Role In Oxidative Phosphorylation Research Articles

Mitochondrial Oxidative Stress Induces Rapid Intermembrane Space/Matrix Translocation of Apurinic/Apyrimidinic Endonuclease 1 Protein through TIM23 Complex

Mitochondria are essential cellular organelles that import the majority of proteins to sustain their function in cellular metabolism and homeostasis. Due to their role in oxidative phosphorylation, mitochondria are constantly affected by oxidative stress. Stability of mitochondrial DNA (mtDNA) is essential for mitochondrial physiology and cellular well-being and for this reason mtDNA lesions have to be rapidly recognized and repaired. Base excision repair (BER) is the main pathway responsible for repairing non-helix distorting base lesions both into the nucleus and in mitochondria. Apurinic/Apyrimidinic Endonuclease 1 (APE1) is a key component of BER pathway and the only protein that can recognize and process an abasic (AP) site. Comprehensions of the mechanisms regulating APE1 intracellular trafficking are still fragmentary. In this study we focused our attention on the mitochondrial form of APE1 protein and how oxidative stress induces its translocation to maintain mtDNA integrity. Our data proved that: (i) the rise of mitochondrial ROS determines a very rapid translocation of APE1 from the intermembrane space (IMS) into the matrix; and (ii) TIM23/PAM machinery complex is responsible for the matrix translocation of APE1. Moreover, our data support the hypothesis that the IMS, where the majority of APE1 resides, could represent a sort of storage site for the protein.

Genetic Modulators of Niclosamide Sensitivity and Resistance in Acute Myeloid Leukemia

Introduction Acute myeloid leukemia (AML) is a malignancy of myeloid progenitor cells that leads to the accumulation of immature blasts in the blood and bone marrow. While the 5-year relative survival rate has increased from 6.4% to 28.7% in the last 40 years, death rates have remained steady at 3-4%. Less toxic, more effective targeted treatments are needed to improve outcomes for patients with AML. Previous studies identified cAMP response element-binding protein (CREB) as a potential therapeutic target for AML therapy. CREB is overexpressed in AML and is associated with poor prognosis. We identified a small molecule, XX-650-23, that inhibits interaction between CREB and its co-activator, CREB Binding Protein. However, XX-650-23 does not have optimal physical chemical properties for clinical application in humans. Niclosamide, an FDA approved anthelmintic drug, shares structural similarity with XX-650-23, and suppresses the proliferation of AML cells in vitro and in vivo by inhibiting CREB-dependent signaling. The aim of this study is to define the molecular pathways that mediate the effects of niclosamide in AML cells. This will allow for more precise selection of patient populations for treatment, identify potential sources of niclosamide resistance and reveal combination therapies that make use of the distinct pathways targeted. Methods To identify genetic factors modulating cellular sensitivity to niclosamide treatment, we performed a CRISPR/Cas9 library screen in the presence or absence of niclosamide. HL60 cells expressing Cas9 were infected with a custom sgRNA lentivirus library of 220 genes from a previous genome-wide shRNA screen. After puromycin selection, HL60 cells expressing pooled sgRNAs were grown in either niclosamide or 0.1% DMSO for 20 days. Genomic DNA was extracted from control and niclosamide-treated cells. Frequency of sgRNA were measured by next generation sequencing and analyzed using the Cas9 high-throughput maximum likelihood estimator (casTLE) algorithm. Results Sixty-five gene hits were found to be significant (p≤0.05). Of the top 220 hits from the previous genome-wide shRNA screen, 26 were identified as positive hits (p ≤0.05) in the CRISPR/Cas9 knock out screen. The CRISPR screen identified genes enriched in a number of pathways including programmed cell death, nucleotide biosynthesis, mitochondrial processes and canonical glycolysis, as measured by gene ontology term analysis. Numerous gene deletions conferred resistance to niclosamide treatment. Genes DHODH (score= 123; effect= 2.9) and HSPA9 (score= 144; effect = 2.5), involved in nucleotide biosynthesis and mitochondrial processes, were significantly enriched in the surviving population. Perturbation of known metabolic pathways sensitized AML cells to niclosamide, including ATP synthase F1 subunit beta (ATP5B) (score= 316; effect= -5.1), hexokinase 2 (HK2) (score= 312; effect= -5.4), phosphofructokinase (PFKP) (score= 195; effect= -1.9) and mitochondrial phosphate carrier protein (SLC25A3) (score= 271; effect= -7.8). SLC25A3 and ATP5B play a role in oxidative phosphorylation and mitochondrial proton transmembrane transport, whereas HK2 and PFKP are key to glycolysis, suggesting that niclosamide's mechanism of action is dependent on energy metabolism. Furthermore, we found that disruption of Bcl-2 (BCL2) was sensitizing to niclosamide treatment (score= 129; effect= -4.7). To confirm the sensitizing effect of Bcl-2 inhibition on niclosamide treatment, HL60 cells were treated with niclosamide and Bc1-2 inhibitor venetoclax. Pretreatment of HL60 cells with niclosamide led to significantly decreased cell viability upon venetoclax treatment (p<0.0001). Simultaneous treatment of HL60 cells with niclosamide and venetoclax had a synergistic effect on cellular viability, with combination index values <1 at ED50 to ED90 (Chou-Talalay method). Overall, our study identified genes with strong signatures for cell death, nucleotide biosynthesis, mitochondrial function and metabolic processes. Compounds inhibiting genes identified in this screen could be combined with niclosamide for synergistic effect. Targets overrepresented in the surviving population represent sources of resistance to niclosamide therapy. This study provides insight into mechanisms of action and resistance in AML cells treated with niclosamide and novel targets for combination therapy. Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: Niclosamide is an anthelmintic, FDA approved to treat tapeworm infections.

Abstract 6150: The role of oxidative phosphorylation in a novel iPSC <->derived model of osteosarcomagenesis

Abstract Osteosarcoma is the most common bone malignancy in childhood and early adolescence. Thirty percent of patients with the rare genetic disorder Type II Rothmund- Thomson Syndrome (RTS) develop osteosarcoma. Patients with Type II RTS have biallelic mutations in the RECQL4 gene, which encodes an ATP dependent DNA helicase. Unfortunately, many attempts to model RTS associated osteosarcoma have not been successful. Here, we describe a patient-derived induced pluripotent stem cell (iPSC) model capable of recapitulating the process of osteosarcomagenesis. The main goal of our inquiry is to develop a new model to study osteosarcoma in order to elucidate cellular pathways important in osteosarcomagenesis that are potential therapeutic targets. RTS patient fibroblasts were reprogrammed to iPSCs, then subsequently differentiated to mesenchymal stem cells. These cells were validated as pluripotent and then further differentiated to early and late osteoblasts, the potential precursors of osteosarcoma. This model of osteoblast development provides an unlimited resource for inquiry into the molecular mechanism of osteosarcomagenesis. Clear advantages of the iPSC derived osteoblasts include the isogenic nature of the cells, as these iPSC-derived osteoblasts retain the genetic fidelity of the patient from which they were derived. We have also isolated matched parental cells and similarly reprogrammed and differentiated them in order to provide ideal matched controls with clinical relevance, as the parents are heterozygous for the same RECQL4 mutation, yet do not develop osteosarcoma. We believe these cells are the ideal model to study the molecular mechanism associated with RTS-associated osteosarcoma. In order to gain better insight into osteosarcoma pathophysiology, we utilized the iPSC-derived osteoblasts to perform RNAseq and subsequent GSEA and DEseq2 analyses. From these transcriptional studies, we have found dysregulation of oxidative phosphorylation in the RTS patient samples compared to their parental controls. We specifically observe differences in complexes I, III, and IV of the electron transport chain. Interestingly, we also observe changes in vacuolar ATPases that are function to increase proton concentration in the cell and within the extracellular environment. This is consistent with phenomena observed in other cancers, which have been observed to have a similar increase in v-ATPases on the plasma membrane. Inhibitors of these complexes provide us with a starting point to address new therapeutic options for patients. Citation Format: Brittany Ellis Jewell, An Xu, Dandan Zhu, Linchao Lu, Ruiying Zhao, Lisa L. Wang, Dung-Fang Lee. The role of oxidative phosphorylation in a novel iPSC <->derived model of osteosarcomagenesis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6150.

The basal transcription factor II H subunit Tfb5 is required for stress response and pathogenicity in the tangerine pathotype of Alternaria alternata.

The basal transcription factor II H (TFIIH) is a multicomponent complex. In the present study, we characterized a TFIIH subunit Tfb5 by analysing loss‐ and gain‐of‐function mutants to gain a better understanding of the molecular mechanisms underlying stress resistance and pathogenicity in the citrus fungal pathogen Alternaria alternata. Tfb5 deficiency mutants (ΔAatfb5) decreased sporulation and pigmentation, and were impaired in the maintenance of colony surface hydrophobicity and cell wall integrity. ΔAatfb5 increased sensitivity to ultraviolet light, DNA‐damaging agents, and oxidants. The expression of Aatfb5 was up‐regulated in the wild type upon infection in citrus leaves, implicating the requirement of Aatfb5 in fungal pathogenesis. Biochemical and virulence assays revealed that ΔAatfb5 was defective in toxin production and cellwall‐degrading enzymes, and failed to induce necrotic lesions on detached citrus leaves. Aatfb5 fused with green fluorescent protein (GFP) was localized in the cytoplasm and nucleus and physically interacted with another subunit, Tfb2, based on yeast two‐hybrid and co‐immunoprecipitation analyses. Transcriptome and Antibiotics & Secondary Metabolite Analysis Shell (antiSMASH) analyses revealed the positive and negative roles of Aatfb5 in the production of various secondary metabolites and in the regulation of many metabolic and biosynthetic processes in A. alternata. Aatfb5 may play a negative role in oxidative phosphorylation and a positive role in peroxisome biosynthesis. Two cutinase‐coding genes (AaCut2 and AaCut15) required for full virulence were down‐regulated in ΔAatfb5. Overall, this study expands our understanding of how A. alternata uses the basal transcription factor to deal with stress and achieve successful infection in the plant host.

A genetic polymorphism that is associated with mitochondrial energy metabolism increases risk of fibromyalgia.

Alterations in cellular energy metabolism have been implicated in chronic pain, suggesting a role for mitochondrial DNA. Previous studies reported associations of a limited number of mitochondrial DNA polymorphisms with specific pain conditions. In this study, we examined the full mitochondrial genomes of people with a variety of chronic pain conditions. A discovery cohort consisting of 609 participants either with or without a complex persistent pain conditions (CPPCs) was examined. Mitochondrial DNA was subjected to deep sequencing for identification of rare mutations, common variants, haplogroups, and heteroplasmy associated with 5 CPPCs: episodic migraine, irritable bowel syndrome, fibromyalgia, vulvar vestibulitis, or temporomandibular disorders. The strongest association found was the presence of the C allele at the single nucleotide polymorphism m.2352T>C (rs28358579) that significantly increased the risk for fibromyalgia (odds ratio [OR] = 4.6, P = 4.3 × 10). This relationship was even stronger in women (OR = 5.1, P = 2.8 × 10), and m.2352T>C was associated with all other CPPCs in a consistent risk-increasing fashion. This finding was replicated in another cohort (OR = 4.3, P = 2.6 × 10) of the Orofacial Pain: Prospective Evaluation and Risk Assessment study consisting of 1754 female participants. To gain insight into the cellular consequences of the associated genetic variability, we conducted an assay testing metabolic reprogramming in human cell lines with defined genotypes. The minor allele C was associated with decreased mitochondrial membrane potential under conditions where oxidative phosphorylation is required, indicating a role of oxidative phosphorylation in pathophysiology of chronic pain. Our results suggest that cellular energy metabolism, modulated by m.2352T>C, contributes to fibromyalgia and possibly other chronic pain conditions.

Reprogramming Oxidative Phosphorylation in Cancer: A Role for RNA-Binding Proteins.

Significance: Cancer is a major disease imposing high personal and economic burden draining large part of National Health Care and Research budgets worldwide. In the last decade, research in cancer has underscored the reprogramming of metabolism to an enhanced aerobic glycolysis as a major trait of the cancer phenotype with great potential for targeted therapy. Recent Advances: Mitochondria are essential organelles in metabolic reprogramming for controlling the production of biological energy through oxidative phosphorylation (OXPHOS) and the supply of metabolic precursors that sustain proliferation. In addition, mitochondria are critical hubs that integrate different signaling pathways that control cellular metabolism and cell fate. The mitochondrial ATP synthase plays a fundamental role in OXPHOS and cellular signaling. Critical Issues: This review overviews mitochondrial metabolism and OXPHOS, and the major changes reported in the expression and function of mitochondrial proteins of OXPHOS in oncogenesis and in cellular differentiation. We summarize the prominent role that RNA-binding proteins (RNABPs) play in the sorting and localized translation of nuclear-encoded mRNAs that help define the mitochondrial cell-type-specific phenotype. Moreover, we emphasize the mechanisms that contribute to restrain the activity and expression of the mitochondrial ATP synthase in carcinomas, and illustrate that the dysregulation of proteins that control energy metabolism correlates with patients' survival. Future Directions: Future research should elucidate the mechanisms and RNABPs that promote the specific alterations of the mitochondrial phenotype in carcinomas arising from different tissues with the final aim of developing new therapeutic strategies to treat cancer.

Oxidative Phosphorylation Regulated By PGC-1α Promotes Multiple Myeloma Progression

Introduction: With the deeper understanding of metabolic reprograming, more and more studies highlight that many tumors heavily rely on mitochondrial oxidative phosphorylation (OXPHOS) for bioenergetics and biosynthesis, and targeting OXPHOS appears to be a promising approach for cancer therapy. Despite multiple metabolism alterations occur in multiple myeloma (MM), the role of OXPHOS in MM is rarely focused on. Many metabolic pathways are orchestrated by certain master co-regulators through transcriptional programs. The expression of OXPHOS gene set was reported to be regulated by peroxisome proliferator activated receptor gamma coactivator 1α (PGC-1α) in some other tissues, which serves as a transcriptional coactivator. Our previous studies demonstrated that PGC-1α was overexpression in MM cell line RPMI-8226 and knockdown of PGC-1α inhibited proliferation. Herein, we aimed to investigate the significance of OXPHOS in MM and clarify whether PGC-1α is the core regulator of OXPHOS in MM. Methods: Transcriptome data of GSE6477, GSE13591, GSE47552 were downloaded from the Gene Expression Omnibus (GEO) database and then removed batch effects. Gene set variation analysis (GSVA) were used to identify pathways enriched in MM. Differential expression analysis was performed and the PGC-1α expression value was extracted. Gene set enrichment analysis (GSEA) was adopted to explore biological functions involved by PGC-1α. RT-PCR was conducted to reflect the influence of PGC-1α inhibitor on OXPHOS gene set. Mitochondrial morphology was exhibited by transmission electron microscopy analysis. Cell counting kit-8 assay was employed to determine cell viability and flow cytometry analysis was used to assess apoptosis. Results: Integrated bioinformatics analysis revealed that OXPHOS pathway was significantly upregulated in MM than in normal donors (Figure A), and aberrantly overexpression of OXPHOS gene set was confirmed in our MM samples at mRNA level (Figure B-C). Moreover, we also demonstrated that aberrantly overexpression of OXPHOS genes in MM were generally associated with poorer survival (Figure D-F), indicating that OXPHOS appears as a potential oncogenic pathway for MM progression. Meanwhile, bioinformatics results showed that PGC-1α was upregulated in 247 MM patients from GEO database (Figure G). In addition, our results indicated that the overexpression of PGC-1α prevailed among 5 MM cell lines with different inherited backgrounds and our several MM samples (Figure H-I). GSEA analysis showed that gene sets of OXPHOS, TCA cycle, respiratory electron transport, ATP synthesis, were all significantly enriched in MM patients with high PGC-1α expression (Figure J-L). The inhibition of PGC-1α have exerted significant inhibition effect on the transcription of OXPHOS gene set in MM. In accordance with that OXPHOS is a major source of ATP, PGC-1α inhibitor resulted in the reduction of ATP levels of MM cells. Meanwhile, treatment of PGC-1α inhibitor triggered shriveled mitochondria with decreased volume, disorganized cristae, and increased electron density of matrix, indicative of impaired mitochondria OXPHOS. In vitro experiments suggested that PGC-1α inhibitor robustly inhibited proliferation of MM cells in a time and dose-dependent manner, with little effect on normal hematopoietic cells. Besides, PGC-1α inhibitor induced MM cells apoptosis significantly. Conclusions: Our investigations reported for the first time that OXPHOS regulated by PGC-1α may serve as a potential mechanism for MM progression. Besides, this study may provide new strategies for the treatment of MM from the perspective of OXPHOS and its core regulator. Figure Disclosures No relevant conflicts of interest to declare.

Role of mitochondria and cardiolipins in growth inhibition of breast cancer cells by retinoic acid

BackgroundAll-trans-retinoic-acid (ATRA) is a promising agent in the prevention/treatment of breast-cancer. There is growing evidence that reprogramming of cellular lipid metabolism contributes to malignant transformation and progression. Lipid metabolism is implicated in cell differentiation and metastatic colonization and it is involved in the mechanisms of sensitivity/resistance to different anti-tumor agents. The role played by lipids in the anti-tumor activity of ATRA has never been studied.MethodsWe used 16 breast cancer cell-lines whose degree of sensitivity to the anti-proliferative action of ATRA is known. We implemented a non-oriented mass-spectrometry based approach to define the lipidomic profiles of each cell-line grown under basal conditions and following treatment with ATRA. To complement the lipidomic data, untreated and retinoid treated cell-lines were also subjected to RNA-sequencing to define the perturbations afforded by ATRA on the whole-genome gene-expression profiles. The number and functional activity of mitochondria were determined in selected ATRA-sensitive and –resistant cell-lines. Bio-computing approaches were used to analyse the high-throughput lipidomic and transcriptomic data.ResultsATRA perturbs the homeostasis of numerous lipids and the most relevant effects are observed on cardiolipins, which are located in the mitochondrial inner membranes and play a role in oxidative-phosphorylation. ATRA reduces the amounts of cardiolipins and the effect is associated with the growth-inhibitory activity of the retinoid. Down-regulation of cardiolipins is due to a reduction of mitochondria, which is caused by an ATRA-dependent decrease in the expression of nuclear genes encoding mitochondrial proteins. This demonstrates that ATRA anti-tumor activity is due to a decrease in the amounts of mitochondria causing deficits in the respiration/energy-balance of breast-cancer cells.ConclusionsThe observation that ATRA anti-proliferative activity is caused by a reduction in the respiration and energy balance of the tumor cells has important ramifications for the therapeutic action of ATRA in breast cancer. The study may open the way to the development of rational therapeutic combinations based on the use of ATRA and anti-tumor agents targeting the mitochondria.

Abstract 2111: Increased heme influx dictates tumorigenic functions of non-small cell lung cancer cells

Abstract Heme, iron protoporphyrin IX, is a central metabolic and signaling molecule that regulates diverse molecular and cellular processes relating to oxygen utilization and metabolism. Heme serves as a cofactor or a prosthetic group for complexes II, III, and IV in electron transport chain. Hence, it has a direct role in oxidative phosphorylation and energy generation in cells. Most human cells can synthesize and uptake heme from circulation. A number of epidemiological studies have shown that high heme intake is associated with an increased risk of cancer, including lung cancer. Our lab has previously shown that an array of non-small cell lung cancer cells (NSCLCs) exhibit high heme synthesis and uptake. This increased heme flux correlates with an enhanced oxygen consumption and ATP production. NSCLCs also exhibited significantly elevated levels of hemoproteins and mitochondrial biogenesis proteins compared to normal lung cell lines. Expression levels of these proteins correlate with increased oxygen consumption and the total heme content of NSCLCs suggesting a possible role of heme in energy metabolism and mitochondrial biogenesis. In order to assess the involvement of heme in tumorigenicity of the NSCLC cell lines, we overexpressed heme synthesis enzyme, ALAS1 or heme transporters, HCP1 or HRG1 using lentivirus system. NSCLC cell lines A549 and H1299 were infected with a lentivirus carrying ALAS1 or HCP1 or HRG1 plasmid. Stable clones were obtained with antibiotic selection and checked for overexpression with western-blots. Cells overexpressing ALAS1, HCP1 and HRG1 showed significantly higher rates of heme influx as well as intensified oxygen consumption and ATP formation compared to eGFP controls. Tumorigenic properties such as invasion, migration and colony formation were also found to be significantly increased in these cell lines compared to their eGFP counterparts. Subcutaneous xenografts generated with overexpression cell lines also showed significantly high tumor growth rates and mass compared to eGFP controls. Our data from both in vitro and in vivo experiments show that heme directly improves the tumorigenic properties of NSCLC cells by substantially improving the respiration rates and energy metabolism. Citation Format: Sagar Shashikant Sohoni, Poorva Ghosh, Li Zhang. Increased heme influx dictates tumorigenic functions of non-small cell lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2111.

Oxygen dependence of glucose sensing: role in glucose homeostasis and related pathology.

In glucose homeostasis, glucose concentration is sensed by its metabolism through glucokinase (GCK) and oxidative phosphorylation. Because oxidative phosphorylation is an integral part of the sensory system, glucose sensing is necessarily dependent on oxygen pressure. Much of the dependence on oxygen is suppressed by location of glucose sensing cells in tissues with well-regulated blood flow. In healthy individuals the oxygen dependence is primarily observed in response to transient global hypoxia events such as during birth or transition to high altitude. The GCK sensing system is, however, used to control release of both insulin and glucagon, the preeminant hormonal regulators of blood glucose, as well as glucose sensitive neuronal activity. Suppression of oxygen delivery to glucose-sensing cells or interference with regulation of tissue blood flow by either local or systemic causes, stresses the glucose regulatory system. This is true whether the stress is imposed locally, such as by altered oxygen delivery to the pancreas, or globally, as in pulmonary insufficiency or exposure to high altitude. It may be expected that chronic application of this stress predisposes individuals to developing diabetes. Type 2 diabetes is a broad class of diseases characterized by disturbance of glucose homeostasis, i.e., having either hyperglycemia and/or decreased sensitivity to insulin. Given the role of oxidative phosphorylation in glucose sensing, tissue oxygen deprivation may predispose individuals to developing diabetes as well as contributing to the disease itself. This is particularly true in age-related diabetes because the incidence of vascular insufficiency increases markedly with increasing age. NEW & NOTEWORTHY Glucose sensing requires glucose metabolism through glycolysis and oxidative phosphorylation. Dependence of the latter on oxygen concentration imposes an oxygen dependence on glucose sensing. We have used a validated computational model to quantify that dependence. Evidence is presented that tissue oxygenation plays an important role in predisposition of individuals to developing type 2 diabetes and in progression of the disease.

Kinetin is Sufficient to Accelerate Mitophagy Flux in H9c2 Cardiac Myoblast Cells

The lysosomal degradation of dysfunctional mitochondria is vital for cellular health. Although widely known for their role in oxidative phosphorylation, mitochondria also generate reactive oxygen species (ROS) that are cytotoxic. Mitochondria themselves are susceptible to ROS damage, which can render them dysfunctional and eventually lead to various modes of cell death. Therefore, to maintain a healthy population of mitochondria, it is crucial for cells to eliminate injured mitochondria through the autophagy‐lysosomal pathway, a process termed mitophagy. Previous studies have identified Kinetin (KTN) as an activator of PTEN‐induced kinase (PINK‐1), a mitochondrial kinase that is activated in response to mitochondrial damage and orchestrates downstream mitophagy processes. While KTN has been shown to induce mitophagy in human‐derived neural cells, its role in cardiomyocytes remains unknown. In the present study, we tested the ability of KTN to induce mitophagy in H9c2 cardiac myoblast cells. The cells were treated with KTN, and a subset of cells was additionally treated with lysosomal protease inhibitors: pepstatin A (pepA) and E64d. Mitochondrial fractions were isolated and the expression levels of LC3‐II (Microtubule‐associated protein 1A/1B‐light chain 3), an established marker of autophagic vesicles, were analyzed using Western blotting. Our results showed that pepA/ E64d treatment led to a much larger increase in LC3‐II levels among KTN‐treated cells relative to the vehicle‐treated cells, suggesting that KTN was sufficient to accelerate mitophagy flux in cardiomyocytes. This result was confirmed by a novel dual fluorescent mitophagy reporter. Our lab is currently investigating whether KTN can protect cardiomyocytes against stress‐induced cellular injury through upregulation of mitophagy.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ataxia-Telangiectasia Mutated is located in cardiac mitochondria and impacts oxidative phosphorylation

The absence of Ataxia-Telangiectasia mutated protein kinase (ATM) is associated with neurological, metabolic and cardiovascular defects. The protein has been associated with mitochondria and its absence results in mitochondrial dysfunction. Furthermore, it can be activated in the cytosol by mitochondrial oxidative stress and mediates a cellular anti-oxidant response through the pentose phosphate pathway (PPP). However, the precise location and function of ATM within mitochondria and its role in oxidative phosphorylation is still unknown. We show that ATM is found endogenously within cardiac myocyte mitochondria under normoxic conditions and is consistently associated with the inner mitochondrial membrane. Acute ex vivo inhibition of ATM protein kinase significantly decreased mitochondrial electron transfer chain complex I-mediated oxidative phosphorylation rate but did not decrease coupling efficiency or oxygen consumption rate during β-oxidation. Chemical inhibition of ATM in rat cardiomyoblast cells (H9c2) significantly decreased the excited-state autofluorescence lifetime of enzyme-bound reduced NADH and its phosphorylated form, NADPH (NAD(P)H; 2.77 ± 0.26 ns compared to 2.57 ± 0.14 ns in KU60019-treated cells). This suggests an interaction between ATM and the electron transfer chain in the mitochondria, and hence may have an important role in oxidative phosphorylation in terminally differentiated cells such as cardiomyocytes.

Fhit\u2013Fdxr interaction in the mitochondria: modulation of reactive oxygen species generation and apoptosis in cancer cells

Fhit protein is lost in cancers of most, perhaps all, cancer types; when restored, it can induce apoptosis and suppress tumorigenicity, as shown in vitro and in mouse tumor models in vivo. Following protein cross-linking and proteomics analyses, we characterized a Fhit protein complex involved in triggering Fhit-mediated apoptosis. The complex includes the heat-shock chaperonin pair, HSP60/10, which is likely involved in importing Fhit into the mitochondria, where it interacts with ferredoxin reductase, responsible for transferring electrons from NADPH to cytochrome P450 via ferredoxin, in electron transport chain complex III. Overexpression of Fhit protein in Fhit-deficient cancer cells modulates the production of intracellular reactive oxygen species, causing increased ROS, following peroxide treatment, with subsequent increased apoptosis of lung cancer cells under oxidative stress conditions; conversely, Fhit-negative cells escape ROS overproduction and ROS-induced apoptosis, likely carrying oxidative damage. Thus, characterization of Fhit-interacting proteins has identified direct effectors of a Fhit-mediated apoptotic signal pathway that is lost in many cancers. This is of translational interest considering the very recent emphasis in a number of high-profile publications, concerning the role of oxidative phosphorylation in the treatment of human cancers, and especially cancer stem cells that rely upon oxidative phosphorylation for survival. Additionally, we have shown that cells from a Fhit-deficient lung cancer cell line, are sensitive to killing by exposure to atovaquone, thought to act as a selective oxidative phosphorylation inhibitor by targeting the CoQ10 dependence of the mitochondrial complex III, while the Fhit-expressing sister clone is resistant to this treatment.

The inner mitochondrial membrane protein ANT1 modulates IL-6 expression via the JNK pathway in macrophages.

Mitochondria are increasingly associated with inflammation. Here, we focus on the relationship between inflammation and adenine nucleotide translocator type 1 (ANT1), which is localized in the mitochondrial inner membrane. ANT1 plays an important role in oxidative phosphorylation, and mutations in the ANT1 gene are responsible for mitochondrial diseases. Ample studies have demonstrated that ANT1 has a critical role in cardiomyocytes and neurons, but little has been reported on its functions in immune cells. We knocked down ANT1 expression in macrophages and examined inflammatory cytokine expression after lipopolysaccharide stimulation. ANT1 knockdown reduces the expression of IL-6. JNK, upstream of IL-6, is downregulated, but other MAP kinases and the NF-κB signaling remain unchanged. These results suggest that ANT1 modulates IL-6 expression through JNK in macrophages.

A Hydrophobic Small Protein, BpOF4_01690, Is Critical for Alkaliphily of Alkaliphilic Bacillus pseudofirmus OF4.

A monocistronic small protein, BpOF4_01690, was annotated in alkaliphilic Bacillus pseudofirmus OF4. It comprises 59 amino acids and is hydrophobic. Importantly, homologs of this protein were identified only in alkaliphiles. In this study, a mutant with a BpOF4_01690 gene deletion (designated Δ01690) exhibited weaker growth than that of the wild type in both malate-based defined and glucose-based defined media under low-sodium conditions at pH 10.5. Additionally, the enzymatic activity of the respiratory chain of Δ01690 was much lower than that of the wild type. These phenotypes were similar to those of a ctaD deletion mutant and an atpB-F deletion mutant. Therefore, we hypothesize that BpOF4_01690 plays a critical role in oxidative phosphorylation under highly alkaline conditions.

Mitochondrial dysfunction induces the invasive phenotype, and cell migration and invasion, through the induction of AKT and AMPK pathways in lung cancer cells.

Mitochondria are well known for their important roles in oxidative phosphorylation, amino acid metabolism, fatty acid oxidation and ion homeostasis. Although the effects of mitochondrial dysfunction on tumorigenesis in various cancer cells have been reported, the correlation between mitochondrial dysfunction and epithelial‑to‑mesenchymal transition (EMT) in lung cancer development and metastasis has not been well elucidated. In the present study, the effects of mitochondrial dysfunction on EMT and migration in lung cancer cells were investigated using inhibitors of mitochondrial respiration, oligomycin A and antimycin A. Oligomycin A and antimycin A induced distinct mesenchymal‑like morphological features in H23, H1793 and A549 lung cancer cells. In addition, they decreased the expression levels of the epithelial marker protein E‑cadherin, but increased the expression levels of the mesenchymal marker proteins Vimentin, Snail and Slug. The results of immunofluorescence staining indicated that oligomycin A and antimycin A downregulated cortical E‑cadherin expression and upregulated the expression of Vimentin. In addition, oligomycin A and antimycin A increased the migration and invasion of A549 lung cancer cells, and promoted the expression levels of phosphorylated (p)‑protein kinase B (AKT) and p‑AMP‑activated protein kinase (AMPK). Notably, the production of reactive oxygen species by oligomycin A and antimycin A did not affect the expression of EMT protein markers. Conversely, treatment with the AKT inhibitor wortmannin and the AMPK inhibitor Compound C upregulated E‑cadherin and downregulated Vimentin expression. These results suggested that oligomycin A and antimycin A may induce migration and invasion of lung cancer cells by inducing EMT via the upregulation of p‑AKT and p‑AMPK expression.

Measurement of Mitochondrial Toxicity Parameters in Embryonic Hippocampus.

Recent discoveries have focused on mitochondria functions in the neuroscience research for approaches to study mitochondria dysfunction in neurodegenerative diseases. Mitochondrion is one of the organelles that is possibly worst affected in cognitive impairments. These are known as "powerhouse" of the cell as they are the main source of generation of ATP through aerobic respiration. They have role in oxidative phosphorylation and metabolism, they play central role in cell differentiation, apoptosis, oxygen sensing and detoxification of reactive oxygen species, innate immunity, mitochondrial matrix calcium, and maintenance of cell quality and regulation of cytoplasmic. There is a relationship between mitochondrial dysfunction and cognitive disorder that may be related to certain neurotoxins or mutations in mitochondrial DNA as well as the nuclear. Evaluating compounds for mitochondrial toxicity is an important capability for evaluation of cognitive effects by drugs. Studying mitochondria isolated from individual mouse brain regions is a challenge because of small amount of the available brain tissue. There are conventional techniques for isolation and purification of mitochondria from ventral midbrain, hippocampus, or striatum. The utilization of alcohol within pregnancy impairs the development of the unborn offspring and can lead to a plethora of anatomical, behavioral, and cognitive abnormalities.In here, a method for isolation of brain mitochondria from mouse is described. The method utilizes a refrigerated tabletop microtube centrifuge, and produces research grade quality mitochondria in amounts sufficient for performing multiple enzymatic and functional assays, thereby eliminating the necessity for pooling mouse brain tissue. A method for measuring ROS measurement, mitochondrial membrane potential, mitochondrial swelling, cytochrome c release, and mtDNA alterations after exposure to drugs is also included.

Alterations of sirtuins in mitochondrial cytochrome c-oxidase deficiency.

BackgroundSirtuins are NAD+ dependent deacetylases, which regulate mitochondrial energy metabolism as well as cellular response to stress. The NAD/NADH-system plays a crucial role in oxidative phosphorylation linking sirtuins and the mitochondrial respiratory chain. Furthermore, sirtuins are able to directly deacetylate and activate different complexes of the respiratory chain. This prompted us to analyse sirtuin levels in skin fibroblasts from patients with cytochrome c-oxidase (COX) deficiency and to test the impact of different pharmaceutical activators of sirtuins (SRT1720, paeonol) to modulate sirtuins and possibly respiratory chain enzymes in patient cells in vitro.MethodsWe assayed intracellular levels of sirtuin 1 and the mitochondrial sirtuins SIRT3 and SIRT4 in human fibroblasts from patients with COX- deficiency. Furthermore, sirtuins were measured after inhibiting complex IV in healthy control fibroblasts by cyanide and after incubation with activators SRT1720 and paeonol. To determine the effect of sirtuin inhibition at the cellular level we measured total cellular acetylation (control and patient cells, with and without treatment) by Western blot.ResultsWe observed a significant decrease in cellular levels of all three sirtuins at the activity, protein and transcriptional level (by 15% to 50%) in COX-deficient cells. Additionally, the intracellular concentration of NAD+ was reduced in patient cells. We mimicked the biochemical phenotype of COX- deficiency by incubating healthy fibroblasts with cyanide and observed reduced sirtuin levels. A pharmacological activation of sirtuins resulted in normalized sirtuin levels in patient cells. Hyper acetylation was also reversible after treatment with sirtuin activators. Pharmacological modulation of sirtuins resulted in altered respiratory chain complex activities.ConclusionsWe found inhibition of situins 1, 3 and 4 at activity, protein and transcriptional levels in fibroblasts from patient with COX-deficiency. Pharmacological activators were able to restore reduced sirtuin levels and thereby modulate respiratory chain activities.

MTOR has a developmental stage-specific role in mitochondrial fitness independent of conventional mTORC1 and mTORC2 and the kinase activity.

The mammalian target of rapamycin (mTOR), present in mTOR complex 1 (mTORC1) and mTORC2, is a serine/threonine kinase that integrates nutrients, growth factors, and cellular energy status to control protein synthesis, cell growth, survival and metabolism. However, it remains elusive whether mTOR plays a developmental stage-specific role in tissue development and whether mTOR can function independent of its complexes and kinase activity. In this study, by inducible genetic manipulation approach, we investigated the role of mTOR and its dependence on mTOR complexes and kinase activity in mitochondrial fitness of early, progenitor stage (lineage-negative; Lin-) versus later, lineage-committed stage (lineage-positive; Lin+) of hematopoietic cells. We found that oxidative phosphorylation (OXPHOS), ATP production and mitochondrial DNA synthesis were decreased in mTOR-/- Lin- cells but increased in mTOR-/- Lin+ cells, suggesting that mTOR plays a developmental stage-specific role in OXPHOS, ATP production and mitochondrial DNA synthesis. In contrast to mTOR deletion, simultaneous deletion of Raptor, a key component of mTORC1, and Rictor, a key component of mTORC2, led to increased mitochondrial DNA in Lin- cells and decreased mitochondrial DNA and ATP production in Lin+ cells, suggesting that mTOR regulates mitochondrial DNA synthesis in Lin- and Lin+ cells and ATP production in Lin+ cells independent of mTORC1 and mTORC2. Similar to mTOR deletion, deletion of Raptor alone attenuated glycolysis and increased mitochondrial mass and mitochondrial membrane potential in Lin- cells and increased mitochondrial mass and OXPHOS in Lin+ cells, whereas deletion of Rictor alone had no effect on these mitochondrial parameters in Lin- and Lin+ cells, suggesting that mTOR regulates glycolysis and mitochondrial membrane potential in Lin- cells, OXPHOS in Lin+ cells, and mitochondrial mass in both Lin- and Lin+ cells dependent on mTORC1, but not mTORC2. Either Raptor deficiency or Rictor deficiency recapitulated mTOR deletion in decreasing OXPHOS in Lin- cells and glycolysis in Lin+ cells, suggesting that mTOR regulates OXPHOS in Lin- cells and glycolysis in Lin+ cells dependent on both mTORC1 and mTORC2. Finally, mice harboring a mTOR kinase dead D2338A knock-in mutant showed decreased glycolysis in Lin+ cells, as seen in mTOR-/- Lin+ cells, but no change in glycolysis in Lin- cells, in contrast to the decreased glycolysis in mTOR-/- Lin- cells, suggesting that mTOR regulates glycolysis in Lin+ cells dependent on its kinase activity, whereas mTOR regulates glycolysis in Lin- cells independent of its kinase activity.

175 The effects of tbq on cardiac intracellular atp levels; role of oxidative phosphorylation and oxidative stress

In a recent study, 2,5-Di-(tert-butyl)−1,4-benzohydroquinone (TBQ) produced a concentration dependent and fully reversible inhibition of the sarcoplasmic reticulum Ca2+ ATPase (SERCA) in rat ventricular myocytes1. While TBQ is a potentially...