core biochemical activities. Using Pif1 overexpression toxicity assays, we determined that the acetyltransferase NuA4 (Esa1) and deacetylase Rpd3 are primarily responsible for dynamically acetylating nuclear Pif1. Mass spectrometry analysis revealed that Pif1 was modified throughout the protein’s sequence on the N-terminus (K118, K129), helicase domain (K525, K639, K725), and C-terminus (K800). Acetylation of Pif1 exacerbated its overexpression toxicity phenotype, which was alleviated upon deletion of its N-terminus. Biochemical assays demonstrated that acetylation of Pif1 stimulated its helicase activity, while maintaining its substrate preferences. Additionally, both the ATPase and DNA binding activities of Pif1 were stimulated upon acetylation. Limited proteolysis assays indicate that acetylation of Pif1 induces a conformational change that may account for its altered enzymatic properties. We propose an acetylation-based model for the regulation of Pif1 activities, addressing how this post translational modification can influence its role as a key player in a multitude of DNA transactions vital to the maintenance of genome integrity.

A DNA polymerization-independent role for mitochondrial DNA polymerase I-like protein C in African trypanosomes.

Abstract: The mitochondrial DNA of Trypanosoma brucei and related parasites is a catenated network containing thousands of minicircles and tens of maxicircles called kinetoplast DNA (kDNA). Replication of the single nucleoid requires at least three DNA polymerases (POLIB, POLIC, and POLID) each having discrete localization near the kDNA during S phase. POLIB and POLID have roles in minicircle replication while the specific role of POLIC in kDNA maintenance is less clear. Here, we use an RNAi-complementation system to dissect the functions of the distinct POLIC domains: the conserved family A DNA polymerase domain (POLA) and the uncharacterized N-terminal region (UCR). While RNAi complementation with wild-type POLIC restored kDNA content and cell cycle localization, active site point mutations in the POLA domain impaired minicircle replication similarly to POLIB and POLID depletions. Complementation with POLA domain alone abolished POLIC foci formation and partially rescued the RNAi phenotype. Furthermore, we provide evidence of a crucial role for the UCR in cell cycle localization that facilitates proper distribution of progeny networks. This is the first report of a DNA polymerase that impacts mitochondrial nucleoid distribution.

Involvement of the mitochondrial nuclease EndoG in the regulation of cell proliferation through the control of reactive oxygen species.

Abstract: The apoptotic nuclease EndoG is involved in mitochondrial DNA replication. Previous results suggested that, in addition to regulate cardiomyocyte hypertrophy, EndoG could be involved in cell proliferation. Here, by using in vivo and cell culture models, we investigated the role of EndoG in cell proliferation. Genetic deletion of Endog both in vivo and in cultured cells or Endog silencing in vitro induced a defect in rodent and human cell proliferation with a tendency of cells to accumulate in the G1 phase of cell cycle and increased reactive oxygen species (ROS) production. The defect in cell proliferation occurred with a decrease in the activity of the AKT/PKB-GSK-3β-Cyclin D axis and was reversed by addition of ROS scavengers. EndoG deficiency did not affect the expression of ROS detoxifying enzymes, nor the expression of the electron transport chain complexes and oxygen consumption rate. Addition of the micropeptide Humanin to EndoG-deficient cells restored AKT phosphorylation and proliferation without lowering ROS levels. Thus, our results show that EndoG is important for cell proliferation through the control of ROS and that Humanin can restore cell division in EndoG-deficient cells and counteracts the effects of ROS on AKT phosphorylation.

Knockdown of TFAM in Tumor Cells Retarded Autophagic Flux through Regulating p53 Acetylation and PISD Expression

Abstract: Mitochondrial transcription factor A (TFAM) is required for mitochondrial DNA replication and transcription, which are essential for mitochondrial biogenesis. Previous studies reported that depleting mitochondrial functions by genetic deletion of TFAM impaired autophagic activities. However, the underlying mechanisms remain largely unknown. In the current study, we identified that knockdown of TFAM repressed the synthesis of autophagy bio-marker LC3-II in tumor cells and decreased the expression of phosphatidyl-serine decarboxylase (PISD). Besides, downregulation of PISD with siRNA reduced the level of LC3-II, indicating that depletion of TFAM retarded autophagy via inhibiting PISD expression. Furthermore, it was found that the tumor repressor p53 could stimulate the transcription and expression of PISD by binding the PISD enhancer. Additionally, the protein stability and transcriptional activity of p53 in TFAM knockdown tumor cells was attenuated, and this was associated with decreased acetylation, especially the acetylation of lysine 382 of p53. Finally, we identified that TFAM knockdown increased the NAD+/NADH ratio in tumor cells. This led to the upregulation of Sirtuin1 (SIRT1), a NAD-dependent protein deacetylase, to deacetylate p53 and attenuated its transcriptional activation on PISD. In summary, our study discovered a new mechanism regarding disturbed autophagy in tumor cells with mitochondrial dysfunction due to the depletion of TFAM.

TYMP Variants Result in Late-Onset Mitochondrial Myopathy With Altered Muscle Mitochondrial DNA Homeostasis

Abstract: Biallelic TYMP variants result in the mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), a juvenile-onset disorder with progressive course and fatal outcome. Milder late-onset (>40 years) form has been rarely described. Gene panel sequencing in a cohort of 60 patients featuring muscle accumulation of mitochondrial DNA (mtDNA) deletions detected TYMP defects in three subjects (5%), two of them with symptom onset in the fifth decade. One of the patients only displayed ptosis and ophthalmoparesis. Biochemical and molecular studies supported the diagnosis. Screening of TYMP is recommended in adult patients with muscle mtDNA instability, even in the absence of cardinal MNGIE features.

Nuclear Pif1 is Post Translationally Modified and Regulated by Lysine Acetylation

Abstract: In S. cerevisiae, the Pif1 helicase functions to impact both nuclear and mitochondrial DNA replication and repair processes. Pif1 is a 5’-3’ helicase, which preferentially unwinds RNA-DNA hybrids and resolves G-quadruplex structures. Further, regulation of Pif1 by phosphorylation negatively impacts its interaction with telomerase during double strand break repair. Here, we report that in addition to phosphorylation, Pif1 is also modified by lysine acetylation, which influences both its cellular and core biochemical activities. Using Pif1 overexpression toxicity assays, we determined that the acetyltransferase NuA4 (Esa1) and deacetylase Rpd3 are primarily responsible for dynamically acetylating nuclear Pif1. Mass spectrometry analysis revealed that Pif1 was modified throughout the protein’s sequence on the N-terminus (K118, K129), helicase domain (K525, K639, K725), and C-terminus (K800). Acetylation of Pif1 exacerbated its overexpression toxicity phenotype, which was alleviated upon deletion of its N-terminus. Biochemical assays demonstrated that acetylation of Pif1 stimulated its helicase activity, while maintaining its substrate preferences. Additionally, both the ATPase and DNA binding activities of Pif1 were stimulated upon acetylation. Limited proteolysis assays indicate that acetylation of Pif1 induces a conformational change that may account for its altered enzymatic properties. We propose an acetylation-based model for the regulation of Pif1 activities, addressing how this post translational modification can influence its role as a key player in a multitude of DNA transactions vital to the maintenance of genome integrity.

Hereditary Parkinson’s disease as a new clinical manifestation of the damaged POLG gene

Abstract: The protein product of the nuclear-encoded POLG gene plays a key role in the maintenance of mitochondrial DNA replication, and its failure causes multi-system diseases with varying severity. The clinical spectrum is extremely wide, and the most common symptoms include ptosis, myoclonus, epilepsy, myopathy, sensory ataxia, parkinsonism, cognitive decline and infertility. Now, it is known that mitochondrial dysfunction in Parkinson's disease plays a key role in the loss of dopaminergic neurons in the substantia nigra. Therefore, changes in the POLG gene may influence the development of various hereditary neurodegenerative diseases, including monogenic parkinsonism. However, only limited information is available on the relationship between Parkinson's disease and POLG gene and until now, there are no available data about the Hungarian population. In our study, we performed a next-generation sequencing study of 67 Hungarian patients with parkinsonism and analyzed the potentially damaging alterations in the POLG gene. 3 patients have been identified with a potential pathogen variant. In this study, we would like to call attention to the fact that during the differential diagnosis of parkinsonism, the possible involvement of POLG gene should be kept in mind. Especially in the presence of additional symptoms, such as ophthalmoparesis, non-vascular white matter lesions, psychiatric comorbidity, and relatively early age of onset, the POLG gene should be taken into consideration. Based on previous data from the literature and our own experience, we have summarized a possible diagnostic approach for POLG-associated parkinsonism. Orv Hetil. 2020; 161(20): 821-828.

Drug repurposing for mitochondrial diseases using a pharmacological model of complex I deficiency in the yeast Yarrowia lipolytica

Abstract: Mitochondrial diseases affect 1 in 5,000 live births around the world. They are caused by inherited or de novo mutations in over 350 nuclear-encoded and mtDNA-encoded genes. There is no approved treatment to stop the progression of any mitochondrial disease despite the enormous global unmet need. Affected families often self-compound cocktails of over-the-counter vitamins and generally recognized as safe nutritional supplements that have not received regulatory approval for efficacy. Finding a new use for an approved drug is called repurposing, an attractive path for mitochondrial diseases because of the reduced safety risks, low costs and fast timelines to a clinic-ready therapy or combination of therapies. Here I describe the first-ever drug repurposing screen for mitochondrial diseases involving complex I deficiency, e.g., Leigh syndrome, using the yeast Yarrowia lipolytica as a model system. Unlike the more commonly used yeast Saccharomyces cerevisiae but like humans, Yarrowia lipolytica has a functional and metabolically integrated respiratory complex I and is an obligate aerobe. In 384-well-plate liquid culture format without shaking, Yarrowia lipolytica cells grown in either glucose-containing media or acetate-containing media were treated with a half-maximal inhibitory concentration (3µM and 6μM, respectively) of the natural product and complex I inhibitor piericidin A. Out of 2,560 compounds in the Microsource Spectrum collection, 24 suppressors of piercidin A reached statistical significance in one or both media conditions. The suppressors include calcium channel blockers nisoldipine, amiodarone and tetrandrine as well as the farnesol-like sesquiterpenoids parthenolide, nerolidol and bisabolol, which may all be modulating mitochondrial calcium homeostasis. Estradiols and synthetic estrogen receptor agonists are the largest class of suppressors that rescue growth of piericidin-A-treated Yarrowia lipolytica cells in both glucose-containing and acetate-containing media. Analysis of structure-activity relationships suggests that estrogens may enhance bioenergetics by evolutionarily conserved interactions with mitochondrial membranes that promote mitochondrial filamentation and mitochondrial DNA replication.

Neu-p11 induces NLRP3 inflammasome-induced pyroptosis via provoking NOX4-mediated mitochondrial oxidative injury in chordoma cells

Abstract: Chordoma is a rarely malignant bone tumor with highly resistance to radiotherapy and chemotherapy. Melatonin has been shown to inhibit tumor cell invasion, metastasis and induce apoptosis in several types of cancer. Activation of melatonin receptor inhibit cancer stemness of chordoma, which suggesting that melatonin receptor agonists have potential therapeutic value for the clinical treatment of chordoma. The present study aimed to investigate the anticancer action and molecular mechanism of Piromelatine (Neu-P11) in chordoma cells, a high-efficacy agonist of melatonin receptor-1/melatonin receptor-2 (MT1/MT2). We used Neu-p11 (1, 10, 100, 1000 M) or vehicle to treat chordoma cell lines U-CH1 and MUG-Chor for 36 hours. CCK-8 assay and LDH activity assay showed that Neu-p11 had dose-dependent cytotoxic effect on chordoma cells. Neu-p11 induced NLRP3 and Cleaved-Caspase-1 expression, while Caspase-1 specific inhibitor Z-YVAD-FMK and NLRP3 specific inhibitor MCC950 independently blocked the cytotoxic effect of Neu-p11 on chordoma cells, indicating that Neu-p11 induced a NLRP3-dependent cell pyroptosis. Neu-p11 promoted NADPH oxidase 4 (NOX4) translocation to mitochondria, resulting in a significant increase in mitochondria-derived ROS and decreased expressions of mitochondrial antioxidant proteins (MnSOD, Sirt3) and mitochondrial DNA replication factor (Tfam), which could be attenuated by NOX4 specific inhibitor GKT137831. Further investigation revealed that Neu-p11 resulted in decreased copy number of mtDNA and increased mtDNA releasing in cytoplasm. Decreased oxygen consumption, reduced membrane potential and ultrastructural damage of mitochondrial were detected in Neu-p11 treated chordoma cells, which could be attenuated by GKT137831. GKT137831 inhibited mitochondria-translocation of NLRP3 and attenuated activation of NLRP3 inflammasome caused by Neu-p11 treatment. Collectively, we demonstrated that Neu-p11 induces mitochondrial translocation of NOX4 leads to oxidative damage of mitochondria, mediating chordoma cells NLRP3-dependent pyroptosis. Neu-p11 may be a a new strategy for chordoma treatment. Graphic abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

A second hybrid-binding domain modulates the activity of Drosophila ribonuclease H1.

Abstract: In eukaryotes, ribonuclease H1 (RNase H1) is involved in the processing and removal of RNA/DNA hybrids in both nuclear and mitochondrial DNA The enzyme comprises a C-terminal catalytic domain and an N-terminal hybrid-binding domain (HBD), separated by a linker of variable length, 115 amino acids in Drosophila melanogaster (Dm) Molecular modelling predicted this extended linker to fold into a structure similar to the conserved HBD Based on a deletion series, both the catalytic domain and the conserved HBD were required for high-affinity binding to heteroduplex substrates, while loss of the novel HBD led to an ∼90% drop in Kcat with a decreased KM, and a large increase in the stability of the RNA/DNA hybrid-enzyme complex, supporting a bipartite-binding model in which the second HBD facilitates processivity Shotgun proteomics following in vivo cross-linking identified single-stranded DNA-binding proteins from both nuclear and mitochondrial compartments, respectively RpA-70 and mtSSB, as prominent interaction partners of Dm RNase H1 However, we were not able to document direct and stable interactions with mtSSB when the proteins were co-overexpressed in S2 cells, and functional interactions between them in vitro were minor

PrimPol (Primase/Polymerase), replicating enzyme – A mini review

Abstract: Polymerases were revealed first in 1970s. Most important to the modest perception the enzyme responsible for nuclear DNA replication that was pol , for DNA repair pol and for mitochondrial DNA replication pol DNA construction and renovation done by DNA polymerases, so directing both the constancy and discrepancy of genetic information. Replication of genome initiate with DNA template-dependent fusion of small primers of RNA. This preliminary phase in replication of DNA demarcated as de novo primer synthesis which is catalyzed by specified polymerases known as primases. Sixteen diverse DNA-synthesizing enzymes about human perspective are devoted to replication, reparation, mutilation lenience, and inconsistency of nuclear DNA. But in dissimilarity, merely one DNA polymerase has been called in mitochondria. It has been suggest that PrimPol is extremely acting the roles by re-priming DNA replication in mitochondria to permit an effective and appropriate way replication to be accomplished. Investigations from a numeral of test site have significantly amplified our appreciative of the role, recruitment and regulation of the enzyme during DNA replication. Though, we are simply just start to increase in value the versatile roles that play PrimPol in eukaryote.