Mitochondrial ATP Synthase Complex Research Articles

A histidine-rich extension of the mitochondrial F0 subunit ATP6 from the ice worm Mesenchytraeus solifugus increases ATP synthase activity in bacteria.

Bioenergetic profiles of psychrophiles across domains of life are unusual in that intracellular ATP levels increase with declining temperature. Whole-transcriptome sequencing of the glacier ice worm Mesenchytraeus solifugus revealed a unique C-terminal extension on the ATP6 protein, which forms part of the proton pore of mitochondrial ATP synthase (Complex V). This extension, positioned near the proton exit pore, comprises alternating histidine residues thought to increase proton flux through Complex V leading to elevated ATP synthesis. To test this hypothesis, we fused the M. solifugus C-terminal extension to Escherichia coli AtpB (the ATP6 orthologue) and observed a ~ 5-fold increase in ATP synthesis. This enhancement was unidirectional as we observed no change to ATP hydrolysis rates. These findings offer an avenue for identifying critical factors associated with ice worm adaptation.

IPF-related new macrophage subpopulations and diagnostic biomarker identification - combine machine learning with single-cell analysis

sIdiopathic pulmonary fibrosis (IPF) is a chronic disease of unknown etiology that lacks a specific treatment. In IPF, macrophages play a key regulatory role as a major component of the lung immune system, especially during inflammation and fibrosis. However, our understanding of the cellular heterogeneity and molecular characterization of macrophages in IPF, as well as their relevance in the clinical setting, is relatively limited. In this study, we analyzed in-depth single-cell transcriptome sequencing (scRNA-seq) data from lung tissues of IPF patients, identified macrophage subpopulations in IPF, and probed their molecular characteristics and biological functions. hdWGCNA identified co-expressed gene modules of a subpopulation of IPF-associated macrophages (IPF-MΦ), and probed the IPF-MΦ by a machine-learning approach. hdWGCNA identified a subpopulation of IPF-associated macrophage subpopulations and probed the IPF-MΦ signature gene (IRMG) for its prognostic value, and a prediction model was developed on this basis. In addition, IPF-MΦ was obtained after recluster analysis of macrophages in IPF lung tissues. Coexpressed gene modules of IPF-MΦ were identified by hdWGCNA. Then, a machine learning approach was utilized to reveal the characteristic genes of IPF-MΦ, and a prediction model was built on this basis. In addition, we discovered a type of macrophage unique to IPF lung tissue named ATP5-MΦ. Its characteristic gene encodes a subunit of the mitochondrial ATP synthase complex, which is closely related to oxidative phosphorylation and proton transmembrane transport, suggesting that ATP5-MΦ may have higher ATP synthesis capacity in IPF lung tissue. This study provides new insights into the pathogenesis of IPF and provides a basis for evaluating disease prognosis and predictive medicine in IPF patients.Graphical

Variants in Human ATP Synthase Mitochondrial Genes: Biochemical Dysfunctions, Associated Diseases, and Therapies.

Mitochondrial ATP synthase (Complex V) catalyzes the last step of oxidative phosphorylation and provides most of the energy (ATP) required by human cells. The mitochondrial genes MT-ATP6 and MT-ATP8 encode two subunits of the multi-subunit Complex V. Since the discovery of the first MT-ATP6 variant in the year 1990 as the cause of Neuropathy, Ataxia, and Retinitis Pigmentosa (NARP) syndrome, a large and continuously increasing number of inborn variants in the MT-ATP6 and MT-ATP8 genes have been identified as pathogenic. Variants in these genes correlate with various clinical phenotypes, which include several neurodegenerative and multisystemic disorders. In the present review, we report the pathogenic variants in mitochondrial ATP synthase genes and highlight the molecular mechanisms underlying ATP synthase deficiency that promote biochemical dysfunctions. We discuss the possible structural changes induced by the most common variants found in patients by considering the recent cryo-electron microscopy structure of human ATP synthase. Finally, we provide the state-of-the-art of all therapeutic proposals reported in the literature, including drug interventions targeting mitochondrial dysfunctions, allotopic gene expression- and nuclease-based strategies, and discuss their potential translation into clinical trials.

Abstract 278: Role of ATPIF1 in glioblastoma cell survival and metabolism

Abstract The ATPase inhibitory factor 1 (ATPIF1) is a 12 kDa protein that inhibits the mitochondrial F1Fo-ATP synthase in the F1domain. It reverses the mitochondrial ATP synthase complex's function, contributing to the onset of oncogenesis. The overexpression of ATPIF1 in various cancers has recently attracted greater attention; however, its biological significance in human pathology, specifically cancer, remains unknown. Based on immunohistochemistry and Western blot analysis of glioblastoma (GBM) patient specimens, we found ATPIF1 is highly expressed in GBMs. Here we showed that mitochondrial fractions of silencing ATPIF1 in U-87 and U251 cells showed decreased expression of ATPIF1, ATP5A, and ATPG1-G2-G3. Next, we showed that manipulation of ATPIF1 showed increased Reactive Oxygen Species (ROS) and Caspase3/7 activity levels compared to untreated cells. ATPIF1 knockdown further caused cell cycle arrest at the G₀/G1stage, and FITC Annexin V analysis showed increased apoptosis. Increased glucose uptake is associated with aggressive oncological behavior, and here we found elevated levels of TCA metabolites in ATPIF1 knockdown cells. ATPIF1 silenced cells had a shift in extracellular acidification rate, accompanied by a simultaneous increase in proton leak, according to the metabolic analysis performed by Seahorse bio-analyzer. Overall, these observations suggest ATPIF1 plays a key role in the progression of GBM. However, further research is needed to verify the precise mechanisms by which ATPIF1 functions in GBM. Citation Format: Maheedhara Reddy Guda, Nelli Mnatsakanyan, Daniel Morris, Swapna Asuthkar, Andrew J. Tsung, Kiran Velpula. Role of ATPIF1 in glioblastoma cell survival and metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 278.

Mitochondrial ATP Synthase and Mild Uncoupling by Butyl Ester of Rhodamine 19, C4R1.

The homeostasis of the transmembrane potential of hydrogen ions in mitochondria is a prerequisite for the normal mitochondrial functioning. However, in different pathological conditions it is advisable to slightly reduce the membrane potential, while maintaining it at levels sufficient to produce ATP that will ensure the normal functioning of the cell. A number of chemical agents have been found to provide mild uncoupling; however, natural proteins residing in mitochondrial membrane can carry this mission, such as proteins from the UCP family, an adenine nucleotide translocator and a dicarboxylate carrier. In this study, we demonstrated that the butyl ester of rhodamine 19, C4R1, binds to the components of the mitochondrial ATP synthase complex due to electrostatic interaction and has a good uncoupling effect. The more hydrophobic derivative C12R1 binds poorly to mitochondria with less uncoupling activity. Mass spectrometry confirmed that C4R1 binds to the β-subunit of mitochondrial ATP synthase and based on molecular docking, a C4R1 binding model was constructed suggesting the binding site on the interface between the α- and β-subunits, close to the anionic amino acid residues of the β-subunit. The association of the uncoupling effect with binding suggests that the ATP synthase complex can provide induced uncoupling.

Genome-Wide Analysis of lncRNA and mRNA Expression in the Uterus of Laying Hens during Aging.

Eggshell plays an essential role in preventing physical damage and microbial invasions. Therefore, the analysis of genetic regulatory mechanisms of eggshell quality deterioration during aging in laying hens is important for the biosecurity and economic performance of poultry egg production worldwide. This study aimed to compare the differences in the expression profiles of long non-coding RNAs (lncRNAs) and mRNAs between old and young laying hens by the method of high-throughput RNA sequencing to identify candidate genes associated with aging in the uterus of laying hens. Overall, we detected 176 and 383 differentially expressed (DE) lncRNAs and mRNAs, respectively. Moreover, functional annotation analysis based on the Gene Ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) databases revealed that DE-lncRNAs and DE-mRNAs were significantly enriched in "phosphate-containing compound metabolic process", "mitochondrial proton-transporting ATP synthase complex", "inorganic anion transport", and other terms related to eggshell calcification and cuticularization. Through integrated analysis, we found that some important genes such as FGF14, COL25A1, GPX8, and GRXCR1 and their corresponding lncRNAs were expressed differentially between two groups, and the results of quantitative real-time polymerase chain reaction (qPCR) among these genes were also in excellent agreement with the sequencing data. In addition, our study found that TCONS_00181492, TCONS_03234147, and TCONS_03123639 in the uterus of laying hens caused deterioration of eggshell quality in the late laying period by up-regulating their corresponding target genes FGF14, COL25A1, and GRXCR1 as well as down-regulating the target gene GPX8 by TCONS_01464392. Our findings will provide a valuable reference for the development of breeding programs aimed at breeding excellent poultry with high eggshell quality or regulating dietary nutrient levels to improve eggshell quality.

Proteomic signatures of infiltrative gastric cancer by proteomic and bioinformatic analysis.

Proteomic signatures of Ming's infiltrative gastric cancer (IGC) remain unknown. To elucidate the molecular characteristics of IGC at the proteomics level. Twelve pairs of IGC and adjacent normal tissues were collected and their proteomes were analyzed by high performance liquid chromatography tandem mass spectrometry. The identified peptides were sequenced de novo and matched against the SwissProt database using Maxquant software. The differentially expressed proteins (DEPs) were screened using |log2(Fold change)| > 1 and P-adj < 0.01 as the thresholds. The expression levels of selected proteins were verified by Western blotting. The interaction network of the DEPs was constructed with the STRING database and visualized using Cytoscape with cytoHubba software. The DEPs were functionally annotated using clusterProfiler, STRING and DAVID for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. P < 0.05 was considered statistically significant. A total of 7361 DEPs were identified, of which 94 were significantly up-regulated and 223 were significantly down-regulated in IGC relative to normal gastric tissues. The top 10 up-regulated proteins were MRTO4, BOP1, PES1, WDR12, BRIX1, NOP2, POLR1C, NOC2L, MYBBP1A and TSR1, and the top 10 down-regulated proteins were NDUFS8, NDUFS6, NDUFA8, NDUFA5, NDUFC2, NDUFB8, NDUFB5, NDUFB9, UQCRC2 and UQCRC1. The up-regulated proteins were enriched for 9 biological processes including DNA replication, ribosome biogenesis and initiation of DNA replication, and the cellular component MCM complex. Among the down-regulated proteins, 17 biological processes were enriched, including glucose metabolism, pyruvic acid metabolism and fatty acid β-oxidation. In addition, the mitochondrial inner membrane, mitochondrial matrix and mitochondrial proton transport ATP synthase complex were among the 6 enriched cellular components, and 11 molecular functions including reduced nicotinamide adenine dinucleotide dehydrogenase activity, acyl-CoA dehydrogenase activity and nicotinamide adenine dinucleotide binding were also enriched. The significant KEGG pathways for the up-regulated proteins were DNA replication, cell cycle and mismatch repair, whereas 18 pathways including oxidative phosphorylation, fatty acid degradation and phenylalanine metabolism were significantly enriched among the down-regulated proteins. The proteins involved in cell cycle regulation, DNA replication and mismatch repair, and metabolism were significantly altered in IGC, and the proteomic profile may enable the discovery of novel biomarkers.

The Marine-Derived Macrolactone Mandelalide A Is an Indirect Activator of AMPK.

The mandelalides are complex macrolactone natural products with distinct macrocycle motifs and a bioactivity profile that is heavily influenced by compound glycosylation. Mandelalides A and B are direct inhibitors of mitochondrial ATP synthase (complex V) and therefore more toxic to mammalian cells with an oxidative metabolic phenotype. To provide further insight into the pharmacology of the mandelalides, we studied the AMP-activated protein kinase (AMPK) energy stress pathway and report that mandelalide A is an indirect activator of AMPK. Wild-type mouse embryonic fibroblasts (MEFs) and representative human non-small cell lung cancer (NSCLC) cells showed statistically significant increases in phospho-AMPK (Thr172) and phospho-ACC (Ser79) in response to mandelalide A. Mandelalide L, which also harbors an A-type macrocycle, induced similar increases in phospho-AMPK (Thr172) and phospho-ACC (Ser79) in U87-MG glioblastoma cells. In contrast, MEFs co-treated with an AMPK inhibitor (dorsomorphin), AMPKα-null MEFs, or NSCLC cells lacking liver kinase B1 (LKB1) lacked this activity. Mandelalide A was significantly more cytotoxic to AMPKα-null MEFs than wild-type cells, suggesting that AMPK activation serves as a protective response to mandelalide-induced depletion of cellular ATP. However, LKB1 status alone was not predictive of the antiproliferative effects of mandelalide A against NSCLC cells. When EGFR status was considered, erlotinib and mandelalide A showed strong cytotoxic synergy in combination against erlotinib-resistant 11-18 NSCLC cells but not against erlotinib-sensitive PC-9 cells. Finally, prolonged exposures rendered mandelalide A, a potent and efficacious cytotoxin, against a panel of human glioblastoma cell types regardless of the underlying metabolic phenotype of the cell. These results add biological relevance to the mandelalide series and provide the basis for their further pre-clinical evaluation as ATP synthase inhibitors and secondary activators of AMPK.

The chimeric gene atp6c confers cytoplasmic male sterility in maize by impairing the assembly of the mitochondrial ATP synthase complex

Cytoplasmic male sterility (CMS) is a powerful tool for the exploitation of hybrid heterosis and the study of signaling and interactions between the nucleus and the cytoplasm. C-type CMS (CMS-C) in maize has long been used in hybrid seed production, but the underlying sterility factor and its mechanism of action remain unclear. In this study, we demonstrate that the mitochondrial gene atp6c confers male sterility in CMS-C maize. The ATP6C protein shows stronger interactions with ATP8 and ATP9 than ATP6 during the assembly of F1Fo-ATP synthase (F-type ATP synthase, ATPase), thereby reducing the quantity and activity of assembled F1Fo-ATP synthase. By contrast, the quantity and activity of the F1' component are increased in CMS-C lines. Reduced F1Fo-ATP synthase activity causes accumulation of excess protons in the inner membrane space of the mitochondria, triggering a burst of reactive oxygen species (ROS), premature programmed cell death of the tapetal cells, and pollen abortion. Collectively, our study identifies a chimeric mitochondrial gene (ATP6C) that causes CMS in maize and documents the contribution of ATP6C to F1Fo-ATP synthase assembly, thereby providing novel insights into the molecular mechanisms of male sterility in plants.

T1121G Point Mutation in the Mitochondrial Gene COX1 Suppresses a Null Mutation in ATP23 Required for the Assembly of Yeast Mitochondrial ATP Synthase.

Nuclear-encoded Atp23 was previously shown to have dual functions, including processing the yeast Atp6 precursor and assisting the assembly of yeast mitochondrial ATP synthase. However, it remains unknown whether there are genes functionally complementary to ATP23 to rescue atp23 null mutant. In the present paper, we screen and characterize three revertants of atp23 null mutant and reveal a T1121G point mutation in the mitochondrial gene COX1 coding sequence, which leads to Val374Gly mutation in Cox1, the suppressor in the revertants. This was verified further by the partial restoration of mitochondrial ATP synthase assembly in atp23 null mutant transformed with exogenous hybrid COX1 T1121G mutant plasmid. The predicted tertiary structure of the Cox1 p.Val374Gly mutation showed no obvious difference from wild-type Cox1. By further chase labeling with isotope [35S]-methionine, we found that the stability of Atp6 of ATP synthase increased in the revertants compared with the atp23 null mutant. Taking all the data together, we revealed that the T1121G point mutation of mitochondrial gene COX1 could partially restore the unassembly of mitochondrial ATP synthase in atp23 null mutant by increasing the stability of Atp6. Therefore, this study uncovers a gene that is partially functionally complementary to ATP23 to rescue ATP23 deficiency, broadening our understanding of the relationship between yeast the cytochrome c oxidase complex and mitochondrial ATP synthase complex.

Proteomic analysis of arylamine N-acetyltransferase 1 knockout breast cancer cells: Implications in immune evasion and mitochondrial biogenesis.

Previous studies have shown that inhibition or depletion of N-acetyltransferase 1 (NAT1) in breast cancer cell lines leads to growth retardation both in vitro and in vivo, suggesting that NAT1 contributes to rapid growth of breast cancer cells. To understand molecular and cellular processes that NAT1 contributes to and generate novel hypotheses in regard to NAT1′s role in breast cancer, we performed an unbiased analysis of proteomes of parental MDA-MB-231 breast cancer cells and two separate NAT1 knockout (KO) cell lines. Among 4890 proteins identified, 737 proteins were found significantly (p < 0.01) upregulated, and 651 proteins were significantly (p < 0.01) downregulated in both NAT1 KO cell lines. We performed enrichment analyses to identify Gene Ontology biological processes, molecular functions, and cellular components that were enriched in each data set. Among the proteins upregulated in NAT1 KO cells, pathways associated with MHC (major histocompatibility complex) I-mediated antigen presentation were significantly enriched. This raises an interesting and new hypothesis that upregulation of NAT1 in breast cancer cells may aid them evade immune detection. Multiple pathways involved in mitochondrial functions were collectively downregulated in NAT1 KO cells, including multiple subunits of mitochondrial ATP synthase (Complex V of the electron transport chain). This was accompanied by a reduction in cell cycle-associated proteins and an increase in pro-apoptotic pathways in NAT1 KO cells, consistent with reported observations that NAT1 KO cells exhibit a slower growth rate both in vitro and in vivo. Thus, mitochondrial dysfunction in NAT1 KO cells likely contributes to growth retardation.

Impairment of Mitochondrial ATP Synthesis Induces RIPK3-dependent Necroptosis in Lung Epithelial Cells During Lung Injury by Lung Inflammation.

Dysfunction of mitochondrial metabolism is implicated in cellular injury and cell death. While mitochondrial dysfunction is associated with lung injury by lung inflammation, the mechanism by which the impairment of mitochondrial ATP synthesis regulates necroptosis during acute lung injury (ALI) by lung inflammation is unclear. Here, we showed that the impairment of mitochondrial ATP synthesis induces receptor interacting serine/threonine kinase 3 (RIPK3)-dependent necroptosis during lung injury by lung inflammation. We found that the impairment of mitochondrial ATP synthesis by oligomycin, an inhibitor of ATP synthase, resulted in increased lung injury and RIPK3 levels in lung tissues during lung inflammation by LPS in mice. The elevated RIPK3 and RIPK3 phosphorylation levels by oligomycin resulted in high mixed lineage kinase domain-like (MLKL) phosphorylation, the terminal molecule in necroptotic cell death pathway, in lung epithelial cells during lung inflammation. Moreover, the levels of protein in bronchoalveolar lavage fluid (BALF) were increased by the activation of necroptosis via oligomycin during lung inflammation. Furthermore, the levels of ATP5A, a catalytic subunit of the mitochondrial ATP synthase complex for ATP synthesis, were reduced in lung epithelial cells of lung tissues from patients with acute respiratory distress syndrome (ARDS), the most severe form of ALI. The levels of RIPK3, RIPK3 phosphorylation and MLKL phosphorylation were elevated in lung epithelial cells in patients with ARDS. Our results suggest that the impairment of mitochondrial ATP synthesis induces RIPK3-dependent necroptosis in lung epithelial cells during lung injury by lung inflammation.

Mitochondrial ATP synthase subunit d, a component of the peripheral stalk, is essential for growth and heat stress tolerance in Arabidopsis thaliana.

As rapid changes in climate threaten global crop yields, an understanding of plant heat stress tolerance is increasingly relevant. Heat stress tolerance involves the coordinated action of many cellular processes and is particularly energy demanding. We acquired a knockout mutant and generated knockdown lines in Arabidopsis thaliana of the d subunit of mitochondrial ATP synthase (gene name: ATPQ, AT3G52300, referred to hereafter as ATPd), a subunit of the peripheral stalk, and used these to investigate the phenotypic significance of this subunit in normal growth and heat stress tolerance. Homozygous knockout mutants for ATPd could not be obtained due to gametophytic defects, while heterozygotes possess no visible phenotype. Therefore, we used RNA interference to create knockdown plant lines for further studies. Proteomic analysis and blue native gels revealed that ATPd downregulation impairs only subunits of the mitochondrial ATP synthase (complex V). Knockdown plants were more sensitive to heat stress, had abnormal leaf morphology, and were severely slow growing compared to wild type. These results indicate that ATPd plays a crucial role in proper function of the mitochondrial ATP synthase holoenzyme, which, when reduced, leads to wide-ranging defects in energy-demanding cellular processes. In knockdown plants, more hydrogen peroxide accumulated and mitochondrial dysfunction stimulon (MDS) genes were activated. These data establish the essential structural role of ATPd and support the importance of complex V in normal plant growth, and provide new information about its requirement for heat stress tolerance.

Visualizing Lung Cellular Adaptations during Combined Ozone and LPS Induced Murine Acute Lung Injury.

Lungs are continually faced with direct and indirect insults in the form of sterile (particles or reactive toxins) and infectious (bacterial, viral or fungal) inflammatory conditions. An overwhelming host response may result in compromised respiration and acute lung injury, which is characterized by lung neutrophil recruitment as a result of the patho-logical host immune, coagulative and tissue remodeling response. Sensitive microscopic methods to visualize and quantify murine lung cellular adaptations, in response to low-dose (0.05 ppm) ozone, a potent environmental pollutant in combination with bacterial lipopolysaccharide, a TLR4 agonist, are crucial in order to understand the host inflammatory and repair mechanisms. We describe a comprehensive fluorescent microscopic analysis of various lung and systemic body compartments, namely the broncho-alveolar lavage fluid, lung vascular perfusate, left lung cryosections, and sternal bone marrow perfusate. We show damage of alveolar macrophages, neutrophils, lung parenchymal tissue, as well as bone marrow cells in correlation with a delayed (up to 36-72 h) immune response that is marked by discrete chemokine gradients in the analyzed compartments. In addition, we present lung extracellular matrix and cellular cytoskeletal interactions (actin, tubulin), mitochondrial and reactive oxygen species, anti-coagulative plasminogen, its anti-angiogenic peptide fragment angiostatin, the mitochondrial ATP synthase complex V subunits, α and β. These surrogate markers, when supplemented with adequate in vitro cell-based assays and in vivo animal imaging techniques such as intravital microscopy, can provide vital information towards understanding the lung response to novel immunomodulatory agents.

Citrate Synthase and OGDH Protein Targets are Linked with Adrenomegaly Under Atherosclerosis Combined with Chronic Stress

Background: Pathological changes of the adrenal gland and the possible underlying molecular mechanisms are currently unclear in the case of atherosclerosis (AS) combined with chronic stress (CS). Methods and Findings: New Zealand white rabbits were used to construct a CS and AS animal model. Proteomics and bioinformatics were employed to identify hub proteins in the adrenal gland related to CS and AS. Hematoxylin–eosin staining was used to visualize pathological changes in adrenal gland. Hub proteins were detected using immunohistochemistry, immunofluorescence assays, and Western blotting. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to analyze the expression of hub protein mRNAs. In addition, a neural network model was constructed. The quantitative relationships among the results of the three techniques were inferred by cubic spline interpolation. Two most significant hub proteins (citrate synthase and OGDH) that were related to CS and AS were identified in the adrenal gland using numerous bioinformatic methods. The hub proteins were mainly enriched in mitochondrial proton transport ATP synthase complex, ATPase activation, and the AMPK signaling pathway. Compared with the control group, the adrenal glands were larger and more disordered, irregular, and necrotic in the AS+CS group. The expression of citrate synthase and OGDH were higher in the AS+CS group than the control group, both at the protein and mRNA levels (P<0.05). There were strong correlations among the cross-sectional areas of adrenal glands, citrate synthase, and OGDH (P<0.05) via Spearman’s rho analysis, receiver operating characteristic curves, a neural network model, and cubic spline interpolation. Conclusions: AS combined with CS plays an important role in the hypothalamic–pituitary–adrenal (HPA) axis, promotes adrenomegaly, increases the release of GC, and may enhance ATP synthesis and energy metabolism in the body through citrate synthase and OGDH gene targets, providing a potential research direction for future, related explorations into this mechanism. Funding: The present study was funded by the National Key R&D Program of China (Grant no. 2020YFC2003000), Chinese Academy of Medical Sciences, CAMS Innovation Fund for Medical Sciences (Grant no. 2018-I2M-1-002.), National Natural Science Foundation of China (Grant no. 31271097, and 51672030). Declaration of Interest: The authors declare that they have no competing interests Ethical Approval: All experiments were approved by Animal Care and Use Committee of the Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences (CAMS) & Peking Union Medical College. All institutional and national guidelines for the care and use of laboratory animals were followed.

Citrate Synthase and OGDH as Potential Biomarkers of Atherosclerosis under Chronic Stress.

Background Pathological changes of the adrenal gland and the possible underlying molecular mechanisms are currently unclear in the case of atherosclerosis (AS) combined with chronic stress (CS). Methods New Zealand white rabbits were used to construct a CS and AS animal model. Proteomics and bioinformatics were employed to identify hub proteins in the adrenal gland related to CS and AS. Hub proteins were detected using immunohistochemistry, immunofluorescence assays, and Western blotting. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to analyze the expression of genes. In addition, a neural network model was constructed. The quantitative relationships were inferred by cubic spline interpolation. Enzymatic activity of mitochondrial citrate synthase and OGDH was detected by the enzymatic assay kit. Function of citrate synthase and OGDH with knockdown experiments in the adrenal cell lines was performed. Furthermore, target genes-TF-miRNA regulatory network was constructed. Coimmunoprecipitation (IP) assay and molecular docking study were used to detect the interaction between citrate synthase and OGDH. Results Two most significant hub proteins (citrate synthase and OGDH) that were related to CS and AS were identified in the adrenal gland using numerous bioinformatic methods. The hub proteins were mainly enriched in mitochondrial proton transport ATP synthase complex, ATPase activation, and the AMPK signaling pathway. Compared with the control group, the adrenal glands were larger and more disordered, irregular, and necrotic in the AS+CS group. The expression of citrate synthase and OGDH was higher in the AS+CS group than in the control group, both at the protein and mRNA levels (P < 0.05). There were strong correlations among the cross-sectional areas of adrenal glands, citrate synthase, and OGDH (P < 0.05) via Spearman's rho analysis, receiver operating characteristic curves, a neural network model, and cubic spline interpolation. Enzymatic activity of citrate synthase and OGDH increased under the situation of atherosclerosis and chronic stress. Through the CCK8 assay, the adrenal cell viability was downregulated significantly after the knockdown experiment of citrate synthase and OGDH. Target genes-TF-miRNA regulatory network presented the close interrelations among the predicted microRNA, citrate synthase and OGDH. After Coimmunoprecipitation (IP) assay, the result manifested that the citrate synthase and OGDH were coexpressed in the adrenal gland. The molecular docking study showed that the docking score of optimal complex conformation between citrate synthase and OGDH was -6.15 kcal/mol. Conclusion AS combined with CS plays a significant role on the hypothalamic–pituitary–adrenal (HPA) axis, promotes adrenomegaly, increases the release of glucocorticoid (GC), and might enhance ATP synthesis and energy metabolism in the body through citrate synthase and OGDH gene targets, providing a potential research direction for future related explorations into this mechanism.

Characterization of the promoter region of bovine ATP5B: roles of MyoD and GATA1 in the regulation of basal transcription

Intramuscular fat (IMF) content is a key determinant of beef quality, making it a key topic of research interest. ATP5B serves as the catalytic component of the mitochondrial ATP synthase complex and plays essential roles in controlling fat contents and oxidative metabolism in bovine skeletal muscle. In this study, we determined that bovine ATP5B was highly expressed in longissimus thoracis. To elucidate the molecular mechanisms involved in bovine ATP5B regulation, we cloned and characterized the promoter region of ATP5B. Applying 5'-rapid amplification of cDNA end analysis (RACE), we identified two transcriptional start sites (TSSs) in its promoter region. Using a series of 5'-deletion promoter plasmids in luciferase reporter assay, we found that the proximal minimal promoter of ATP5B was located within the region −539/220 relative to the TSS. Site-directed mutation in combination with chromatin immunoprecipitation (ChIP) assays demonstrated that MyoD and GATA1 binding to the promoter region drives bovine ATP5B transcription. Taken together, these results provide new insight into the regulatory mechanisms of ATP5B transcription in mediating the IMF content of beef.

High‐Throughput Imaging of Kidney Cell Function to Elucidate Unknown Mechanisms of Antiretroviral Drug Toxicity

Globally, the World Health Organization estimates that 38 million people live with human immunodeficiency virus (HIV) infection and 257 million with hepatitis B virus (HBV). Toxicity from antiretroviral drugs is a major cause of kidney disease in these individuals. Tenofovir disoproxil fumarate (TDF) is a first line therapy for both HIV and HBV. TDF induces functional defects in the kidney proximal tubule (PT) for reasons that are unknown, partly due to a lack of appropriate experimental models to investigate this. TDF nephrotoxicity is characterized by two major clinical phenotypes: approximately 25% of patients develop defects in PT solute transport (Labarga et al., 2009); whilst 1% develop severe damage characterized by the appearance of enlarged and dysmorphic mitochondria in the PT (Hall et al., 2011). The aim of our study was to establish realistic in vitro models of TDF toxicity, to further investigate the underlying metabolic phenotypes.Experiments were performed on human‐derived PT cells (RPTEC/TERT1) grown to confluence. A high‐throughput imaging pipeline was established using automated microscopy and machine learning based image analysis. Solute transport was assessed by measuring the number/size of domes formed in the monolayer. Metabolism was assessed by antibody staining for mitochondrial morphology and a marker of autophagy (LC3).We screened a number of different treatment regimes, and identified two that produced phenotypes matching those reported in patients. Treatment with 300 μM TDF for 24 hours induced a decrease in solute transport, but without changing cell number or producing mitochondrial defects. In contrast, treatment with 500 μM TDF for 24 hours caused mitochondrial enlargement and activated autophagy. Further experiments were then performed with these treatment protocols to investigate the underlying metabolic phenotypes. TDF produced a dose dependent decrease in ATP, despite an increase in glycolysis. Baseline oxygen consumption rate (OCR) was also decreased, with a blunted response to oligomycin. However, a substantial increase in OCR occurred in response to the uncoupler FCCP. Moreover, metabolomics analysis revealed no evidence of major defects in the citric acid cycle, beta‐oxidation or urea cycle.Taken together, these findings are consistent with a partial inhibition of the mitochondrial ATP synthase (complex V). Of note, TDF is phosphorylated within cells to produce Tenofovir diphosphate (TFVpp), a structural analogue of ATP. In subsequent experiments using an established in vitro assay of complex V activity, we observed a dose dependent inhibition with TFVpp.In summary, we have developed a high‐throughput imaging pipeline of human‐derived PT cells to generate realistic in vitro models of functional TDF toxicity. Metabolic characterization of these models revealed a phenotype consistent with complex V inhibition, which might explain toxicity observed in patients, since PT solute transport in vivo is almost entirely dependent on mitochondrial ATP production.Support or Funding InformationSwiss National Science Foundation and Swiss National Centre of Competence in Research (NCCR) Kidney Control of Homeostasis.

ATP synthase F1 subunits recruited to centromeres by CENP-A are required for male meiosis

The histone H3 variant CENP-A epigenetically defines the centromere and is critical for chromosome segregation. Here we report an interaction between CENP-A and subunits of the mitochondrial ATP synthase complex in the germline of male Drosophila. Furthermore, we report that knockdown of CENP-A, as well as subunits ATPsyn-α, -βlike (a testis-specific paralogue of ATPsyn-β) and -γ disrupts sister centromere cohesion in meiotic prophase I. We find that this disruption is likely independent of reduced ATP levels. We identify that ATPsyn-α and -βlike localise to meiotic centromeres and that this localisation is dependent on the presence of CENP-A. We show that ATPsyn-α directly interacts with the N-terminus of CENP-A in vitro and that truncation of its N terminus perturbs sister centromere cohesion in prophase I. We propose that the CENP-A N-terminus recruits ATPsyn-α and -βlike to centromeres to promote sister centromere cohesion in a nuclear function that is independent of oxidative phosphorylation.

Efficient termination of nuclear lncRNA transcription promotes mitochondrial genome maintenance

Most DNA in the genomes of higher organisms does not code for proteins. RNA Polymerase II (Pol II) transcribes non-coding DNA into long non-coding RNAs (lncRNAs), but biological roles of lncRNA are unclear. We find that mutations in the yeast lncRNA CUT60 result in poor growth. Defective termination of CUT60 transcription causes read-through transcription across the ATP16 gene promoter. Read-through transcription localizes chromatin signatures associated with Pol II elongation to the ATP16 promoter. The act of Pol II elongation across this promoter represses functional ATP16 expression by a Transcriptional Interference (TI) mechanism. Atp16p function in the mitochondrial ATP-synthase complex promotes mitochondrial DNA stability. ATP16 repression by TI through inefficient termination of CUT60 therefore triggers mitochondrial genome loss. Our results expand the functional and mechanistic implications of non-coding DNA in eukaryotes by highlighting termination of nuclear lncRNA transcription as mechanism to stabilize an organellar genome.