Subunits Of Respiratory Complexes Research Articles

Insights into conformational changes in cytochrome bduring the early steps of its maturation.

Membrane proteins carrying redox cofactors are key subunits of respiratory chain complexes, yet the exact path of their folding and maturation remains poorly understood. Here, using cryo-EM and structure prediction via Alphafold2, we generated models of early assembly intermediates of cytochrome b (Cytb), a central subunit of complex III. The predicted structure of the first assembly intermediate suggests how the binding of Cytb to the assembly factor Cbp3-Cbp6 imposes an open configuration to facilitate the acquisition of its heme cofactors. Moreover, structure predictions of the second intermediate indicate how hemes get stabilized by binding of the assembly factor Cbp4, with a concomitant weakening of the contact between Cbp3-Cbp6 and Cytb, preparing for the release of the fully hemylated protein from the assembly factors.

OTUB1/NDUFS2 axis promotes pancreatic tumorigenesis through protecting against mitochondrial cell death

Pancreatic cancer is one of the most fatal cancers in the world. A growing number of studies have begun to demonstrate that mitochondria play a key role in tumorigenesis. Our previous study reveals that NDUFS2 (NADH: ubiquinone oxidoreductase core subunit S2), a core subunit of the mitochondrial respiratory chain complex I, is upregulated in Pancreatic adenocarcinoma (PAAD). However, its role in the development of PAAD remains unknown. Here, we showed that NDUFS2 played a critical role in the survival, proliferation and migration of pancreatic cancer cells by inhibiting mitochondrial cell death. Additionally, protein mass spectrometry indicated that the NDUFS2 was interacted with a deubiquitinase, OTUB1. Overexpression of OTUB1 increased NDUFS2 expression at the protein level, while knockdown of OTUB1 restored the effects in vitro. Accordingly, overexpression and knockdown of OTUB1 phenocopied those of NDUFS2 in pancreatic cancer cells, respectively. Mechanically, NDUFS2 was deubiquitinated by OTUB1 via K48-linked polyubiquitin chains, resulted in an elevated protein stability of NDUFS2. Moreover, the growth of OTUB1-overexpressed pancreatic cancer xenograft tumor was promoted in vivo, while the OTUB1-silenced pancreatic cancer xenograft tumor was inhibited in vivo. In conclusion, we revealed that OTUB1 increased the stability of NDUFS2 in PAAD by deubiquitylation and this axis plays a pivotal role in pancreatic cancer tumorigenesis and development.

Mitochondrial reactive oxygen species impact human fibroblast responses to protracted γ-ray exposures

Purpose: Continuous exposure to ionizing radiation at a low dose rate poses significant health risks to humans on deep space missions, prompting the need for mechanistic studies to identify countermeasures against its deleterious effects. Mitochondria are a major subcellular locus of radiogenic injury, and may trigger secondary cellular responses through the production of reactive oxygen species (mtROS) with broader biological implications. Methods and Materials: To determine the contribution of mtROS to radiation-induced cellular responses, we investigated the impacts of protracted γ-ray exposures (IR; 1.1 Gy delivered at 0.16 mGy/min continuously over 5 days) on mitochondrial function, gene expression, and the protein secretome of human HCA2-hTERT fibroblasts in the presence and absence of a mitochondria-specific antioxidant mitoTEMPO (MT; 5 µM). Results: IR increased fibroblast mitochondrial oxygen consumption (JO2) and H2O2 release rates (JH2O2) under energized conditions, which corresponded to higher protein expression of NADPH Oxidase (NOX) 1, NOX4, and nuclear DNA-encoded subunits of respiratory chain Complexes I and III, but depleted mtDNA transcripts encoding subunits of the same complexes. This was associated with activation of gene programs related to DNA repair, oxidative stress, and protein ubiquination, all of which were attenuated by MT treatment along with radiation-induced increases in JO2 and JH2O2. IR also increased secreted levels of interleukin-8 and Type I collagens, while decreasing Type VI collagens and enzymes that coordinate assembly and remodeling of the extracellular matrix. MT treatment attenuated many of these effects while augmenting others, revealing complex effects of mtROS in fibroblast responses to IR. Conclusion: These results implicate mtROS production in fibroblast responses to protracted radiation exposure, and suggest potentially protective effects of mitochondrial-targeted antioxidants against radiogenic tissue injury in vivo.

Identification and characterization of a new pathologic mutation in a large Leber hereditary optic neuropathy pedigree

BackgroundMost patients suffering from Leber hereditary optic neuropathy carry one of the three classic pathologic mutations, but not all individuals with these genetic alterations develop the disease. There are different risk factors that modify the penetrance of these mutations. The remaining patients carry one of a set of very rare genetic variants and, it appears that, some of the risk factors that modify the penetrance of the classical pathologic mutations may also affect the phenotype of these other rare mutations.ResultsWe describe a large family including 95 maternally related individuals, showing 30 patients with Leber hereditary optic neuropathy. The mutation responsible for the phenotype is a novel transition, m.3734A > G, in the mitochondrial gene encoding the ND1 subunit of respiratory complex I. Molecular-genetic, biochemical and cellular studies corroborate the pathogenicity of this genetic change.ConclusionsWith the study of this family, we confirm that, also for this very rare mutation, sex and age are important factors modifying penetrance. Moreover, this pedigree offers an excellent opportunity to search for other genetic or environmental factors that additionally contribute to modify penetrance.

Abstract 659: TR-107 and erastin produce synergistic inhibition of colorectal cancer cell viability in vitro

Abstract Introduction: Ferroptosis is an emerging potential target for the treatment of colorectal cancer (CRC) and has been shown to sensitize cancer cells to combinatory chemotherapy. Our studies show that the TR-107 compound, an agonist of ClpP - the proteolytic subunit of mitochondrial maintenance complex - can synergize with Erastin, an inducer of ferroptosis cell death. TR-107 potently disrupts mitochondrial proteostasis and attenuates oxidative phosphorylation, resulting in excessive oxidative stress. Here, we show that Erastin synergizes with TR-107 activity by downregulating the cofactor of GPX4, a major intracellular antioxidant defense resulting in CRC cell apoptosis. Methods: We first evaluated the efficacy of TR-107 against eight CRC cell lines belonging to four established consensus molecular subtypes. Cells were treated with control (DMSO) or TR-107 (10 nM, 50 nM, 1 µM) for 24, 48, and 72 hours. We determined IC50 values with Cell Titer-Glo, cell proliferation with the Beckman Coulter cell counter, cell cycle with flow cytometry, OXPHOS activity with Seahorse Real-Time ATP Rate assay, mitochondrial protein levels with western blot and proteomics, and differential gene expression using RNA sequencing and RT-PCR. Subsequently, we assessed the efficacy of TR-107 and Erastin co-treatment. Cells were treated with DMSO, TR-107 (10, 50 nM), Erastin (1, 10 µM), or combinations of the abovementioned concentrations. Cell viability was measured 72 hours post-treatment with Cell Titer-Glo. Synergy scores (HSA) were calculated with Synergyfinder on RStudio. Ferroptosis-related protein expression 24 hours after co-treatment was assessed with western blot and densitometry. Results: TR-107 IC50 values ranged from 2-70 nM, producing a dose- and time-dependent inhibitory effect in cell proliferation. Western blot showed that 50 nM TR-107 abolished the expression of critical mitochondrial proteins ClpX, TFAM, and TUFM. Seahorse data showed a significant reduction in OXPHOS activity. Multi-omics revealed downregulation of respiratory chain complex subunits, mtDNA transcription/translation factors, and upregulation of mitophagy and ferroptosis-related pathways in treated cells. Co-treatment with Erastin produced synergistic HSA scores in 63% (5/8) of assessed CRC cell lines. RKO and NCI-H508 were highly sensitive to Erastin treatment, suggesting that lower Erastin concentrations may synergize with TR-107 and thus reduce dose toxicity. Western blot analysis 24 hours post-treatment showed a loss of GPX4 protein levels in co-treated cells compared to 50 nM TR-107 treatment. This suggests a rapid downregulation of intracellular antioxidant defense that may induce ferroptosis sooner and more effectively than single-agent treatment. Conclusion: In vitro results suggest that co-treating with TR-107 and Erastin produces a significant, synergistic reduction in CRC cell viability. Citation Format: Michael Giarrizzo, Joseph F. LaComb, Hetvi R. Patel, Agnieszka B. Bialkowska, Rohan Reddy, Lee M. Graves, Edwin J. Iwanowicz. TR-107 and erastin produce synergistic inhibition of colorectal cancer cell viability in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 659.

PPR596 Is Required for nad2 Intron Splicing and Complex I Biogenesis in Arabidopsis.

Mitochondria are essential organelles that generate energy via oxidative phosphorylation. Plant mitochondrial genome encodes some of the respiratory complex subunits, and these transcripts require accurate processing, including C-to-U RNA editing and intron splicing. Pentatricopeptide repeats (PPR) proteins are involved in various organellar RNA processing events. PPR596, a P-type PPR protein, was previously identified to function in the C-to-U editing of mitochondrial rps3 transcripts in Arabidopsis. Here, we demonstrate that PPR596 functions in the cis-splicing of nad2 intron 3 in mitochondria. Loss of the PPR596 function affects the editing at rps3eU1344SS, impairs nad2 intron 3 splicing and reduces the mitochondrial complex I's assembly and activity, while inducing alternative oxidase (AOX) gene expression. This defect in nad2 intron splicing provides a plausible explanation for the slow growth of the ppr595 mutants. Although a few P-type PPR proteins are involved in RNA C-to-U editing, our results suggest that the primary function of PPR596 is intron splicing.

A Drosophila model of mitochondrial disease phenotypic heterogeneity.

Mutations in genes that affect mitochondrial function cause primary mitochondrial diseases. Mitochondrial diseases are highly heterogeneous and even patients with the same mitochondrial disease can exhibit broad phenotypic heterogeneity, which is poorly understood. Mutations in subunits of mitochondrial respiratory complex I cause complex I deficiency, which can result in severe neurological symptoms and death in infancy. However, some complex I deficiency patients present with much milder symptoms. The most common nuclear gene mutated in complex I deficiency is the highly conserved core subunit NDUFS1. To model the phenotypic heterogeneity in complex I deficiency, we used RNAi lines targeting the Drosophila NDUFS1 homolog ND-75 with different efficiencies. Strong knockdown of ND-75 in Drosophila neurons resulted in severe behavioural phenotypes, reduced lifespan, altered mitochondrial morphology, reduced endoplasmic reticulum (ER)-mitochondria contacts and activation of the unfolded protein response (UPR). By contrast, weak ND-75 knockdown caused much milder behavioural phenotypes and changes in mitochondrial morphology. Moreover, weak ND-75 did not alter ER-mitochondria contacts or activate the UPR. Weak and strong ND-75 knockdown resulted in overlapping but distinct transcriptional responses in the brain, with weak knockdown specifically affecting proteosome activity and immune response genes. Metabolism was also differentially affected by weak and strong ND-75 knockdown including gamma-aminobutyric acid (GABA) levels, which may contribute to neuronal dysfunction in ND-75 knockdown flies. Several metabolic processes were only affected by strong ND-75 knockdown including the pentose phosphate pathway and the metabolite 2-hydroxyglutarate (2-HG), suggesting 2-HG as a candidate biomarker of severe neurological mitochondrial disease. Thus, our Drosophila model provides the means to dissect the mechanisms underlying phenotypic heterogeneity in mitochondrial disease.

Searching for Frataxin Function: Exploring the Analogy with Nqo15, the Frataxin-like Protein of Respiratory Complex I from Thermus thermophilus.

Nqo15 is a subunit of respiratory complex I of the bacterium Thermus thermophilus, with strong structural similarity to human frataxin (FXN), a protein involved in the mitochondrial disease Friedreich's ataxia (FRDA). Recently, we showed that the expression of recombinant Nqo15 can ameliorate the respiratory phenotype of FRDA patients' cells, and this prompted us to further characterize both the Nqo15 solution's behavior and its potential functional overlap with FXN, using a combination of in silico and in vitro techniques. We studied the analogy of Nqo15 and FXN by performing extensive database searches based on sequence and structure. Nqo15's folding and flexibility were investigated by combining nuclear magnetic resonance (NMR), circular dichroism, and coarse-grained molecular dynamics simulations. Nqo15's iron-binding properties were studied using NMR, fluorescence, and specific assays and its desulfurase activation by biochemical assays. We found that the recombinant Nqo15 isolated from complex I is monomeric, stable, folded in solution, and highly dynamic. Nqo15 does not share the iron-binding properties of FXN or its desulfurase activation function.

Multi-level profiling unravels mitochondrial dysfunction in myotonic dystrophy type 2

Myotonic dystrophy type 2 (DM2) is an autosomal-dominant multisystemic disease with a core manifestation of proximal muscle weakness, muscle atrophy, myotonia, and myalgia. The disease-causing CCTG tetranucleotide expansion within the CNBP gene on chromosome 3 leads to an RNA-dominated spliceopathy, which is currently untreatable. Research exploring the pathophysiological mechanisms in myotonic dystrophy type 1 has resulted in new insights into disease mechanisms and identified mitochondrial dysfunction as a promising therapeutic target. It remains unclear whether similar mechanisms underlie DM2 and, if so, whether these might also serve as potential therapeutic targets. In this cross-sectional study, we studied DM2 skeletal muscle biopsy specimens on proteomic, molecular, and morphological, including ultrastructural levels in two separate patient cohorts consisting of 8 (explorative cohort) and 40 (confirmatory cohort) patients. Seven muscle biopsy specimens from four female and three male DM2 patients underwent proteomic analysis and respiratory chain enzymology. We performed bulk RNA sequencing, immunoblotting of respiratory chain complexes, mitochondrial DNA copy number determination, and long-range PCR (LR-PCR) to study mitochondrial DNA deletions on six biopsies. Proteomic and transcriptomic analyses revealed a downregulation of essential mitochondrial proteins and their respective RNA transcripts, namely of subunits of respiratory chain complexes I, III, and IV (e.g., mt-CO1, mt-ND1, mt-CYB, NDUFB6) and associated translation factors (TACO1). Light microscopy showed mitochondrial abnormalities (e.g., an age-inappropriate amount of COX-deficient fibers, subsarcolemmal accumulation) in most biopsy specimens. Electron microscopy revealed widespread ultrastructural mitochondrial abnormalities, including dysmorphic mitochondria with paracrystalline inclusions. Immunofluorescence studies with co-localization of autophagy (p62, LC-3) and mitochondrial marker proteins (TOM20, COX-IV), as well as immunohistochemistry for mitophagy marker BNIP3 indicated impaired mitophagic flux. Immunoblotting and LR-PCR did not reveal significant differences between patients and controls. In contrast, mtDNA copy number measurement showed a reduction of mtDNA copy numbers in the patient group compared to controls. This first multi-level study of DM2 unravels thus far undescribed functional and structural mitochondrial abnormalities. However, the molecular link between the tetranucleotide expansion and mitochondrial dysfunction needs to be further elucidated.

The mitochondriogenic but not the immunosuppressant effects of mTOR inhibitors prompt neuroprotection and delay disease evolution in a mouse model of progressive multiple sclerosis

IntroductionPurportedly, the progression of multiple sclerosis (MS) occurs when neurodegenerative processes due to derangement of axonal bioenergetics take over the autoimmune response. However, a clear picture of the causative interrelationship between autoimmunity and axonal mitochondrial dysfunction in progressive MS (PMS) pathogenesis waits to be provided. MethodsIn the present study, by adopting the NOD mouse model of PMS, we compared the pharmacological effects of the immunosuppressants dexamethasone and fingolimod with those of mTOR inhibitors rapamycin and everolimus that, in addition to immunosuppression, also regulate mitochondrial functioning. Female Non-Obese Diabetic (NOD) mice were immunized with MOG35–55 and treated with drugs to evaluate functional, immune and mitochondrial parameters during disease evolution. ResultsWe found that dexamethasone and fingolimod did not affect the pattern of progression as well as survival. Conversely, mTOR inhibitors rapamycin and everolimus delayed disease progression and robustly extended survival of immunized mice. The same effects were obtained when treatment was delayed by 30 days after immunization. Remarkably, dexamethasone and fingolimod prompted the same degree of immunosuppression of rapamycin within both spleen and spinal cord of mice. However, only rapamycin prompted mitochondriogenesis by increasing mitochondrial content, and expression of several mitochondrial respiratory complex subunits, thereby preventing mtDNA reduction in the spinal cords of immunized mice. These pharmacodynamic effects were not reproduced in healthy NOD mice, suggesting a disease context-dependent pharmacodynamic effect. DiscussionData corroborate the key role of mitochondriogenesis to treatment of MS progression, and for the first time disclose the translational potential of mTOR inhibitors in PMS therapy.

Integrated metabolomics and transcriptomics to reveal biomarkers and mitochondrial metabolic dysregulation of premature ovarian insufficiency.

The metabolic characteristics of premature ovarian insufficiency (POI), a reproductive endocrine disease characterized by abnormal sex hormone metabolism and follicle depletion, remain unclear. Metabolomics is a powerful tool for exploring disease phenotypes and biomarkers. This study aims to identify metabolic markers and construct diagnostic models, and elucidate the underlying pathological mechanisms for POI. Non-targeted metabolomics was utilized to characterize the plasma metabolic profile of 40 patients. The metabolic markers were identified through bioinformatics and machine learning, and constructed an optimal diagnostic model by classified multi-model analysis. Enzyme-linked immunosorbent assay (ELISA) was used to verify antioxidant indexes, mitochondrial enzyme complexes, and ATP levels. Finally, integrated transcriptomics and metabolomics were used to reveal the dysregulated pathways and molecular regulatory mechanisms of POI. The study identified eight metabolic markers significantly correlated with ovarian reserve function. The XGBoost diagnostic model was developed based on six machine learning models, demonstrating its robust diagnostic performance and clinical applicability through the evaluation of receiver operating characteristic (ROC) curve, decision curve analysis (DCA), calibration curve, and precise recall (PR) curve. Multi-omics analysis showed that mitochondrial respiratory chain electron carrier (CoQ10) and enzyme complex subunits were down-regulated in POI. ELISA validation revealed an elevation in oxidative stress markers and a reduction in the activities of antioxidant enzymes, CoQ10, and mitochondrial enzyme complexes in POI. Our findings highlight that mitochondrial dysfunction and energy metabolism disorders are closely related to the pathogenesis of POI. The identification of metabolic markers and predictive models holds significant implications for the diagnosis, treatment, and monitoring of POI.

Different Mutations in SF3B1 Gene Have Specific Transcriptomic Characteristics in Myelodysplastic Neoplasms

Myelodysplastic neoplasms (MDS) are hematopoietic stem cell (HSC) disorders characterized by ineffective hematopoiesis, peripheral cytopenia, and increased tendency to leukemic transformation. Somatic mutations in spliceosome machinery occur in approx. 50% of MDS patients. Among these, up to 30% of MDS patients carry a mutation in SF3B1. According to the 2022 classification system, MDS-SF3B1 is now considered as separate MDS subtype. Mutated SF3B1 perturbs mitochondrial function, resulting in apoptosis and ineffective erythropoiesis. The most common mutation of SF3B1, K700E, accounts for 50% of the variants, with additional codons (such as 666 and 662) acting as hotspot sites. Different mutations seem to be associated with distinct clinicopathological features. However, the prognostic implication of different SF3B1 mutations in MDS remains controversial and needs further clarification. This study investigated differences in clinical and molecular features of MDS-SF3B1 with different types of SF3B1 mutations. To investigate a direct impact of different point mutations in SF3B1, the study was based on four isogenic NALM6 cell lines, the wildtype (wt) and three cell lines, each with different SF3B1 mutation (CRISPR/Cas9-edited K700E, K666N, and H662Q). To understand phenotypic manifestations of the mutations in the disease, we also analyzed clinical and transcriptional characteristics of 146 lower-risk MDS patients. Using RNA-seq in the cell lines and CD34+ BM patient cells (17 patients with no mutation, 11 patients with K700E SF3B1 mutation, 5 patients with K666N/E SF3B1 mutation), we investigated effects of different SF3B1 mutations on RNA splicing events and gene expression levels. Initially, outcome analysis confirmed the previously described observation that patients with a SF3B1 mutation have favorable outcomes compared to those with other splicing factor mutations. Further, the analysis showed that specific mutations in SF3B1 tend to lead to different prognoses; patients with K700E had longer survival compared to those with other SF3B1 mutations (mean progression-free survival was 64.8 vs. 29.1 months). Analysis of RNA splicing in NALM6 showed that K666N and H662Q mutations share similar characteristics whereas K700E mutation is more similar to wt-cells. Most significant changes were found in retained introns (RI); increased RI inclusion levels were detected in K666N/H662Q cells compared to K700E/wt cells. In patients, RI events were also strongly increased in K666N/E compared to K700E samples (Fig. 1). A significant overlap was identified between cell line and patient data; out of 173 genes with higher RI inclusion level in K666N-NALM6 cells, 77 genes had higher RI inclusion also in patients. Interestingly, 12 out of these 77 genes were related to mitochondrial functions (e.g., CREBZF, DDX1, MRPL23, MUTYH, and RAF1). RNA-seq data further showed significant alternations in gene expression levels in the four isogenic cell lines and also in patient samples with different mutations. Surprisingly, a more pronounced difference was detected in gene expression between patients with different SF3B1 mutations than between patients with no mutation and with any SF3B1 mutation (irrespective of its type), suggesting that specific SF3B1 mutations result in different phenotypes. Pathway analyses showed that deregulated genes in samples with K700E vs. other hotspot mutations were significantly enriched in electron transport, cell cycle, proliferation, quiescence, and HSC differentiation. Importantly, expression of multiple mitochondria-related genes was altered between K700E and K666N/H662Q NALM6 cells (334 genes, FDR < 0.01; Fig. 2), indicating different metabolic characteristics of the cells with non-K700E mutations. For example, several subunits of mitochondrial respiratory complexes I and IV were upregulated in K700E cells, pointing to differences in energetic metabolism. Altogether, our data support the hypothesis that only K700E mutation of SF3B1 may have a positive prognostic value and that other SF3B1 mutations may influence cells differently, specifically affecting their mitochondrial functions. Based on our results, we conclude that prognostic implication of different SF3B1 mutations in MDS will need additional refinements in future. Supported by AZV CR (NU20-03-00412), GA CR (20-19162S), and MH CZ-DRO (00023736).

The clinical, myopathological, and genetic analysis of 155 Chinese mitochondrial ophthalmoplegia patients with mitochondrial DNA single large deletions.

Progressive external ophthalmoplegia (PEO) is a common subtype of mitochondrial encephalomyopathy. The study aimed to investigate the relationship between mitochondrial DNA (mtDNA) abnormalities, muscle pathology, and clinical manifestations in Chinese patients with single large-scale mtDNA deletion presenting with PEO. This is a retrospective single-center study. Patients with PEO who had a single large deletion in mitochondrial DNA were included in this study. The associations were analyzed between mtDNA deletion patterns, myopathological changes, and clinical characteristics. In total, 155 patients with mitochondrial PEO carrying single large-scale mtDNA mutations were enrolled, including 137 chronic progressive external ophthalmoplegia (CPEO) and 18 Kearns-Sayre syndrome (KSS) patients. The onset ages were 9.61 ± 4.12 in KSS and 20.15 ± 9.06 in CPEO. The mtDNA deletions ranged from 2225 bp to 9131 bp, with m.8470_13446del being the most common. The KSS group showed longer deletions than the CPEO group (p = 0.004). Additionally, a higher number of deleted genes encoding respiratory chain complex subunits (p = 0.001) and tRNA genes (p = 0.009) were also observed in the KSS group. A weak negative correlation between the mtDNA deletion size and ages of onset (p < 0.001, r = -0.369) was observed. The proportion of ragged red fibers, ragged blue fibers, and cytochrome c negative fibers did not correlate significantly with onset ages (p > 0.05). However, a higher percentage of abnormal muscle fibers corresponds to an increased prevalence of exercise intolerance, limb muscle weakness, dysphagia, and cerebellar ataxia. We reported a large Chinese cohort consisting of mitochondrial PEO patients with single large-scale mtDNA deletions. Our results demonstrated that the length and locations of mtDNA deletions may influence onset ages and clinical phenotypes. The severity of muscle pathology could not only indicate diagnosis but also may be associated with clinical manifestations beyond the extraocular muscles.

High Fat Diet-Induced Obesity Dysregulates Splenic B Cell Mitochondrial Activity.

Diet-induced obesity impairs mitochondrial respiratory responses in tissues that are highly metabolically active, such as the heart. However, less is known about the impact of obesity on the respiratory activity of specific cell types, such as splenic B cells. B cells are of relevance, as they play functional roles in obesity-induced insulin resistance, inflammation, and responses to infection. Here, we tested the hypothesis that high-fat-diet (HFD)-induced obesity could impair the mitochondrial respiration of intact and permeabilized splenic CD19+ B cells isolated from C57BL/6J mice and activated ex vivo with lipopolysaccharide (LPS). High-resolution respirometry was used with intact and permeabilized cells. To reveal potential mechanistic targets by which HFD-induced obesity dysregulates B cell mitochondria, we conducted proteomic analyses and 3D serial block face scanning electron microscopy (SBFEM). High-resolution respirometry revealed that intact LPS-stimulated B cells of obese mice, relative to controls, displayed lower ATP-linked, as well as maximal uncoupled, respiration. To directly investigate mitochondrial function, we used permeabilized LPS-stimulated B cells, which displayed increased H2O2 emission and production with obesity. We also examined oxidative phosphorylation efficiency simultaneously, which revealed that oxygen consumption and ATP production were decreased in LPS-stimulated B cells with obesity relative to controls. Despite minimal changes in total respiratory complex abundance, in LPS-stimulated B cells of obese mice, three of the top ten most downregulated proteins were all accessory subunits of respiratory complex I. SBFEM showed that B cells of obese mice, compared to controls, underwent no change in mitochondrial cristae integrity but displayed increased mitochondrial volume that was linked to bioenergetic function. Collectively, these results establish a proof of concept that HFD-induced obesity dysregulates the mitochondrial bioenergetic metabolism of activated splenic B cells.

Targeted knockout of a conserved plant mitochondrial gene by genome editing

Fusion proteins derived from transcription activator-like effectors (TALEs) have emerged as genome editing tools for mitochondria. TALE nucleases (TALENs) have been applied to delete chimaeric reading frames and duplicated (redundant) genes but produced complex genomic rearrangements due to the absence of non-homologous end-joining. Here we report the targeted deletion of a conserved mitochondrial gene, nad9, encoding a subunit of respiratory complex I. By generating a large number of TALEN-mediated mitochondrial deletion lines, we isolated, in addition to mutants with rearranged genomes, homochondriomic mutants harbouring clean nad9 deletions. Characterization of the knockout plants revealed impaired complex I biogenesis, male sterility and defects in leaf and flower development. We show that these defects can be restored by expressing a functional Nad9 protein from the nuclear genome, thus creating a synthetic cytoplasmic male sterility system. Our data (1) demonstrate the feasibility of using genome editing to study mitochondrial gene functions by reverse genetics, (2) highlight the role of complex I in plant development and (3) provide proof-of-concept for the construction of synthetic cytoplasmic male sterility systems for hybrid breeding by genome editing.

In fission yeast, 65 non-essential mitochondrial proteins related to respiration and stress become essential in low-glucose conditions.

Mitochondria perform critical functions, including respiration, ATP production, small molecule metabolism, and anti-oxidation, and they are involved in a number of human diseases. While the mitochondrial genome contains a small number of protein-coding genes, the vast majority of mitochondrial proteins are encoded by nuclear genes. In fission yeast Schizosaccharomyces pombe, we screened 457 deletion (del) mutants deficient in nuclear-encoded mitochondrial proteins, searching for those that fail to form colonies in culture medium containing low glucose (0.03-0.1%; low-glucose sensitive, lgs), but that proliferate in regular 2-3% glucose medium. Sixty-five (14%) of the 457 deletion mutants displayed the lgs phenotype. Thirty-three of them are defective either in dehydrogenases, subunits of respiratory complexes, the citric acid cycle, or in one of the nine steps of the CoQ10 biosynthetic pathway. The remaining 32 lgs mutants do not seem to be directly related to respiration. Fifteen are implicated in translation, and six encode transporters. The remaining 11 function in anti-oxidation, amino acid synthesis, repair of DNA damage, microtubule cytoskeleton, intracellular mitochondrial distribution or unknown functions. These 32 diverse lgs genes collectively maintain mitochondrial functions under low (1/20-1/60× normal) glucose concentrations. Interestingly, 30 of them have homologues associated with human diseases.

ФУНКЦИОНАЛЬНАЯ СОСТОЯТЕЛЬНОСТЬ И ПОЛИМОРФИЗМ ГЕНОМА МИТОХОНДРИЙ ЛЕЙКОЦИТОВ ПРИ ИШЕМИЧЕСКОЙ БОЛЕЗНИ СЕРДЦА, ОСЛОЖНЕННОЙ НАРУШЕНИЯМИ РИТМА

Introduction. Ischemic heart disease (CHD) is the most common pathology in the structure of cardiovascular diseases. Ischemia is a trigger for the occurrence of cardiac arrhythmias (HRDs), requiring the implantation of cardioverter-defibrillators. The functioning of cardiomyocytes is closely associated with respiratory processes in mitochondria (Mtx). Normally, all Mtx in the human body carry the same genome - mitochondrial DNA (mtDNA), respectively, the functional capabilities of all Mtx within the body are similar. Carriage of polymorphic variants of genes encoding subunits of respiratory chain complexes and rRNA may affect the functionality of Mthx. Target. To study the respiratory function of Mtx and the features of mtDNA of leukocytes in patients with coronary artery disease, accompanied by life-threatening NRS. Material and methods. Peripheral venous blood was taken from patients with coronary artery disease (n=45) and ischemic heart disease with HRS (n=120). Mtx was obtained by differential centrifugation in a sucrose gradient. Oxygen consumption rates (OCR) (nM O2/min/mg protein) Mthx were measured in two energy states: V3, the presence of oxidation substrate(s) and phosphorylation substrate (ADP) in the incubation buffer; V4 - after the consumption of ADP in the buffer. Succinate and a mixture of pyruvate and malate were used as oxidation substrates. To assess the functional reserve of Mtx, OPC was determined in the presence and in the absence of saturated palmitic acid (PA) in buffer. The coefficient of conjugation of oxidative processes and phosphorylation was calculated - respiratory control (RC) (V3/V4). The mtDNA haplogroup was established, as well as the carriage of polymorphic variants of genes encoding subunits of the respiratory chain complexes and ribosomal RNA (rRNA) (12S and 16S subunits). Statistical evaluation was carried out in the STATISTICA 13 program using the Mann-Whitney test for independent samples and the Wilcoxon test for dependent data groups. Data are presented as medians and quartiles (Me (Q1; Q3). Differences were considered statistically significant at p&lt;0.05. Results. SPK in V3, V4 and DC of mitochondria in CAD and CAD with HRS did not have significant differences in both buffers without PC. When PA was added to the buffer, a significant increase in SEC was observed in Mth patients with isolated CAD. Thus, in the V3 state, SPK in pyruvate-malate buffer increased from 127.7 (85.3; 196.4) to 198.3 (162.7; 234.9) nM O2/min/mg of protein (p=0, 04), and in the V4 state from (46.6 (38.5; 56.3) to 64.4 (60.3; 87.7) nM O2/min/mg protein (p=0.02). the use of succinate buffer and PC, the mitochondrial SPK in patients with uncomplicated coronary artery disease increased slightly (p = 0.07) from 125.5 (64.4; 172.3) to 184.3 (47.3; 406.3) and from 44, 2 (27.5; 71.9) to 86.1 (36.9; 159.4) nM O2 / min / mg protein in V3 and V4, respectively. In the group of CAD with HRS, the addition of PA to the buffer did not affect the SEC in V3 and V4, as well as DC when using both buffers.mtDNA genotyping found that the main haplogroup in patients with IHD and HRS is haplogroup H - 41%.In 90% of cases, patients in this group were carriers of missense polymorphisms of 16S rRNA genes, which correlates with earlier studies of mtDNA in patients with uncomplicated CAD.In almost half of the cases, tRNA polymorphisms were detected in patients with CAD with HRS.Rarely (less than 40%), missense polymorphisms of the respiratory chain complex I genes were found. Polymorphisms of the genes for cytochrome B, cytochrome c oxidase, ATP synthase, and 12S rRNA turned out to be minor in frequency of occurrence. The frequencies of occurrence of all these polymorphic variants of the genes were similar to those in patients with uncomplicated coronary artery disease. The mtDNA of patients with HRS was 1.5 times more likely to have multiple missense polymorphisms in the protein genes of two different respiratory chain complexes. For genes with missense substitutions in three complexes and substitutions simultaneously in 12S and 16S rRNA, the differences between the groups were 1.6 and 1.7 times. Conclusion. Patients with CAD complicated by HRS are characterized by a reduced functional reserve of mitochondria, in contrast to patients with uncomplicated CAD. This may be due to the carriage of polymorphic variants of the mtDNA genes responsible for coding subunits of proteins of the Mth electron transport chain and mitochondrial rRNA. Also, in patients with CAD and HRS, compared with patients with uncomplicated CAD, the combined carriage of several polymorphic variants of the genes encoding these proteins and rRNA was more often detected.

Cardiolipin prolongs the lifetimes of respiratory proteins in Drosophila flight muscle

Respiratory complexes and cardiolipins have exceptionally long lifetimes. The fact that they co-localize in mitochondrial cristae raises the questions of whether their longevities have a common cause and whether the longevity of OXPHOS proteins is dependent on cardiolipin. To address these questions, we developed a method to measure side-by-side the half-lives of proteins and lipids in wildtype Drosophila and cardiolipin deficient mutants. We fed adult flies with stable isotope-labeled precursors (13C615N2-lysine or 13C6-glucose) and determined the relative abundance of heavy isotopomers in protein and lipid species by mass spectrometry. To minimize confounding effects of tissue regeneration, we restricted our analysis to the thorax, the bulk of which consists of post-mitotic flight muscles. Analysis of 680 protein and 45 lipid species showed that the subunits of respiratory complexes I-V and the carriers for phosphate and ADP/ATP were among the longest-lived proteins (average half-life of 48±16 days) while the molecular species of cardiolipin were the longest-lived lipids (average half-life of 27±6 days). The remarkable longevity of these crista residents was not shared by all mitochondrial proteins, especially not by those residing in the matrix and the inner boundary membrane. Ablation of cardiolipin synthase, which causes replacement of cardiolipin by phosphatidylglycerol, and ablation of tafazzin, which causes partial replacement of cardiolipin by monolyso-cardiolipin, decreased the lifetimes of the respiratory complexes. Ablation of tafazzin also decreased the lifetimes of the remaining cardiolipin species. These data suggest that an important function of cardiolipin in mitochondria is to protect respiratory complexes from degradation.

Mitochondrial genome study in blood of maternally inherited ALS cases

BackgroundALS is a heterogeneous disease in which different factors such as mitochondrial phenotypes act in combination with a genetic predisposition. This study addresses the question of whether homoplasmic (total mitochondrial genome of a sample is affected) and/or heteroplasmic mutations (wildtype and mutant mitochondrial DNA molecules coexist) might play a role in familial ALS. Blood was drawn from familial ALS patients with a possible maternal pattern of inheritance according to their pedigrees, which was compared to blood of ALS patients without maternal association as well as age-matched controls. In two cohorts, we analyzed the mitochondrial genome from whole blood or isolated white blood cells and platelets using a resequencing microarray (Affymetrix MitoChip v2.0) that is able to detect homoplasmic and heteroplasmic mitochondrial DNA mutations and allows the assessment of low-level heteroplasmy.ResultsWe identified an increase in homoplasmic ND5 mutations, a subunit of respiratory chain complex I, in whole blood of ALS patients that allowed maternal inheritance. This effect was more pronounced in patients with bulbar onset. Heteroplasmic mutations were significantly increased in different mitochondrial genes in platelets of patients with possible maternal inheritance. No increase of low-level heteroplasmy was found in maternal ALS patients.ConclusionOur results indicate a contribution of homoplasmic ND5 mutations to maternally associated ALS with bulbar onset. Therefore, it might be conceivable that specific maternally transmitted rather than randomly acquired mitochondrial DNA mutations might contribute to the disease process. This stands in contrast with observations from Alzheimer’s and Parkinson’s diseases showing an age-dependent accumulation of unspecific mutations in mitochondrial DNA.

Exploring biomarkers of premature ovarian insufficiency based on oxford nanopore transcriptional profile and machine learning

Premature ovarian insufficiency (POI) is a reproductive endocrine disorder characterized by infertility and perimenopausal syndrome, with a highly heterogeneous genetic etiology and its mechanism is not fully understood. Therefore, we utilized Oxford Nanopore Technology (ONT) for the first time to characterize the full-length transcript profile, and revealed biomarkers, pathway and molecular mechanisms for POI by bioinformatics analysis and machine learning. Ultimately, we identified 272 differentially expressed genes, 858 core genes, and 25 hub genes by analysis of differential expression, gene set enrichment, and protein–protein interactions. Seven candidate genes were identified based on the intersection features of the random forest and Boruta algorithm. qRT-PCR results indicated that COX5A, UQCRFS1, LCK, RPS2 and EIF5A exhibited consistent expression trends with sequencing data and have potential as biomarkers. Additionally, GSEA analysis revealed that the pathophysiology of POI is closely associated with inhibition of the PI3K-AKT pathway, oxidative phosphorylation and DNA damage repair, as well as activation of inflammatory and apoptotic pathways. Furthermore, we emphasize that downregulation of respiratory chain enzyme complex subunits and inhibition of oxidative phosphorylation pathways play crucial roles in the pathophysiology of POI. In conclusion, our utilization of long-read sequencing has refined the annotation information within the POI transcriptional profile. This valuable data provides novel insights for further exploration into molecular regulatory networks and potential biomarkers associated with POI.