mtDNA-encoded Proteins Research Articles

The POLG Variant c.678G>C; p.(Gln226His) Is Associated with Mitochondrial Abnormalities in Fibroblasts Derived from a Patient Compared to a First-Degree Relative

Background: The nuclear-encoded enzyme polymerase gamma (Pol-γ) is crucial in the replication of the mitochondrial genome (mtDNA), which in turn is vital for mitochondria and hence numerous metabolic processes and energy production in eukaryotic cells. Variants in the POLG gene, which encodes the catalytic subunit of Pol-γ, can significantly impair Pol-γ enzyme function. Pol-γ-associated disorders are referred to as POLG-spectrum disorders (POLG-SDs) and are mainly autosomal-recessively inherited. Clinical manifestations include muscle weakness and fatigue, and severe forms of the disease can lead to premature death in infancy, childhood, and early adulthood, often associated with seizures, liver failure, or intractable epilepsy. Here, we analyzed fibroblasts from a compound heterozygous patient with the established pathogenic variant c.2419C>T; p.(Arg807Cys) and a previously undescribed variant c.678G>C; p.(Gln226His) with a clinical manifestation compatible with POLG-SDs, sensory ataxic neuropathy, and infantile muscular atrophy. We conducted a battery of functional studies for Pol-γ and mitochondrial dysfunction on the patient’s fibroblasts, to test whether the novel variant c.678G>C; p.(Gln226His) may be causative in human disease. Aims/Methods: We analyzed skin-derived fibroblasts in comparison to a first-degree relative (the mother of the patient), an asymptomatic carrier harboring only the established c.2419C>T; p.(Arg807Cys) mutation. Assessments of mitochondrial function included measurements of mtDNA content, mRNA levels of mitochondrial genes, mitochondrial mass, and mitochondrial morphology. Case Presentation and Results: A 13-year-old male presented with symptoms starting at three years of age, including muscle weakness and atrophy in the lower extremities and facial muscles, which later extended to the upper limbs, voice, and back muscles, without further progression. The patient also reported fatigue and muscle pain after physical activity, with no sensory deficits. Extensive diagnostic tests such as electromyography, nerve conduction studies, muscle biopsy, and MRI were unremarkable. Exome sequencing revealed that he carried the compound heterozygous variants in POLG c.678G>C; p.(Gln226His) and c.2419C>T; p.(Arg807Cys), but no other potential genetic pathogenic causes. In comparison to a first-degree relative (his mother) who only carried the c.2419C>T; p.(Arg807Cys) pathogenic mutation, in vitro analyses revealed a significant reduction in mtDNA content (~50%) and mRNA levels of mtDNA-encoded proteins. Mitochondrial mass was reduced by approximately 20%, and mitochondrial interconnectivity within cells was impaired, as determined by fluorescence microscopy and mitochondrial staining. Conclusions: Our findings suggest that the c.678G>C; p.(Gln226His) variant, in conjunction with the c.2419C>T; p.(Arg807Cys) mutation, may compromise mtDNA replication and mitochondrial function and could result in clinically significant mitochondriopathy. As this study is based on one patient compared to a first-degree relative (but with an identical mitochondrial genome), the pathogenicity of c.678G>C; p.(Gln226His) of POLG should be confirmed in future studies, in particular, in conjunction with other POLG-variants.

Decreased Mrpl42 expression exacerbates myocardial ischemia and reperfusion injury by inhibiting mitochondrial translation

Mammalian mitochondrial DNA (mtDNA) encodes a total of 13 proteins, all of which are subunits of enzyme complexes of the oxidative phosphorylation. The mtDNA-encoded protein synthesis depends on the mitochondrial ribosomal proteins (MRPs), which assemble to form a specialized form of ribosome. Some mtDNA-encoded proteins have been reported to be reduced after myocardial ischemic injury. However, the molecular mechanisms responsible for this decrease and whether this decrease is involved in myocardial ischemia/reperfusion (I/R) injury remains unknown. Here, we found that the mtDNA-encoded protein levels were significantly decreased after I/R injury, while the mRNA levels of these genes were either increased or had no significant change. Subsequently, by querying and analyzing public database resources, we found that the expression of many mitochondrial translation-related proteins tended to decrease after myocardial infarction injury, and the reduction in the expression of these proteins was most obvious for Mrpl42. Furthermore, we found that cardiac Mrpl42 knockdown aggravated I/R-induced cardiac contractile dysfunction and cardiomyocyte death, while restoring Mrpl42 expression in the heart reduced I/R injury. Mrpl42 knockdown impaired the translation of mtDNA-encoded genes, ultimately led to aberrations in mitochondrial morphology and respiratory function. In addition, we found that the decrease in the expression of Mrpl42 after I/R injury was caused by the downregulation of Nrf2, which directly regulates Mrpl42 transcription. Our study revealed that ischemic downregulation of Mrpl42 expression and subsequent inhibition of mitochondrial translation contribute to cardiac I/R injury. Targeting Mrpl42 may be a novel therapeutic intervention for cardiac I/R injury and myocardial infarction.

Impact of mtDNA-encoded proteins arising from cytosolic translation (mPACT)

Mitochondria are multifunctional organelles that regulate energy homeostasis, apoptosis, cell signaling and many biosynthetic pathways. Mitochondria have their own genomes that reportedly contain 37 well-known genes, including 13 protein-coding genes, 2 genes encoding ribosomal RNAs (rRNAs) and 22 genes encoding transfer RNAs (tRNAs). RNA is essential for most biological functions which can act as a translation template for protein production or perform the function itself to directly regulate cellular processes. In addition to those functioning inside mitochondria, mitochondria can regulate many important biological processes through exporting RNAs including long non-coding mitochondrial RNAs (ncmtRNAs), circRNAs, rRNAs, tRNAs and mRNAs.

Selection promotes age-dependent degeneration of the mitochondrial genome.

Somatic mutations in mitochondrial genomes (mtDNA) accumulate exponentially during aging. Using single cell sequencing, we characterize the spectrum of age-accumulated mtDNA mutations in mouse and human liver and identify directional forces that accelerate the accumulation of mutations beyond the rate predicted by a neutral model. "Driver" mutations that give genomes a replicative advantage rose to high cellular abundance and carried along "passenger" mutations, some of which are deleterious. In addition, alleles that alter mtDNA-encoded proteins selectively increased in abundance overtime, strongly supporting the idea of a "destructive" selection that favors genomes lacking function. Overall, this combination of selective forces acting in hepatocytes promotes somatic accumulation of mutations in coding regions of mtDNA that are otherwise conserved in evolution. We propose that these selective processes could contribute to the population prevalence of mtDNA mutations, accelerate the course of heteroplasmic mitochondrial diseases and promote age-associated erosion of the mitochondrial genome.

Alterations in coenzyme Q10 status in a cybrid line harboring the 3243A>G mutation of mitochondrial DNA is associated with abnormal mitochondrial bioenergetics and dysregulated mitochondrial biogenesis

Mitochondrial DNA (mtDNA) mutations, including the m.3243A>G mutation that causes mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), are associated with secondary coenzyme Q10 (CoQ10) deficiency. We previously demonstrated that PPARGC1A knockdown repressed the expression of PDSS2 and several COQ genes. In the present study, we compared the mitochondrial function, CoQ10 status, and levels of PDSS and COQ proteins and genes between mutant cybrids harboring the m.3243A>G mutation and wild-type cybrids. Decreased mitochondrial energy production, defective respiratory function, and reduced CoQ10 levels were observed in the mutant cybrids. The ubiquinol-10:ubiquinone-10 ratio was lower in the mutant cybrids, indicating blockage of the electron transfer upstream of CoQ, as evident from the reduced ratio upon rotenone treatment and increased ratio upon antimycin A treatment in 143B cells. The mutant cybrids exhibited downregulation of PDSS2 and several COQ genes and upregulation of COQ8A. In these cybrids, the levels of PDSS2, COQ3-a isoform, COQ4, and COQ9 were reduced, whereas those of COQ3-b and COQ8A were elevated. The mutant cybrids had repressed PPARGC1A expression, elevated ATP5A levels, and reduced levels of mtDNA-encoded proteins, nuclear DNA-encoded subunits of respiratory enzyme complexes, MNRR1, cytochrome c, and DHODH, but no change in TFAM, TOM20, and VDAC1 levels. Alterations in the CoQ10 level in MELAS may be associated with mitochondrial energy deficiency and abnormal gene regulation. The finding of a reduction in the ubiquinol-10:ubiquinone-10 ratio in the MELAS mutant cybrids differs from our previous discovery that cybrids harboring the m.8344A>G mutation exhibit a high ubiquinol-10:ubiquinone-10 ratio.

Mitochondrial DNA control of gene expression and cell function in HeLa cybrids of HeLa ρ0 cells with transferred mtDNA of A549 cells

Asthma is a disease characterized by airway inflammation and hyperreactivity. Mitochondria have their own DNA (mtDNA) that encodes essential components of Electron Transport Chain complexes and is inherited exclusively maternally. Variants in mtDNA (haplogroups) are linked to risks and severity of other diseases but have not been studied in asthma. We hypothesized that mitochondrial genome can regulate expression of nDNA genes important in asthma pathophysiology. Haplogroup H mtDNA from A549 lung epithelial cells was transferred to mtDNA-deleted HeLa cells (ρ0) to create HeLa[H] cybrids that have identical nuclei but varied mitochondrial genome as compared to HeLa cells with haplogroup L mtDNA. Cells were analyzed for mtDNA copy number by quantitative polymerase chain reaction (qPCR), energy metabolism by monitoring the cellular respiration and glycolysis using Seahorse analyzer, and mtDNA sequencing for haplogroup. Single-cell RNA sequencing technology, SeqGeq bioinformatics program, and western blot were used to analyze differential gene expression and involved pathways among these cells. ρ0 cells have mtDNA copy number <1, while cybrids have mtDNA copy number similar to A549 cells and different from HeLa cells {mtDNA copy number (mean±SD), HeLa 636±115, ρ0 0.13±0.08, cybrid 1956±354, A549 1738±504, n≥2 replicate experiments, ANOVA p<0.001}. ρ0 have no mitochondrial oxygen consumption and maintain energy generation by increased glycolysis, whereas cybrids have similar oxygen consumption and glycolytic rates to HeLa and A549 {mitochondrial basal oxygen consumption pmole/min/mg protein (mean±SD), HeLa 7.78±0.43, ρ0 0.51±0.56, cybrid 6.76±2.34, A549 4.86±0.41, n≥3, ANOVA p<0.001; glycolysis mpH/min/mg protein (mean±SD), HeLa 0.91±0.08, ρ0 1.96±0.16, cybrid 0.52±0.14, A549 0.72±0.48, n≥3, ANOVA p<0.001}. Compared to HeLa, ρ0 cells lack expression of all 13 mtDNA-encoded proteins and have changes in nuclear-encoded DNA (nDNA) gene expression (4,047 nDNA up, and 5,552 nDNA down). Switching from L to H haplogroup results in greater changes in gene expression (9,320 nDNA up, 4,376 nDNA down, 9 mtDNA up, and 4 mtDNA down). Compared to HeLa, cybrids have greater expression of genes important in bioenergetics and asthma, i.e., TCA pathway genes (aconitase, isocitrate dehydrogenase, oxoglutarate dehydrogenase, succinate dehydrogenase), cytokines, and inducible NO synthase (iNOS) pathway genes. Overall, these studies suggest that mitochondria are not only important for energy generation but relevant to cell signaling, inflammation, and metabolism in asthma. This work is supported by the National Heart, Lung, and Blood Institute HL081064, HL103453. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Mitochondrial DNA Haplogroup K Is Protective Against Autism Spectrum Disorder Risk in Populations of European Ancestry

Accumulative evidence indicates a critical role of mitochondrial function in autism spectrum disorders (ASD), implying that ASD risk may be linked to mitochondrial dysfunction due to DNA (mtDNA) variations. Although a few studies have explored the association between mtDNA variations and ASD, the role of mtDNA in ASD is still unclear. Here, we aimed to investigate whether mitochondrial DNA haplogroups are associated with the risk of ASD. Two European cohorts and an Ashkenazi Jewish (AJ) cohort were analyzed, including 2,062 ASD patients in comparison with 4,632 healthy controls. DNA samples were genotyped using Illumina HumanHap550/610 and Illumina 1M arrays, inclusive of mitochondrial markers. Mitochondrial DNA (mtDNA) haplogroups were identified from genotyping data using HaploGrep2. A mitochondrial genome imputation pipeline was established to detect mtDNA variants. We conducted a case-control study to investigate potential associations of mtDNA haplogroups and variants with the susceptibility to ASD. We observed that the ancient adaptive mtDNA haplogroup K was significantly associated with decreased risk of ASD by the investigation of 2 European cohorts including a total of 2,006 cases and 4,435 controls (odds ratio= 0.64, P=1.79× 10-5), and we replicated this association in an Ashkenazi Jewish (AJ) cohort including 56 cases and 197 controls (odds ratio= 0.35, P= 9.46× 10-3). Moreover, we demonstrate that the mtDNA variants rs28358571, rs28358584, and rs28358280 are significantly associated with ASD risk. Further expression quantitative trait loci (eQTLs) analysis indicated that the rs28358584 and rs28358280 genotypes are associated with expression levels of nearby genes in brain tissues, suggesting those mtDNA variants may confer risk for ASD via regulation of expression levels of genes encoded by the mitochondrial genome. This study helps to shed light on the contribution of mitochondria in ASD and provides new insights into the genetic mechanism underlying ASD, suggesting the potential involvement of mtDNA-encoded proteins in the development of ASD. Increasing evidence indicates that mitochondrial dysfunction may be linked to autism spectrum disorder (ASD). This study investigated potential associations of mitochondrial DNA (mtDNA) variants in 2 European and Ashkenazi Jewish cohorts including 2,062 individuals with ASD and 4,632 healthy controls. Researchers found that the ancient mtDNA haplogroup K was linked to a reduced risk of ASD in both European and Ashkenazi Jewish populations. Additionally, specific mtDNA variants were associated with ASD risk and were shown to influence the expression of nearby genes in the brain. These findings highlight the potential involvement of mtDNA in ASD development, offering new insights into the genetic mechanisms underlying the disorder.

In silico prospecting of the mtDNA of Macrobrachium amazonicum from transcriptome data

BackgroundMacrobrachium amazonicum is a freshwater prawn widely distributed in South America that is undergoing speciation, so the denomination “M. amazonicum complex” is used for it. The mitochondrial cytochrome c oxidase subunit I (COI) gene has been used to elucidate this speciation, but heteroplasmies and pseudogenes have been recorded, making separation difficult. Obtaining genes from cDNA (RNA) rather than genomic DNA is an effective tool to mitigate those two types of occurrences. The aim of this study was to assemble in silico the mitochondrial DNA (mtDNA) of the Amazonian coastal population of M. amazonicum inhabiting the state of Pará.ResultsSequences were obtained from the prawn’s transcriptome using the de novo approach. Six libraries of cDNA from the androgen gland, hepatopancreas, and muscle tissue were used. The mtDNA of M. amazonicum was 14,960 bp in length. It contained 13 protein-coding genes, 21 complete transfer RNAs, and the 12S and 16S subunits of ribosomal RNA. All regions were found on the light strand except tRNAGln, which was on the heavy strand. The control region (D-loop) was not recovered, making for a gap of 793 bp. The cladogram showed the formation of the well-defined Macrobrachium clade, with high support value in the established branches (91–100). The three-dimensional spatial conformation of the mtDNA-encoded proteins showed that most of them were mainly composed of major α-helices that typically shows in those proteins inserted in the membrane (mitochondrial).ConclusionsIt was possible to assemble a large part of the mitochondrial genome of M. amazonicum in silico using data from other genomes deposited in GenBank and to validate it through the similarities between its COI and 16S genes and those from animals of the same region deposited in GenBank. Depositing the M. amazonicum mtDNA sequences in GenBank may help solve the taxonomic problems recorded for the species, in addition to providing complete sequences of candidate coding genes for use as biomarkers in ecological studies.

Validation of the male-specific ORF of the paternally-transmitted mtDNA in Mytilus edulis as a protein-coding gene

There appears to be an additional set of sex-specific mtDNA-encoded proteins in bivalve species with doubly uniparental mitochondrial inheritance that may be involved in the transmission of the female and male mitogenomes. In the marine mussel Mytilus edulis, the translation of the female-specific open reading frame (F-ORF) was demonstrated but the translation of the male-specific ORF (M-ORF) remains to be shown. Here we validate the male-specific ORF of the paternal mitogenome in M. edulis as a protein-coding gene. The M-ORF protein was detected only in male gonads and localized in sperm mitochondria and acrosome, suggesting that it is involved in a key sperm function in Mytilus edulis.

The mitochondrial ribosomal protein mRpL4 regulates Notch signaling.

Mitochondrial ribosomal proteins (MRPs) assemble as specialized ribosome to synthesize mtDNA-encoded proteins, which are essential for mitochondrial bioenergetic and metabolic processes. MRPs are required for fundamental cellular activities during animal development, but their roles beyond mitochondrial protein translation are poorly understood. Here, we report a conserved role of the mitochondrial ribosomal protein L4 (mRpL4) in Notch signaling. Genetic analyses demonstrate that mRpL4 is required in the Notch signal-receiving cells to permit target gene transcription during Drosophila wing development. We find that mRpL4 physically and genetically interacts with the WD40 repeat protein wap and activates the transcription of Notch signaling targets. We show that human mRpL4 is capable of replacing fly mRpL4 during wing development. Furthermore, knockout of mRpL4 in zebrafish leads to downregulated expression of Notch signaling components. Thus, we have discovered a previously unknown function of mRpL4 during animal development.

Mitochondria-Targeted Gene Silencing Facilitated by Mito-CPDs.

Mitochondrial DNA (mtDNA) plays an essential role in maintaining normal cellular activities. Its heteroplasmic mutations are known to cause various genetic diseases. Current genetic engineering strategies, such as those based on RNA interference (RNAi) and antisense technology, are difficult to genetically alter mtDNA, however, due to the inability of highly negatively charged oligonucleotides to translocate across the double-membrane mitochondria. We report herein a universal mitochondria-targeted gene-delivery approach by using cell-penetrating poly(disulfide)s (CPDs). Novel CPD-based mitochondrial transporters, named Mito-CPDs, were synthesized by using triphenylphosphonium (TPP)-fused propagating monomers containing either disulfide or diselenide backbones. Upon spontaneous complex formation with an oligonucleotide (single- or double-stranded), the resulting nanoscale Mito-CPD@Oligo exhibited excellent properties in common biological media. While the intracellular gene-delivery efficiency of these Mito-CPDs was comparable to that of commercial transfection agents, their unique mitochondria-localized properties enabled effective release of the loaded cargo inside these organelles. Subsequent mitochondrial delivery of siRNA and antisense oligonucleotides against suitable mtDNA-encoded proteins showed successful down-regulation of target protein expression, leading to profound effects on mitochondrial functions. Mito-CPDs thus provide a useful tool for future investigations of mitochondrial biology and treatment of mitochondria-related diseases.

Mitochondrial DNA abnormalities and metabolic syndrome.

Metabolic syndrome (MetS) is a complex pathological condition that involves disrupted carbohydrate, protein, and fat metabolism in the human body, and is a major risk factor for several chronic diseases, including diabetes, cardiovascular disease, and cerebrovascular disease. While the exact pathogenesis of metabolic syndrome is not yet fully understood, there is increasing evidence linking mitochondrial dysfunction, which is closely related to the mitochondrial genome and mitochondrial dynamics, to the development of this condition. Recent advancements in genetic sequencing technologies have allowed for more accurate detection of mtDNA mutations and other mitochondrial abnormalities, leading to earlier diagnosis and intervention in patients with metabolic syndrome. Additionally, the identification of specific mechanisms by which reduced mtDNA copy number and gene mutations, as well as abnormalities in mtDNA-encoded proteins and mitochondrial dynamics, contribute to metabolic syndrome may promote the development of novel therapeutic targets and interventions, such as the restoration of mitochondrial function through the targeting of specific mitochondrial defects. Additionally, advancements in genetic sequencing technologies may allow for more accurate detection of mtDNA mutations and other mitochondrial abnormalities, leading to earlier diagnosis and intervention in patients with MetS. Therefore, strategies to promote the restoration of mitochondrial function by addressing these defects may offer new options for treating MetS. This review provides an overview of the research progress and significance of mitochondrial genome and mitochondrial dynamics in MetS.

A comparative analysis of mitochondrial ORFs provides new insights on expansion of mitochondrial genome size in Arcidae

BackgroundArcidae, comprising about 260 species of ark shells, is an ecologically and economically important lineage of bivalve mollusks. Interestingly, mitochondrial genomes of several Arcidae species are 2–3 times larger than those of most bilaterians, and are among the largest bilaterian mitochondrial genomes reported to date. The large mitochondrial genome size is mainly due to expansion of unassigned regions (regions that are functionally unassigned). Previous work on unassigned regions of Arcidae mtDNA genomes has focused on nucleotide-level analyses to observe sequence characteristics, however the origin of expansion remains unclear.ResultsWe assembled six new mitogenomes and sequenced six transcriptomes of Scapharca broughtonii to identify conserved functional ORFs that are transcribed in unassigned regions. Sixteen lineage-specific ORFs with different copy numbers were identified from seven Arcidae species, and 11 of 16 ORFs were expressed and likely biologically active. Unassigned regions of 32 Arcidae mitogenomes were compared to verify the presence of these novel mitochondrial ORFs and their distribution. Strikingly, multiple structural analyses and functional prediction suggested that these additional mtDNA-encoded proteins have potential functional significance. In addition, our results also revealed that the ORFs have a strong connection to the expansion of Arcidae mitochondrial genomes and their large-scale duplication play an important role in multiple expansion events. We discussed the possible origin of ORFs and hypothesized that these ORFs may originate from duplication of mitochondrial genes.ConclusionsThe presence of lineage-specific mitochondrial ORFs with transcriptional activity and potential functional significance supports novel features for Arcidae mitochondrial genomes. Given our observation and analyses, these ORFs may be products of mitochondrial gene duplication. These findings shed light on the origin and function of novel mitochondrial genes in bivalves and provide new insights into evolution of mitochondrial genome size in metazoans.

Inhibition of human peptide deformylase by actinonin sensitizes glioblastoma cells to temozolomide chemotherapy

Glioblastoma multiforme (GBM) is a common intracranial primary tumor of the central nervous system with high malignancy, poor prognosis, and short survival. Studies have shown that mitochondrial energy metabolism plays an important role in GBM chemotherapy resistance, suggesting that interrupting mitochondrial oxidative phosphorylation (OXPHOS) may improve GBM treatment. Human peptide deformylase (HsPDF) is a mitochondrial deformylase that removes the formylated methionine from the N-terminus of proteins encoded by mitochondrial DNA (mtDNA), thereby contributing to correct protein folding and participating in the assembly of the electron respiratory chain complex. In this study, we found that the expression of mtDNA-encoded proteins was significantly downregulated after treatment of GBM cells U87MG and LN229 with the HsPDF inhibitor, actinonin. In combination with temozolomide, a preferred chemotherapeutic medicine for GBM, the OXPHOS level decreased, mitochondrial protein homeostasis was unbalanced, mitochondrial fission increased, and the integrated stress response was activated to promote mitochondrial apoptosis. These findings suggest that HsPDF inhibition is an important strategy for overcoming chemoresistance of GBM cells.

Modeling Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-Like Episodes Syndrome Using Patient-Derived Induced Neurons Generated by Direct Reprogramming.

Mitochondrial diseases are a heterogeneous group of rare genetic disorders caused by mutations in nuclear or mitochondrial DNA (mtDNA). These diseases are frequently multisystemic, although mainly affect tissues that require large amounts of energy such as the brain. Mutations in mitochondrial transfer RNA (mt-tRNA) lead to defects in protein translation that may compromise some or all mtDNA-encoded proteins. Mitochondrial Encephalomyopathy, Lactic Acidosis and Stroke-like episodes (MELAS) syndrome is mainly caused by the m.3243A>G mutation in the mt-tRNALeu(UUR) (MT-TL1) gene. Owing to the lack of proper animal models, several cellular models have been developed to study the disease, providing insight in the pathophysiological mechanisms of MELAS. In this study, we show a successful direct conversion of MELAS patient-derived fibroblasts into induced neurons (iNs) for the first time, as well as an electrophysiological characterization of iNs cocultured with astrocytes. In addition, we performed bioenergetics analysis to study the consequences of m.3243A>G mutation in this neuronal model of MELAS syndrome.

Unbiased proteomics, histochemistry, and mitochondrial DNA copy number reveal better mitochondrial health in muscle of high-functioning octogenarians.

Master athletes (MAs) prove that preserving a high level of physical function up to very late in life is possible, but the mechanisms responsible for their high function remain unclear. We performed muscle biopsies in 15 octogenarian world-class track and field MAs and 14 non-athlete age/sex-matched controls (NA) to provide insights into mechanisms for preserving function in advanced age. Muscle samples were assessed for respiratory compromised fibers, mitochondrial DNA (mtDNA) copy number, and proteomics by liquid-chromatography mass spectrometry. MA exhibited markedly better performance on clinical function tests and greater cross-sectional area of the vastus lateralis muscle. Proteomics analysis revealed marked differences, where most of the ~800 differentially represented proteins in MA versus NA pertained to mitochondria structure/function such as electron transport capacity (ETC), cristae formation, mitochondrial biogenesis, and mtDNA-encoded proteins. In contrast, proteins from the spliceosome complex and nuclear pore were downregulated in MA. Consistent with proteomics data, MA had fewer respiratory compromised fibers, higher mtDNA copy number, and an increased protein ratio of the cristae-bound ETC subunits relative to the outer mitochondrial membrane protein voltage-dependent anion channel. There was a substantial overlap of proteins overrepresented in MA versus NA with proteins that decline with aging and that are higher in physically active than sedentary individuals. However, we also found 176 proteins related to mitochondria that are uniquely differentially expressed in MA. We conclude that high function in advanced age is associated with preserving mitochondrial structure/function proteins, with underrepresentation of proteins involved in the spliceosome and nuclear pore complex. Whereas many of these differences in MA appear related to their physical activity habits, others may reflect unique biological (e.g., gene, environment) mechanisms that preserve muscle integrity and function with aging. Funding for this study was provided by operating grants from the Canadian Institutes of Health Research (MOP 84408 to TT and MOP 125986 to RTH). This work was supported in part by the Intramural Research Program of the National Institute on Aging, NIH, Baltimore, MD, USA.

Yme2, a putative RNA recognition motif and AAA+ domain containing protein, genetically interacts with the mitochondrial protein export machinery.

The mitochondrial respiratory chain is composed of nuclear as well as mitochondrial-encoded subunits. A variety of factors mediate co-translational integration of mtDNA-encoded proteins into the inner membrane. In Saccharomyces cerevisiae, Mdm38 and Mba1 are ribosome acceptors that recruit the mitochondrial ribosome to the inner membrane, where the insertase Oxa1, facilitates membrane integration of client proteins. The protein Yme2 has previously been shown to be localized in the inner mitochondrial membrane and has been implicated in mitochondrial protein biogenesis, but its mode of action remains unclear. Here, we show that multiple copies of Yme2 assemble into a high molecular weight complex. Using a combination of bioinformatics and mutational analyses, we find that Yme2 possesses an RNA recognition motif (RRM), which faces the mitochondrial matrix and a AAA+ domain that is located in the intermembrane space. We further show that YME2 genetically interacts with MDM38, MBA1 and OXA1, which links the function of Yme2 to the mitochondrial protein biogenesis machinery.

The mtDNA mutation spectrum in the PolG mutator mouse reveals germline and somatic selection

BackgroundMitochondrial DNA (mtDNA) codes for products necessary for electron transport and mitochondrial gene translation. mtDNA mutations can lead to human disease and influence organismal fitness. The PolG mutator mouse lacks mtDNA proofreading function and rapidly accumulates mtDNA mutations, making it a model for examining the causes and consequences of mitochondrial mutations. Premature aging in PolG mice and their physiology have been examined in depth, but the location, frequency, and diversity of their mtDNA mutations remain understudied. Identifying the locations and spectra of mtDNA mutations in PolG mice can shed light on how selection shapes mtDNA, both within and across organisms.ResultsHere, we characterized somatic and germline mtDNA mutations in brain and liver tissue of PolG mice to quantify mutation count (number of unique mutations) and frequency (mutation prevalence). Overall, mtDNA mutation count and frequency were the lowest in the D-loop, where an mtDNA origin of replication is located, but otherwise uniform across the mitochondrial genome. Somatic mtDNA mutations have a higher mutation count than germline mutations. However, germline mutations maintain a higher frequency and were also more likely to be silent. Cytosine to thymine mutations characteristic of replication errors were the plurality of basepair changes, and missense C to T mutations primarily resulted in increased protein hydrophobicity. Unlike wild type mice, PolG mice do not appear to show strand asymmetry in mtDNA mutations. Indel mutations had a lower count and frequency than point mutations and tended to be short, frameshift deletions.ConclusionsOur results provide strong evidence that purifying selection plays a major role in the mtDNA of PolG mice. Missense mutations were less likely to be passed down in the germline, and they were less likely to spread to high frequencies. The D-loop appears to have resistance to mutations, either through selection or as a by-product of replication processes. Missense mutations that decrease hydrophobicity also tend to be selected against, reflecting the membrane-bound nature of mtDNA-encoded proteins. The abundance of mutations from polymerase errors compared with reactive oxygen species (ROS) damage supports previous studies suggesting ROS plays a minimal role in exacerbating the PolG phenotype, but our findings on strand asymmetry provide discussion for the role of polymerase errors in wild type organisms. Our results provide further insight on how selection shapes mtDNA mutations and on the aging mechanisms in PolG mice.

Mitochondrial DNA and MitomiR Variations in Pancreatic Cancer: Potential Diagnostic and Prognostic Biomarkers.

Pancreatic cancer is an aggressive disease with poor prognosis. Only about 15–20% of patients diagnosed with pancreatic cancer can undergo surgical resection, while the remaining 80% are diagnosed with locally advanced or metastatic pancreatic ductal adenocarcinoma (PDAC). In these cases, chemotherapy and radiotherapy only confer marginal survival benefit. Recent progress has been made in understanding the pathobiology of pancreatic cancer, with a particular effort in discovering new diagnostic and prognostic biomarkers, novel therapeutic targets, and biomarkers that can predict response to chemo- and/or radiotherapy. Mitochondria have become a focus in pancreatic cancer research due to their roles as powerhouses of the cell, important subcellular biosynthetic factories, and crucial determinants of cell survival and response to chemotherapy. Changes in the mitochondrial genome (mtDNA) have been implicated in chemoresistance and metastatic progression in some cancer types. There is also growing evidence that changes in microRNAs that regulate the expression of mtDNA-encoded mitochondrial proteins (mitomiRs) or nuclear-encoded mitochondrial proteins (mitochondria-related miRs) could serve as diagnostic and prognostic cancer biomarkers. This review discusses the current knowledge on the clinical significance of changes of mtDNA, mitomiRs, and mitochondria-related miRs in pancreatic cancer and their potential role as predictors of cancer risk, as diagnostic and prognostic biomarkers, and as molecular targets for personalized cancer therapy.

Searching for signatures of positive selection in cytochrome b gene associated with subterranean lifestyle in fast-evolving arvicolines (Arvicolinae, Cricetidae, Rodentia)

BackgroundMitochondrial genes encode proteins involved in oxidative phosphorylation. Variations in lifestyle and ecological niche can be directly reflected in metabolic performance. Subterranean rodents represent a good model for testing hypotheses on adaptive evolution driven by important ecological shifts. Voles and lemmings of the subfamily Arvicolinae (Rodentia: Cricetidae) provide a good example for studies of adaptive radiation. This is the youngest group within the order Rodentia showing the fastest rates of diversification, including the transition to the subterranean lifestyle in several phylogenetically independent lineages.ResultsWe evaluated the signatures of selection in the mitochondrial cytochrome b (cytB) gene in 62 Arvicolinae species characterized by either subterranean or surface-dwelling lifestyle by assessing amino acid sequence variation, exploring the functional consequences of the observed variation in the tertiary protein structure, and estimating selection pressure. Our analysis revealed that: (1) three of the convergent amino acid substitutions were found among phylogenetically distant subterranean species and (2) these substitutions may have an influence on the protein complex structure, (3) cytB showed an increased ω and evidence of relaxed selection in subterranean lineages, relative to non-subterranean, and (4) eight protein domains possess increased nonsynonymous substitutions ratio in subterranean species.ConclusionsOur study provides insights into the adaptive evolution of the cytochrome b gene in the Arvicolinae subfamily and its potential implications in the molecular mechanism of adaptation. We present a framework for future characterizations of the impact of specific mutations on the function, physiology, and interactions of the mtDNA-encoded proteins involved in oxidative phosphorylation.