Mitochondrial ATP Synthase Research Articles

Discovery and in vivo evaluation of novel inhibitors of the hydrolytic activity of mitochondrial F1Fo ATP synthase to alleviate myocardial ischemia reperfusion injury

Abstract Background F1Fo ATP-synthase is the mitochondrial complex responsible for ATP production. During myocardial ischemia, ATP synthase reverses its activity to hydrolyze ATP leading to energetic deficit and cardiac injury. We have previously discovered via in-silico virtual screening selective hydrolase inhibitors with pyrazole-pyridine scaffold and verified their inhibitory effect on hydrolysis on isolated murine mitochondria. Purpose In the present work we aimed to 1) synthesize novel hydrolase inhibitors, 2) select the best candidates to evaluate their effect against myocardial ischemia (I) reperfusion (R) injury in vivo and 3) investigate the underlying molecular mechanisms of cardioprotection. Methods The inhibitory effect on ATP hydrolysis for all compounds was evaluated on isolated murine heart mitochondria and at a cellular level on H9C2 cells in the presence of rotenone where their ability to maintain membrane potential was monitored. The previously described ATP hydrolase inhibitor BTB06584 (BTB) was used in vitro and in vivo as a reference and three novel compounds were selected for in vivo evaluation namely 1117, 1119 and STK-71. In the first cohort, C57Bl6 mice were randomized into 5 groups that received intravenously: 1) vehicle, 2) 1117, 3)1119, 4) STK-71 and 5) BTB. The administration was performed 10 min prior to 30 min of I and 2 hours of R and infarct size (IS) was determined. The dose of each compound was based on the effective concentration needed at a cellular level to inhibit the ATP hydrolysis. In a second cohort of mice, the best candidate and vehicle were given 10 min after the induction of I and IS was measured to test the intervention in a more translational setting. Finally, in the third cohort, the cardioprotective mechanism was investigated at the 10th min of R. Results We discovered three candidates that inhibit the ATP hydrolytic activity with comparable IC50 values to BTB. These three compounds inhibited ATP hydrolysis on the H9C2 cells at 50μΜ while BTB was inactive at the same concentration. In vivo, the three novel inhibitors reduced IS compared to the control group with STK-71 having the most remarkable cardioprotective effect while BTB did not result in attenuation of IS. STK-71 when given after the induction of I, also reduced IS indicating that its cardioprotective effect is pertained in a translational protocol. Regarding the mechanism of cardioprotection, STK-71 increased T173 phosphorylation and activation of PKA which led to the downstream activation of phospholamban. STK-71 also led to the significant activation of the RISK pathway, increased the expression of the antiapoptotic protein Bcl-Xl and reduced apoptosis. Conclusion We discovered a novel pyrazolopyridine derivative that acts as an inhibitor of ATP hydrolysis and reduces myocardial IS in vivo. Its cardioprotective mechanism involves the activation of PKA signaling and alleviation of apoptosis.

Oxidative phosphorylation rather than glycolysis is the primary energy source for sperm motility in the mussels Mytilus edulis

Broadcast-spawning marine mussels rely on high sperm motility for successful fertilization in the dynamic seawater environment. Mitochondria are typically considered the primary source of ATP generation via oxidative phosphorylation (OXPHOS); however, the ATP generation pathways of mussel sperm have not been fully characterized. To better understand the importance of both OXPHOS and glycolysis for mussel sperm function, we conducted experiments inhibiting these pathways in sperm from Mytilus edulis. Our results indicate that oligomycin, an inhibitor of the mitochondrial ATP synthase, immediately decreased sperm motility rate, velocity, and ATP content, while 2-deoxy-d-glucose, a glycolysis inhibitor, had no effect. The OXPHOS inhibitor rotenone also partially reduced sperm motility rate and velocity. Interestingly, no evidence was found for the inhibitors' effects on the content of energy-rich compounds (lipids, carbohydrates, and proteins) in the mussels' sperm, indicating only modest energy demand to fuel sperm motility. Based on these findings, we conclude that OXPHOS is the primary energy source for sperm motility in marine mussels. Our study sheds light on the intricacies of mussel sperm physiology and highlights the importance of understanding the energy requirements for successful fertilization in broadcast-spawning marine invertebrates.

Melatonin regulates mitochondrial enzymes and ascorbate–glutathione system during plant responses to drought stress through involving endogenous calcium

The study presented here was carried out to evaluate the effect of melatonin (Mel; N-acetyl-5-methoxytryptamine) on the arugula (Eruca sativa Mill.) plants grown under drought stress (DS). The results of the study show that DS significantly reduced the activity of mitochondrial ATP synthase and ATPase. Exposure of plants to DS caused a significant increase in the activity of NADPH oxidase and glycolate oxidase that resulted in the excess generation of superoxide (O2•−) and hydrogen peroxide (H2O2) radicals, respectively. Creation of oxidative stress caused substantial damage to membrane lipids, leakage of electrolytes and reduced the hydration level of the drought-stressed plants. The damaging effects of DS were probably carried out because of lower activity of ATP synthase and ATPase, weak performance of antioxidant defense system and insufficient accumulation of osmolytes. Nevertheless, supplementation of drought stressed plants with 30 µM Mel was remarkable in improving the activity of ATP synthase and ATPase with a concomitant activation of ascorbate–glutathione system, antioxidant enzymes, and accumulation of osmolytes. However, Mel inhibited the activity of NADPH oxidase and glycolate oxidase. This combined effect of events protected the plants from DS-induced impairments through reducing oxidative and osmotic stress as witnessed by a reduction in lipid peroxidation, electrolyte leakage and improvement in hydration status of the plants even under DS. On the contrary, inclusion of calcium (Ca2+) chelator overturned the positive effect of Mel on the stressed plants, that validates the involvement of endogenous Ca2+ during Mel-mediated plant adaptive responses to drought. The results of the study demonstrate that Mel involves Ca2+ in the orchestration of plant adaptive responses to DS by exerting regulatory control over the activity of mitochondrial enzymes and the function of the antioxidant defense system.

Acetate mitigates cardiac mitochondrial dysfunction in experimental model of polycystic ovarian syndrome by modulating GPCR41/43 and PROKR1

The role of short chain fatty acid, acetate in cardiac mitochondrial dysfunction especially in PCOS individuals is unknown. Therefore, the present study investigated the modulatory role of GPCRs (41 and 43) by acetate on cardiac mitochondrial status in PCOS rat model. Eight-week-old female Wistar rats were randomly allotted into four groups (n = 5). Polycystic ovarian syndrome was induced by administering letrozole (1 mg/kg p.o.) once daily for 21 days, thereafter the animals were treated with 200 mg/kg (oral gavage) of acetate for six weeks. Letrozole-induced PCOS rats showed elevated circulating testosterone and anti-mullerian hormone, with multiple ovarian cysts. In addition, these rats also manifested insulin resistance, hyperinsulinemia, and increased plasma triglyceride (TG), TG/HDLc and decreased HDLc, as well as elevated level of cardiac TG, glycogen, glycogen synthase, and plasma/cardiac NF-kB, TNF-α, and SDF-1. Cardiac MDA and caspase-6 increased, while plasma/cardiac NrF2 decreased in PCOS animals. A decrease in mitochondrial ATP synthase, ATP/AMP ratio, CPT2 and SDH, and increased HDAC2 were observed in PCOS rats with decreased level of GPCR 41 and 43 when compared with control. Immunohistochemical evaluation of cardiac tissue also showed decrease expression of PROKR1 in PCOS rats compared with control rats. However, treatment with acetate reversed these systemic, cardiac and mitochondrial anomalies. The present results suggest the therapeutic benefit of acetate, an HDAC2i against cardiac mitochondrial dysfunction in PCOS rat model, by attenuating cardiac inflammation, oxidative stress and apoptosis and these effects are accompanied by modulation of GPCR41 and 43 as well as increased expression of PROKR1.

Cationic Proteins Rich in Lysine Residue Trigger Formation of Non-bilayer Lipid Phases in Model and Biological Membranes: Biophysical Methods of Study.

Cationic membrane-active toxins are the most abundant group of proteins in the venom of snakes and insects. Cationic proteins such as cobra venom cytotoxin and bee venom melittin are known for their pharmacological reactions including anticancer and antimicrobial effects which arise from the toxin-induced alteration in the dynamics and structure of plasma membranes and membranes of organelles. It has been established that these cationic toxins trigger the formation of non-bilayer lipid phase transitions in artificial and native mitochondrial membranes. Remarkably, the toxin-induced formation of non-bilayer lipid phase increases at certain conditions mitochondrial ATP synthase activity. This observation opens an intriguing avenue for using cationic toxins in the development of novel drugs for the treatment of cellular energy deficiency caused by aging and diseases. This observation also warrants a thorough investigation of the molecular mechanism(s) of lipid phase polymorphisms triggered by cationic proteins. This article presents a review on the application of powerful biophysical methods such as resonance spectroscopy (31P-, 1H-, 2H-nuclear magnetic resonance, and electron paramagnetic resonance), luminescence, and differential scanning microcalorimetry in studies of non-bilayer lipid phase transitions triggered by cationic proteins in artificial and biological membranes. A phenomenon of the triggered by cationic proteins the non-bilayer lipid phase transitions occurring within 10-2-10-11s is discussed in the context of potential pharmacological applications of cationic proteins. Next to the ATP dimer is an inverted micelle made of cardiolipin that serves as a vehicle for the transport of H+ ions from the intra-crista space to the matrix. It is proposed that such inverted micelles are triggered by the high density of H+ ions and the cationic proteins rich in lysine residue which compete with the conserved lysine residues of the ATP synthase rotor for binding to cardiolipin in the inner mitochondrial membrane and perturb the bilayer lipid packing of cristae. Phospholipids with a blue polar head represent cardiolipin and those with a red polar head represent other phospholipids found in the crista membrane.

The pro-oncogenic protein IF1 does not contribute to the Warburg effect and is not regulated by PKA in cancer cells

The endogenous inhibitor of mitochondrial F1Fo-ATPase (ATP synthase), IF1, has been shown to exert pro-oncogenic actions, including reprogramming of cellular energy metabolism (Warburg effect). The latter action of IF1 has been reported to be hampered by its PKA-dependent phosphorylation, but both reprogramming of metabolism and PKA-dependent phosphorylation are intensely debated. To clarify these critical issues, we prepared stably IF1-silenced clones and compared their bioenergetics with that of the three parental IF1-expressing cancer cell lines. All functional parameters: respiration rate, ATP synthesis rate (OXPHOS), and mitochondrial membrane potential were similar in IF1-silenced and control cells, clearly indicating that IF1 cannot inhibit the ATP synthase in cancer cells when the enzyme works physiologically. Furthermore, all cell types exposed to PKA modulators and energized with NAD+-dependent substrates or succinate showed similar OXPHOS rate regardless of the presence or absence of IF1. Therefore, our results rule out that IF1 action is modulated by its PKA-dependent phosphorylated/dephosphorylated state. Notably, cells exposed to a negative PKA modulator and energized with NAD+-dependent substrates showed a significant decrease of the OXPHOS rate matching previously reported inactivation of complex I. Overall, this study definitively demonstrates that IF1 inhibits neither mitochondrial ATP synthase nor OXPHOS in normoxic cancer cells and does not contribute to the Warburg effect. Thus, currently the protection of cancer cells from severe hypoxia/anoxia and apoptosis remain the only unquestionable actions of IF1 as pro-oncogenic factor that may be exploited to develop therapeutic approaches.

A Homozygous MAN2B1 Missense Mutation in a Doberman Pinscher Dog with Neurodegeneration, Cytoplasmic Vacuoles, Autofluorescent Storage Granules, and an α-Mannosidase Deficiency.

A 7-month-old Doberman Pinscher dog presented with progressive neurological signs and brain atrophy suggestive of a hereditary neurodegenerative disorder. The dog was euthanized due to the progression of disease signs. Microscopic examination of tissues collected at the time of euthanasia revealed massive accumulations of vacuolar inclusions in cells throughout the central nervous system, suggestive of a lysosomal storage disorder. A whole genome sequence generated with DNA from the affected dog contained a likely causal, homozygous missense variant in MAN2B1 that predicted an Asp104Gly amino acid substitution that was unique among whole genome sequences from over 4000 dogs. A lack of detectable α-mannosidase enzyme activity confirmed a diagnosis of a-mannosidosis. In addition to the vacuolar inclusions characteristic of α-mannosidosis, the dog exhibited accumulations of autofluorescent intracellular inclusions in some of the same tissues. The autofluorescence was similar to that which occurs in a group of lysosomal storage disorders called neuronal ceroid lipofuscinoses (NCLs). As in many of the NCLs, some of the storage bodies immunostained strongly for mitochondrial ATP synthase subunit c protein. This protein is not a substrate for α-mannosidase, so its accumulation and the development of storage body autofluorescence were likely due to a generalized impairment of lysosomal function secondary to the accumulation of α-mannosidase substrates. Thus, it appears that storage body autofluorescence and subunit c accumulation are not unique to the NCLs. Consistent with generalized lysosomal impairment, the affected dog exhibited accumulations of intracellular inclusions with varied and complex ultrastructural features characteristic of autophagolysosomes. Impaired autophagic flux may be a general feature of this class of disorders that contributes to disease pathology and could be a target for therapeutic intervention. In addition to storage body accumulation, glial activation indicative of neuroinflammation was observed in the brain and spinal cord of the proband.

ATP yield of plant respiration: potential, actual and unknown.

The ATP yield of plant respiration (ATP/hexose unit respired) quantitatively links active heterotrophic processes with substrate consumption. Despite its importance, plant respiratory ATP yield is uncertain. The aim here was to integrate current knowledge of cellular mechanisms with inferences required to fill knowledge gaps to generate a contemporary estimate of respiratory ATP yield and identify important unknowns. A numerical balance sheet model combining respiratory carbon metabolism and electron transport pathways with uses of the resulting transmembrane electrochemical proton gradient was created and parameterized for healthy, non-photosynthesizing plant cells catabolizing sucrose or starch to produce cytosolic ATP. Mechanistically, the number of c subunits in the mitochondrial ATP synthase Fo sector c-ring, which is unquantified in plants, affects ATP yield. A value of 10 was (justifiably) used in the model, in which case respiration of sucrose potentially yields about 27.5 ATP/hexose (0.5 ATP/hexose more from starch). Actual ATP yield often will be smaller than its potential due to bypasses of energy-conserving reactions in the respiratory chain, even in unstressed plants. Notably, all else being optimal, if 25 % of respiratory O2 uptake is via the alternative oxidase - a typically observed fraction - ATP yield falls 15 % below its potential. Plant respiratory ATP yield is smaller than often assumed (certainly less than older textbook values of 36-38 ATP/hexose) leading to underestimation of active-process substrate requirements. This hinders understanding of ecological/evolutionary trade-offs between competing active processes and assessments of crop growth gains possible through bioengineering of processes that consume ATP. Determining the plant mitochondrial ATP synthase c-ring size, the degree of any minimally required (useful) bypasses of energy-conserving reactions in the respiratory chain, and the magnitude of any 'leaks' in the inner mitochondrial membrane are key research needs.

What Are the Implications of Cr(III) Serving as an Inhibitor of the Beta Subunit of Mitochondrial ATP Synthase?

A recent report has shown the active site of the beta subunit of mitochondrial ATP synthase is probably the site of action of Cr(III) action, independent of the insulin signaling pathway. This works appears to answer an important question about the mode of action of Cr(III) at a molecular level when supplied in supra-nutritional levels to rodents. However, as with any good research, the research also raises several questions. The relationship between this study and the results of rodent studies of chromium supplementation and between this study and the current understanding the chromium(III) transport and detoxification system are put into perspective.

IF1 promotes oligomeric assemblies of sluggish ATP synthase and outlines the heterogeneity of the mitochondrial membrane potential

The coexistence of two pools of ATP synthase in mitochondria has been largely neglected despite in vitro indications for the existence of reversible active/inactive state transitions in the F1-domain of the enzyme. Herein, using cells and mitochondria from mouse tissues, we demonstrate the existence in vivo of two pools of ATP synthase: one active, the other IF1-bound inactive. IF1 is required for oligomerization and inactivation of ATP synthase and for proper cristae formation. Immunoelectron microscopy shows the co-distribution of IF1 and ATP synthase, placing the inactive “sluggish” ATP synthase preferentially at cristae tips. The intramitochondrial distribution of IF1 correlates with cristae microdomains of high membrane potential, partially explaining its heterogeneous distribution. These findings support that IF1 is the in vivo regulator of the active/inactive state transitions of the ATP synthase and suggest that local regulation of IF1-ATP synthase interactions is essential to activate the sluggish ATP synthase.

Comparative Clustering (CompaCt) of eukaryote complexomes identifies novel interactions and sheds light on protein complex evolution.

Complexome profiling allows large-scale, untargeted, and comprehensive characterization of protein complexes in a biological sample using a combined approach of separating intact protein complexes e.g., by native gel electrophoresis, followed by mass spectrometric analysis of the proteins in the resulting fractions. Over the last decade, its application has resulted in a large collection of complexome profiling datasets. While computational methods have been developed for the analysis of individual datasets, methods for large-scale comparative analysis of complexomes from multiple species are lacking. Here, we present Comparative Clustering (CompaCt), that performs fully automated integrative analysis of complexome profiling data from multiple species, enabling systematic characterization and comparison of complexomes. CompaCt implements a novel method for leveraging orthology in comparative analysis to allow systematic identification of conserved as well as taxon-specific elements of the analyzed complexomes. We applied this method to a collection of 53 complexome profiles spanning the major branches of the eukaryotes. We demonstrate the ability of CompaCt to robustly identify the composition of protein complexes, and show that integrated analysis of multiple datasets improves characterization of complexes from specific complexome profiles when compared to separate analyses. We identified novel candidate interactors and complexes in a number of species from previously analyzed datasets, like the emp24, the V-ATPase and mitochondrial ATP synthase complexes. Lastly, we demonstrate the utility of CompaCt for the automated large-scale characterization of the complexome of the mosquito Anopheles stephensi shedding light on the evolution of metazoan protein complexes. CompaCt is available from https://github.com/cmbi/compact-bio.

Performance benchmarking microplate-immunoassays for quantifying target-specific cysteine oxidation reveals their potential for understanding redox-regulation and oxidative stress.

The antibody-linked oxi-state assay (ALISA) for quantifying target-specific cysteine oxidation can benefit specialist and non-specialist users. Specialists can benefit from time-efficient analysis and high-throughput target and/or sample n-plex capacities. The simple and accessible "off-the-shelf" nature of ALISA brings the benefits of oxidative damage assays to non-specialists studying redox-regulation. Until performance benchmarking establishes confidence in the "unseen" microplate results, ALISA is unlikely to be widely adopted. Here, we implemented pre-set pass/fail criteria to benchmark ALISA by robustly evaluating immunoassay performance in diverse biological contexts. ELISA-mode ALISA assays were accurate, reliable, and sensitive. For example, the average inter-assay CV for detecting 20%- and 40%-oxidised PRDX2 or GAPDH standards was 4.6% (range: 3.6-7.4%). ALISA displayed target-specificity. Immunodepleting the target decreased the signal by ∼75%. Single-antibody formatted ALISA failed to quantify the matrix-facing alpha subunit of the mitochondrial ATP synthase. However, RedoxiFluor quantified the alpha subunit displaying exceptional performance in the single-antibody format. ALISA discovered that (1) monocyte-to-macrophage differentiation amplified PRDX2-specific cysteine oxidation in THP-1cells and (2) exercise increased GAPDH-specific cysteine oxidation in human erythrocytes. The "unseen" microplate data were "seen-to-be-believed" via orthogonal visually displayed immunoassays like the dimer method. Finally, we established target (n=3) and sample (n=100) n-plex capacities in ∼4h with 50-70min hands-on time. Our work showcases the potential of ALISA to advance our understanding of redox-regulation and oxidative stress.

The Mitochondrial ATP Synthase/IF1 Axis in Cancer Progression: Targets for Therapeutic Intervention.

Cancer poses a significant global health problem with profound personal and economic implications on National Health Care Systems. The reprograming of metabolism is a major trait of the cancer phenotype with a clear potential for developing effective therapeutic strategies to combat the disease. Herein, we summarize the relevant role that the mitochondrial ATP synthase and its physiological inhibitor, ATPase Inhibitory Factor 1 (IF1), play in metabolic reprogramming to an enhanced glycolytic phenotype. We stress that the interplay in the ATP synthase/IF1 axis has additional functional roles in signaling mitohormetic programs, pro-oncogenic or anti-metastatic phenotypes depending on the cell type. Moreover, the same axis also participates in cell death resistance of cancer cells by restrained mitochondrial permeability transition pore opening. We emphasize the relevance of the different post-transcriptional mechanisms that regulate the specific expression and activity of ATP synthase/IF1, to stimulate further investigations in the field because of their potential as future targets to treat cancer. In addition, we review recent findings stressing that mitochondria metabolism is the primary altered target in lung adenocarcinomas and that the ATP synthase/IF1 axis of OXPHOS is included in the most significant signature of metastatic disease. Finally, we stress that targeting mitochondrial OXPHOS in pre-clinical mouse models affords a most effective therapeutic strategy in cancer treatment.

IF1 ablation prevents ATP synthase oligomerization, enhances mitochondrial ATP turnover and promotes an adenosine-mediated pro-inflammatory phenotype

ATPase Inhibitory Factor 1 (IF1) regulates the activity of mitochondrial ATP synthase. The expression of IF1 in differentiated human and mouse cells is highly variable. In intestinal cells, the overexpression of IF1 protects against colon inflammation. Herein, we have developed a conditional IF1-knockout mouse model in intestinal epithelium to investigate the role of IF1 in mitochondrial function and tissue homeostasis. The results show that IF1-ablated mice have increased ATP synthase/hydrolase activities, leading to profound mitochondrial dysfunction and a pro-inflammatory phenotype that impairs the permeability of the intestinal barrier compromising mouse survival upon inflammation. Deletion of IF1 prevents the formation of oligomeric assemblies of ATP synthase and alters cristae structure and the electron transport chain. Moreover, lack of IF1 promotes an intramitochondrial Ca2+ overload in vivo, minimizing the threshold to Ca2+-induced permeability transition (mPT). Removal of IF1 in cell lines also prevents the formation of oligomeric assemblies of ATP synthase, minimizing the threshold to Ca2+-induced mPT. Metabolomic analyses of mice serum and colon tissue highlight that IF1 ablation promotes the activation of de novo purine and salvage pathways. Mechanistically, lack of IF1 in cell lines increases ATP synthase/hydrolase activities and installs futile ATP hydrolysis in mitochondria, resulting in the activation of purine metabolism and in the accumulation of adenosine, both in culture medium and in mice serum. Adenosine, through ADORA2B receptors, promotes an autoimmune phenotype in mice, stressing the role of the IF1/ATP synthase axis in tissue immune responses. Overall, the results highlight that IF1 is required for ATP synthase oligomerization and that it acts as a brake to prevent ATP hydrolysis under in vivo phosphorylating conditions in intestinal cells.

Aurora kinase A/AURKA functionally interacts with the mitochondrial ATP synthase to regulate energy metabolism and cell death

Cancer cells often hijack metabolic pathways to obtain the energy required to sustain their proliferation. Understanding the molecular mechanisms underlying cancer cell metabolism is key to fine-tune the metabolic preference of specific tumors, and potentially offer new therapeutic strategies. Here, we show that the pharmacological inhibition of mitochondrial Complex V delays the cell cycle by arresting breast cancer cell models in the G0/G1 phase. Under these conditions, the abundance of the multifunctional protein Aurora kinase A/AURKA is specifically lowered. We then demonstrate that AURKA functionally interacts with the mitochondrial Complex V core subunits ATP5F1A and ATP5F1B. Altering the AURKA/ATP5F1A/ATP5F1B nexus is sufficient to trigger G0/G1 arrest, and this is accompanied by decreased glycolysis and mitochondrial respiration rates. Last, we discover that the roles of the AURKA/ATP5F1A/ATP5F1B nexus depend on the specific metabolic propensity of triple-negative breast cancer cell lines, where they correlate with cell fate. On one hand, the nexus induces G0/G1 arrest in cells relying on oxidative phosphorylation as the main source of energy. On the other hand, it allows to bypass cell cycle arrest and it triggers cell death in cells with a glycolytic metabolism. Altogether, we provide evidence that AURKA and mitochondrial Complex V subunits cooperate to maintain cell metabolism in breast cancer cells. Our work paves the way to novel anti-cancer therapies targeting the AURKA/ATP5F1A/ATP5F1B nexus to lower cancer cell metabolism and proliferation.

P-372 RNA sequencing studies evaluating oocyte quality in women with endometriosis: a meta-analysis

Abstract Study question Can a network-based integrative analysis (NBIA) meta-analysis approach be used to identify clinically relevant cumulus-oocyte complex biological pathway and gene targets in women with endometriosis? Summary answer Bi-level meta-analysis (BLMA) of multiple RNAseq studies shows enrichment of both inflammatory and mitochondrial pathway-related differential gene expression within oocyte, granulosa and cumulus cells. What is known already Endometriosis-related infertility is multifactorial, and studies suggest that impaired oocyte quality plays a significant role. The close relationship between granulosa, cumulus cells and developing oocytes makes them ideal candidates for studying non-invasive biomarkers for oocyte quality. As high-throughput sequencing becomes more accessible, the volume of RNAseq molecular data in public databases such as Gene Expression Omnibus (GEO) has increased. Combined analysis of multiple datasets theoretically increases study power due to larger number of samples. Comparison of differentially expressed genes (DEGs) and biological pathways across multiple independent studies therefore requires a ‘meta-analysis’ framework that can effectively integrate data from different sources. Study design, size, duration We identified relevant RNAseq datasets through NCBI public functional genomics data repository Gene Expression Omnibus (GEO). Datasets were split into “test” and “control” groups referring to models of oocyte incompetence and competence. Accession numbers GSEXXX or PRJNXXX represent unique identifiers for RNAseq data files contained within a given NCBI GEO series or BioProject record, respectively. Five human RNAseq datasets (PRJNA514416, GSE168214, PRJNA216966, PRJNA727838 and GSE81579) were identified and DEG lists generated for each individual dataset. Participants/materials, setting, methods Normalisation and differential expression between RNAseq datasets was carried out in R using the Bioconductor DESeq2 package. DEGs were identified as those whose P-value was adjusted using a false discovery rate less than 0.5, adjusted using Benjamini-Hochberg procedure. Log2 fold-change ratio (logFC) of each DEG was used to identify significantly up and downregulated genes. The NBIA pipeline (Nguyen et al, 2020) examines over-representation of significantly impacted genes in a pathway as per KEGG pathway analysis. Main results and the role of chance Upregulated DEGs were identified in mitochondrial membrane ATP synthase pathways, (ATP5G1, ATP5G2, ATP5J2 and ATP5H). KEGG Pathway terms “Parkinson’s”, “Huntington’s”, and “Alzheimer disease” were also enriched, due to many genes partaking in oxidative phosphorylation in the context of these pathologies. Functional analysis also revealed significantly down-regulated genes related to inflammatory pathways (CXCL1, 2, 3, 5,6 and 8), which have a role in innate immunity. Although surprising, in our analysis, downregulated functions based on KEGG functional pathway analysis, observed that innate immune response, cellular defense response, chemokine-mediated signaling were significantly downregulated. BLMA over-representation analysis (ORA) and impact analysis (IA) analysis showed that the Amoebiasis pathway was enriched expressed across all datasets. Genes involved in this pathway include interleukin (IL), CXCL1, CXCL2, CXCL3, SERPINB, TGFB1 and TGFB2, and its biological process is related to natural killer (NK) cell mediated cytotoxicity. Overall, the enrichment of immune and inflammation-related pathways revealed by BLMA meta-analysis underscores the relevance of inflammation to follicular function. Indeed, ovulation is inextricably linked with inflammation. Other significantly enriched pathways highlighted by this meta-analysis are related to infection (path:hsa05134 Legionellosis; path:hsa05140 Leishmaniasis) immune response (path:hsa04145 Phagosome; path:hsa04610 Complement and coagulation cascades) and inflammation (path:hsa05323 Rheumatoid arthritis). Limitations, reasons for caution Methods used availed of publicly available GEO datasets and bioinformatics software (Bioconductor, R and DAVID). Although useful tools, these approaches are open to inter-user variation. Cell types compared across studies were oocytes, granulosa and cumulus cells. Controlled ovarian stimulation may also be open to influence based on stimulation protocol used. Wider implications of the findings Enrichment of inflammatory and mitochondrial pathways in our GO analysis is compelling. Given the role of mitochondria in oocyte developmental competence, this reinforces the role of impaired oocyte competence in women with endometriosis. Our study identifies several genes that require further elucidation as potential predictors of oocyte quality in endometriosis. Trial registration number N/A

574-P: A Ketogenic Diet Improves Aerobic Capacity in Hyperglycemic Mice by Sex-Dependent Mechanisms

Hyperglycemia is associated with lower aerobic exercise capacity (VO2peak) in humans and rodent models. We tested whether reducing blood glucose levels with a dietary intervention can restore VO2peak in hyperglycemic mice. Male and female CD-1 mice were injected with low dose streptozotocin (STZ) to induce moderate hyperglycemia (>200 mg/dL). Hyperglycemic mice were randomized to receive a standard diet (STZ-CHOW) or a ketogenic diet (STZ-KETO). KETO significantly reduced blood glucose in STZ-treated mice. Next, both groups underwent similar volumes of exercise training by voluntary wheel running, or remained sedentary for 8 wks. In males, VO2peak during maximal exercise testing was similar in sedentary mice, regardless of diet. However, with training, VO2peak was significantly improved in STZ-KETO, but not STZ-CHOW males (see Figure). In females, KETO significantly increased VO2peak in sedentary mice, with no additional increase in response to training. Improved VO2peak with KETO in females was associated with a 50% increase in skeletal muscle mitochondrial ATP synthase protein. In contrast, ATP synthase was not regulated by diet in males. Our data demonstrate that a ketogenic diet can improve VO2peak in mice with hyperglycemia. In males, improved VO2peak with KETO was exercise-dependent, while in females, the beneficial effects of KETO on VO2peak and mitochondrial content were exercise-independent. Disclosure P.Pattamaprapanont: None. M.Formato: None. R.Nogueira soares: None. S.J.Lessard: None.

The Ancestral Shape of the Access Proton Path of Mitochondrial ATP Synthases Revealed by a Split Subunit-a.

The passage of protons across membranes through F1Fo-ATP synthases spins their rotors and drives the synthesis of ATP. While the principle of torque generation by proton transfer is known, the mechanisms and routes of proton access and release and their evolution are not fully understood. Here, we show that the entry site and path of protons in the lumenal half channel of mitochondrial ATP synthases are largely defined by a short N-terminal α-helix of subunit-a. In Trypanosoma brucei and other Euglenozoa, the α-helix is part of another polypeptide chain that is a product of subunit-a gene fragmentation. This α-helix and other elements forming the proton pathway are widely conserved across eukaryotes and in Alphaproteobacteria, the closest extant relatives of mitochondria, but not in other bacteria. The α-helix blocks one of two proton routes found in Escherichia coli, resulting in a single proton entry site in mitochondrial and alphaproteobacterial ATP synthases. Thus, the shape of the access half channel predates eukaryotes and originated in the lineage from which mitochondria evolved by endosymbiosis.

PHB3 Is Required for the Assembly and Activity of Mitochondrial ATP Synthase in Arabidopsis.

Mitochondrial ATP synthase is a multiprotein complex, which consists of a matrix-localized F1 domain (F1-ATPase) and an inner membrane-embedded Fo domain (Fo-ATPase). The assembly process of mitochondrial ATP synthase is complex and requires the function of many assembly factors. Although extensive studies on mitochondrial ATP synthase assembly have been conducted on yeast, much less study has been performed on plants. Here, we revealed the function of Arabidopsis prohibitin 3 (PHB3) in mitochondrial ATP synthase assembly by characterizing the phb3 mutant. The blue native PAGE (BN-PAGE) and in-gel activity staining assays showed that the activities of ATP synthase and F1-ATPase were significantly decreased in the phb3 mutant. The absence of PHB3 resulted in the accumulation of the Fo-ATPase and F1-ATPase intermediates, whereas the abundance of the Fo-ATPase subunit a was decreased in the ATP synthase monomer. Furthermore, we showed that PHB3 could interact with the F1-ATPase subunits β and δ in the yeast two-hybrid system (Y2H) and luciferase complementation imaging (LCI) assay and with Fo-ATPase subunit c in the LCI assay. These results indicate that PHB3 acts as an assembly factor required for the assembly and activity of mitochondrial ATP synthase.

The ABI5-dependent downregulation of mitochondrial ATP synthase OSCP subunit facilitates apple necrotic mosaic virus infection.

Apple necrotic mosaic virus (ApNMV) is significantly associated with apple mosaic disease in China. However, the mechanisms of ApNMV infection, as well as host defense against the virus, are still poorly understood. Mitochondrial ATP synthase plays a fundamental role in the regulation of plant growth and development. However, the mechanism of mitochondrial ATP synthase in response to virus infection remains to be defined. In the present study, a yeast two-hybrid (Y2H) screening revealed that the apple mitochondrial ATP synthase OSCP subunit (MdATPO) interacts with ApNMV coat protein (CP). It was further verified that overexpression of MdATPO in Nicotiana benthamiana inhibited viral accumulation. In contrast, silencing of NbATPO facilitated viral accumulation, indicating that ATPO plays a defensive role during ApNMV infection. Further investigation demonstrated that ApNMV infection accelerated abscisic acid (ABA) accumulation, and ABA negatively regulated ATPO transcription, which was related to the capacity of ABA insensitive 5 (ABI5) to bind to the ABA-responsive elements (ABREs) of the ATPO promoter. Altogether, our results indicated that transcription factor ABI5 negatively regulated ATPO transcription by directly binding its promoter, leading to the sensitivity of apple and N. benthamiana to ApNMV infection. The current presentation facilitates our comprehensive understanding of the intricate responses of host to ApNMV.