bims-mibica Biomed News
on Mitochondrial bioenergetics in cancer
Issue of 2021–09–26
forty-six papers selected by
Kelsey Fisher-Wellman, East Carolina University



  1. EMBO J. 2021 Sep 20. e108648
      So-called ρ0 cells lack mitochondrial DNA and are therefore incapable of aerobic ATP synthesis. How cells adapt to survive ablation of oxidative phosphorylation remains poorly understood. Complexome profiling analysis of ρ0 cells covered 1,002 mitochondrial proteins and revealed changes in abundance and organization of numerous multiprotein complexes including previously not described assemblies. Beyond multiple subassemblies of complexes that would normally contain components encoded by mitochondrial DNA, we observed widespread reorganization of the complexome. This included distinct changes in the expression pattern of adenine nucleotide carrier isoforms, other mitochondrial transporters, and components of the protein import machinery. Remarkably, ablation of mitochondrial DNA hardly affected the complexes organizing cristae junctions indicating that the altered cristae morphology in ρ0 mitochondria predominantly resulted from the loss of complex V dimers required to impose narrow curvatures to the inner membrane. Our data provide a comprehensive resource for in-depth analysis of remodeling of the mitochondrial complexome in response to respiratory deficiency.
    Keywords:  OXPHOS; complexome profiling; mitochondria; mtDNA; rho0 cells
    DOI:  https://doi.org/10.15252/embj.2021108648
  2. Front Cell Dev Biol. 2021 ;9 720656
      Mitochondria are double-membrane organelles that contain their own genome, the mitochondrial DNA (mtDNA), and reminiscent of its endosymbiotic origin. Mitochondria are responsible for cellular respiration via the function of the electron oxidative phosphorylation system (OXPHOS), located in the mitochondrial inner membrane and composed of the four electron transport chain (ETC) enzymes (complexes I-IV), and the ATP synthase (complex V). Even though the mtDNA encodes essential OXPHOS components, the large majority of the structural subunits and additional biogenetical factors (more than seventy proteins) are encoded in the nucleus and translated in the cytoplasm. To incorporate these proteins and the rest of the mitochondrial proteome, mitochondria have evolved varied, and sophisticated import machineries that specifically target proteins to the different compartments defined by the two membranes. The intermembrane space (IMS) contains a high number of cysteine-rich proteins, which are mostly imported via the MIA40 oxidative folding system, dependent on the reduction, and oxidation of key Cys residues. Several of these proteins are structural components or assembly factors necessary for the correct maturation and function of the ETC complexes. Interestingly, many of these proteins are involved in the metalation of the active redox centers of complex IV, the terminal oxidase of the mitochondrial ETC. Due to their function in oxygen reduction, mitochondria are the main generators of reactive oxygen species (ROS), on both sides of the inner membrane, i.e., in the matrix and the IMS. ROS generation is important due to their role as signaling molecules, but an excessive production is detrimental due to unwanted oxidation reactions that impact on the function of different types of biomolecules contained in mitochondria. Therefore, the maintenance of the redox balance in the IMS is essential for mitochondrial function. In this review, we will discuss the role that redox regulation plays in the maintenance of IMS homeostasis as well as how mitochondrial ROS generation may be a key regulatory factor for ETC biogenesis, especially for complex IV.
    Keywords:  MIA; ROS; biogenesis; mitochondria; protein import; redox signaling; respiratory chain assembly
    DOI:  https://doi.org/10.3389/fcell.2021.720656
  3. Methods Mol Biol. 2022 ;2363 77-84
      We here describe measurements of respiratory enzymes in situ, which can be done on very small cell samples and make mitochondrial isolation unnecessary. The method is based on the ability of the fungal peptide alamethicin to permeate biological membranes from the net positively charged side, and form nonspecific ion channels. These channels allow rapid transport of substrates and products across the plasma membrane, the inner mitochondrial membrane, and the inner plastid envelope. In this way, mitochondrial enzyme activities can be studied without disrupting the cells. The enzymes can be investigated in their natural proteinaceous environment and the activity of enzymes, also those sensitive to detergents or to dilution, can be quantified on a whole cell basis. We here present protocols for in situ measurement of two mitochondrial enzymatic activities: malate oxidation measured as oxygen consumption by the electron transport chain, which is sensitive to detergents, and NAD+-isocitrate dehydrogenase, a tricarboxylic acid cycle enzyme that dissociates upon dilution.
    Keywords:  Alamethicin; BY-2 cells; Electron transport chain; Mitochondria; Tricarboxylic acid cycle
    DOI:  https://doi.org/10.1007/978-1-0716-1653-6_7
  4. J Biol Chem. 2021 Sep 17. pii: S0021-9258(21)01006-1. [Epub ahead of print] 101204
      Impairments in mitochondrial energy metabolism have been implicated in human genetic diseases associated with mitochondrial and nuclear DNA mutations, neurodegenerative and cardiovascular disorders, diabetes, and aging. Alteration in mitochondrial complex I structure and activity has been shown to play a key role in Parkinson's disease and ischemia/reperfusion tissue injury, but significant difficulty remains in assessing the content of this enzyme complex in a given sample. The present study introduces a new method utilizing native polyacrylamide gel electrophoresis in combination with flavin fluorescence scanning to measure the absolute content of complex I, as well as α-ketoglutarate dehydrogenase complex (KGDHC), in any preparation. We show that the complex I content is 19±1 pmol/mg of protein in the brain mitochondria, while varies up to 10-fold in different mouse tissues. Together with the measurements of NADH-dependent specific activity, our method also allows accurate determination of complex I catalytic turnover which was calculated as 104 min-1 for NADH:ubiquinone reductase in mouse brain mitochondrial preparations. KGDHC content was determined to be 65±5 and 123±9 pmol/mg protein for mouse brain and bovine heart mitochondria, respectively. Our approach can also be extended to cultured cells, and we demonstrated that about 90 × 103 complex I molecules are present in a single HEK293 cell. The ability to determine complex I content should provide a valuable tool to investigate the enzyme status in samples after in vivo treatment in mutant organisms, cells in culture, or human biopsies.
    Keywords:  enzyme turnover; flavin adenine dinucleotide; flavin mononucleotide; fluorescence; ketoglutarate dehydrogenase complex; mitochondrial respiratory chain complex I; stoichiometry
    DOI:  https://doi.org/10.1016/j.jbc.2021.101204
  5. FASEB J. 2021 Oct;35(10): e21933
      In obesity, skeletal muscle mitochondrial activity changes to cope with increased nutrient availability. Autophagy has been proposed as an essential mechanism involved in the regulation of mitochondrial metabolism. Still, the contribution of autophagy to mitochondrial adaptations in skeletal muscle during obesity is unknown. Here, we show that in response to high-fat diet (HFD) feeding, distinct skeletal muscles in mice exhibit differentially regulated autophagy that may modulate mitochondrial activity. We observed that after 4 and 40 weeks of high-fat diet feeding, OXPHOS subunits and mitochondrial DNA content increased in the oxidative soleus muscle. However, in gastrocnemius muscle, which has a mixed fiber-type composition, the mitochondrial mass increased only after 40 weeks of HFD feeding. Interestingly, fatty acid-supported mitochondrial respiration was enhanced in gastrocnemius, but not in soleus muscle after a 4-week HFD feeding. This increased metabolic profile in gastrocnemius was paralleled by preserving autophagy flux, while autophagy flux in soleus was reduced. To determine the role of autophagy in this differential response, we used an autophagy-deficient mouse model with partial deletion of Atg7 specifically in skeletal muscle (SkM-Atg7+/- mice). We observed that Atg7 reduction resulted in diminished autophagic flux in skeletal muscle, alongside blunting the HFD-induced increase in fatty acid-supported mitochondrial respiration observed in gastrocnemius. Remarkably, SkM-Atg7+/- mice did not present increased mitochondria accumulation. Altogether, our results show that HFD triggers specific mitochondrial adaptations in skeletal muscles with different fiber type compositions, and that Atg7-mediated autophagy modulates mitochondrial respiratory capacity but not its content in response to an obesogenic diet.
    Keywords:  Atg7; fatty acids; obesity; skeletal muscle fiber
    DOI:  https://doi.org/10.1096/fj.202001593RR
  6. Methods Mol Biol. 2022 ;2363 85-100
      Transport of tricarboxylic acid (TCA) cycle substrates across mitochondrial membranes and their subsequent oxidative decarboxylation in the matrix provide reductants for respiration-coupled ATP synthesis. These processes are typically assessed together through the ability of mitochondria to consume oxygen or release carbon dioxide, however, this approach fails to assess or separate the complexity of transport and the subsequent metabolism of substrates and products. In this chapter, we provide a strategy for simultaneously measuring substrate transport and utilization by isolated mitochondria using a mass spectrometry-based technique. The results of cofeeding of isolated mitochondria with unlabeled malate and uniformly labeled pyruvate is used as an example. Mitochondria fed with substrates are separated from the extramitochondrial space by centrifugation through a single layer of silicone oil. Analysis of mitochondrial pellet and reaction supernatant enable quantitation of substrate import and product export. This method also allows an estimation of the contribution of different enzymatic pathways to the formation of a specific product. This assay opens opportunities to verify carrier functions in organello and to identify the substrate preferences of mitochondrial transporters of unknown function using targeted and/or untargeted metabolomics approaches.
    Keywords:  Metabolism; Mitochondria; Selective reaction monitoring (SRM) mass spectrometry; Silicone oil centrifugation; Transport
    DOI:  https://doi.org/10.1007/978-1-0716-1653-6_8
  7. Methods Mol Biol. 2022 ;2363 215-234
      Mitochondria are central hubs of redox biochemistry in the cell. An important role of mitochondrial carbon metabolism is to oxidize respiratory substrates and to pass the electrons down the mitochondrial electron transport chain to reduce oxygen and to drive oxidative phosphorylation. During respiration, reactive oxygen species are produced as a side reaction, some of which in turn oxidize cysteine thiols in proteins. Hence, the redox status of cysteine-containing mitochondrial proteins has to be controlled by the mitochondrial glutathione and thioredoxin systems, which draw electrons from metabolically derived NADPH. The redox status of mitochondrial cysteines can undergo fast transitions depending on the metabolic status of the cell, as for instance at early seed germination. Here, we describe a state-of-the-art method to quantify redox state of protein cysteines in isolated Arabidopsis seedling mitochondria of controlled metabolic and respiratory state by MS2-based redox proteomics using the isobaric thiol labeling reagent Iodoacetyl Tandem Mass Tag™ (iodoTMT). The procedure is also applicable to isolated mitochondria of other plant and nonplant systems.
    Keywords:  Cysteine; Iodoacetyl Tandem Mass Tag; Isobaric labeling; LC-MS/MS; MaxQuant; Mitochondria; Redox; Thiols
    DOI:  https://doi.org/10.1007/978-1-0716-1653-6_16
  8. NPJ Regen Med. 2021 Sep 24. 6(1): 58
      Mitochondria are cellular organelles critical for numerous cellular processes and harboring their own circular mitochondrial DNA (mtDNA). Most mtDNA associated disorders (either deletions, mutations, or depletion) lead to multisystemic disease, often severe at a young age, with no disease-modifying therapies. Mitochondria have a capacity to enter eukaryotic cells and to be transported between cells. We describe a method of ex vivo augmentation of hematopoietic stem and progenitor cells (HSPCs) with normal exogenous mitochondria, termed mitochondrial augmentation therapy (MAT). Here, we show that MAT is feasible and dose dependent, and improves mitochondrial content and oxygen consumption of healthy and diseased HSPCs. Ex vivo mitochondrial augmentation of HSPCs from a patient with a mtDNA disorder leads to superior human engraftment in a non-conditioned NSGS mouse model. Using a syngeneic mouse model of accumulating mitochondrial dysfunction (Polg), we show durable engraftment in non-conditioned animals, with in vivo transfer of mitochondria to recipient hematopoietic cells. Taken together, this study supports MAT as a potential disease-modifying therapy for mtDNA disorders.
    DOI:  https://doi.org/10.1038/s41536-021-00167-7
  9. Mol Cell. 2021 Sep 16. pii: S1097-2765(21)00695-X. [Epub ahead of print]81(18): 3848-3865.e19
      Metabolic rewiring and redox balance play pivotal roles in cancer. Cellular senescence is a barrier for tumorigenesis circumvented in cancer cells by poorly understood mechanisms. We report a multi-enzymatic complex that reprograms NAD metabolism by transferring reducing equivalents from NADH to NADP+. This hydride transfer complex (HTC) is assembled by malate dehydrogenase 1, malic enzyme 1, and cytosolic pyruvate carboxylase. HTC is found in phase-separated bodies in the cytosol of cancer or hypoxic cells and can be assembled in vitro with recombinant proteins. HTC is repressed in senescent cells but induced by p53 inactivation. HTC enzymes are highly expressed in mouse and human prostate cancer models, and their inactivation triggers senescence. Exogenous expression of HTC is sufficient to bypass senescence, rescue cells from complex I inhibitors, and cooperate with oncogenic RAS to transform primary cells. Altogether, we provide evidence for a new multi-enzymatic complex that reprograms metabolism and overcomes cellular senescence.
    Keywords:  MDH1; ME1; NAD; NADPH; PC; cellular senescence; hypoxia; metabolon; mitochondrial dysfunction; p53
    DOI:  https://doi.org/10.1016/j.molcel.2021.08.028
  10. Proc Natl Acad Sci U S A. 2021 09 28. pii: e2106950118. [Epub ahead of print]118(39):
      Combining mass spectrometry-based chemical cross-linking and complexome profiling, we analyzed the interactome of heart mitochondria. We focused on complexes of oxidative phosphorylation and found that dimeric apoptosis-inducing factor 1 (AIFM1) forms a defined complex with ∼10% of monomeric cytochrome c oxidase (COX) but hardly interacts with respiratory chain supercomplexes. Multiple AIFM1 intercross-links engaging six different COX subunits provided structural restraints to build a detailed atomic model of the COX-AIFM12 complex (PDBDEV_00000092). An application of two complementary proteomic approaches thus provided unexpected insight into the macromolecular organization of the mitochondrial complexome. Our structural model excludes direct electron transfer between AIFM1 and COX. Notably, however, the binding site of cytochrome c remains accessible, allowing formation of a ternary complex. The discovery of the previously overlooked COX-AIFM12 complex and clues provided by the structural model hint at potential roles of AIFM1 in oxidative phosphorylation biogenesis and in programmed cell death.
    Keywords:  AIFM1; COX; complexome profiling; cross-linking mass spectrometry; mitochondria
    DOI:  https://doi.org/10.1073/pnas.2106950118
  11. Mol Cell. 2021 Sep 16. pii: S1097-2765(21)00501-3. [Epub ahead of print]81(18): 3878-3878.e1
      Metabolic networks support cancer cell survival, proliferation, and malignant progression. Cancer cells take up large amounts of nutrients such as glucose and glutamine whose metabolism provides the energy, reducing equivalents, and biosynthetic precursors required to meet the biosynthetic demands of proliferation. Intermediates of glycolysis and the tricarboxylic acid (TCA) cycle provide critical building blocks for synthesis of non-essential amino acids, nucleotides, and fatty acids. To view this SnapShot, open or download the PDF.
    DOI:  https://doi.org/10.1016/j.molcel.2021.06.021
  12. Methods Mol Biol. 2022 ;2363 199-213
      Mitochondria actively participate in oxygenic metabolism and are one of the major sources of reactive oxygen species (ROS) production in plant cells. However, instead of measuring ROS concentrations in organelles it is more worthwhile to observe active ROS generation or downstream oxidation products, because the steady state level of ROS is easily buffered. Here, we describe how to measure the in vitro production of superoxide anion radicals (O2·-) by mitochondria and the release of O2·- into the cytosol. A method to determine glutathione, which is the most abundant mitochondrial low-mass antioxidant, is presented since changes in the redox state of glutathione can be indicative of the oxidative action of ROS. The identification of oxidative damage to mitochondrial components is the ultimate symptom that ROS homeostasis is not under control. We present how to determine the extent of oxidation of membrane lipids and the carbonylation of mitochondrial proteins. In summary, oxidative stress symptoms have to be analyzed at different levels, including ROS production, scavenging capacity, and signs of destruction, which only together can be considered markers of mitochondrial ROS status.
    Keywords:  Carbonylated proteins; Glutathione; Lipid peroxidation; Mitochondria; Mitochondrial integrity; Oxidized proteins; Reactive oxygen species; Superoxide anion radical
    DOI:  https://doi.org/10.1007/978-1-0716-1653-6_15
  13. J Biol Chem. 2021 Sep 21. pii: S0021-9258(21)01027-9. [Epub ahead of print] 101224
      Energy metabolism and extracellular matrix function together orchestrate and maintain tissue organization, but crosstalk between these processes is poorly understood. Here, we used single cell RNA-seq (scRNA-seq) analysis to uncover the importance of the mitochondrial respiratory chain for extracellular matrix homeostasis in mature cartilage. This tissue produces large amounts of a specialized extracellular matrix to promote skeletal growth during development and maintain mobility throughout life. A combined approach of high-resolution scRNA-seq, mass spectrometry/matrisome analysis, and atomic force microscopy was applied to mutant mice with cartilage-specific inactivation of respiratory chain function. This genetic inhibition in cartilage results in the expansion of a central area of 1-month-old mouse femur head cartilage, showing disorganized chondrocytes and increased deposition of extracellular matrix material. scRNA-seq analysis identified a cell cluster-specific decrease in mitochondrial DNA-encoded respiratory chain genes and a unique regulation of extracellular matrix-related genes in nonarticular chondrocytes. These changes were associated with alterations in extracellular matrix composition, a shift in collagen/non-collagen protein content, and an increase of collagen crosslinking and ECM stiffness. These results demonstrate that mitochondrial respiratory chain dysfunction is a key factor that can promote ECM integrity and mechanostability in cartilage and presumably also in many other tissues.
    Keywords:  Extracellular matrix; MMP10; THBS1; atomic force microscopy; matrisome; matrix metalloproteinase (MMP); mitochondria; mitochondrial respiratory chain; single cell RNA sequencing; transcriptomics
    DOI:  https://doi.org/10.1016/j.jbc.2021.101224
  14. Nat Metab. 2021 Sep;3(9): 1259-1274
      Changes in maternal diet and metabolic defects in mothers can profoundly affect health and disease in their progeny. However, the biochemical mechanisms that induce the initial reprogramming events at the cellular level have remained largely unknown owing to limitations in obtaining pure populations of quiescent oocytes. Here, we show that the precocious onset of mitochondrial respiratory quiescence causes a reprogramming of progeny metabolic state. The premature onset of mitochondrial respiratory quiescence drives the lowering of Drosophila oocyte NAD+ levels. NAD+ depletion in the oocyte leads to reduced methionine cycle production of the methyl donor S-adenosylmethionine in embryos and lower levels of histone H3 lysine 27 trimethylation, resulting in enhanced intestinal lipid metabolism in progeny. In addition, we show that triggering cellular quiescence in mammalian cells and chemotherapy-resistant human cancer cell models induces cellular reprogramming events identical to those seen in Drosophila, suggesting a conserved metabolic mechanism in systems reliant on quiescent cells.
    DOI:  https://doi.org/10.1038/s42255-021-00450-3
  15. Open Biol. 2021 Sep;11(9): 210168
      The genome of mitochondria, called mtDNA, is a small circular DNA molecule present at thousands of copies per human cell. MtDNA is packaged into nucleoprotein complexes called nucleoids, and the density of mtDNA packaging affects mitochondrial gene expression. Genetic processes such as transcription, DNA replication and DNA packaging alter DNA topology, and these topological problems are solved by a family of enzymes called topoisomerases. Within mitochondria, topoisomerases are involved firstly in the regulation of mtDNA supercoiling and secondly in disentangling interlinked mtDNA molecules following mtDNA replication. The loss of mitochondrial topoisomerase activity leads to defects in mitochondrial function, and variants in the dual-localized type IA topoisomerase TOP3A have also been reported to cause human mitochondrial disease. We review the current knowledge on processes that alter mtDNA topology, how mtDNA topology is modulated by the action of topoisomerases, and the consequences of altered mtDNA topology for mitochondrial function and human health.
    Keywords:  DNA topology; mitochondria; mitochondrial DNA; mitochondrial disease; topoisomerases
    DOI:  https://doi.org/10.1098/rsob.210168
  16. Methods Mol Biol. 2022 ;2363 121-152
      While the routine mitochondrial extraction and isolation protocols have not fundamentally changed since the introduction of density gradients, the approaches we use to examine the proteome have. The initial characterisation of mitochondrial proteomes was carried out using two-dimensional gel electrophoresis in 2001 and gel spot mass spectrometry have now largely been superseded as the throughput and sensitivity of commercial mass spectrometers increases. Whist many of these early studies established the components of the mitochondrial proteome, as gels were replaced by gel free approaches the numbers of confirmed components rapidly increased. In this chapter we present gel-based approaches for the separation and concentration of mitochondrial proteins for their characterization by mass spectrometry. We also describe two gel-free approaches which can be used to quantity the degree of contamination arising during the isolation of mitochondria. These approaches are equally suitable for studies comparing one treatment to another.
    Keywords:  Gel fractionation; Gel purification; Mass spectrometry; Mitochondria; Proteomics; Selected reaction monitoring (SRM) mass spectrometry
    DOI:  https://doi.org/10.1007/978-1-0716-1653-6_11
  17. Mitochondrion. 2021 Sep 15. pii: S1567-7249(21)00122-7. [Epub ahead of print]
      Human mitochondrial diseases are a group of heterogeneous diseases caused by defects in oxidative phosphorylation, due to mutations in mitochondrial (mtDNA) or nuclear DNA. The diagnosis of mitochondrial disease is challenging since mutations in multiple genes can affect mitochondrial function, there is considerable clinical variability and a poor correlation between genotype and phenotype. Herein we assessed mitochondrial function in peripheral blood mononuclear cells (PBMCs) and platelets from volunteers without known metabolic pathology and patients with mitochondrial disease. Oxygen consumption rates were evaluated and respiratory parameters indicative of mitochondrial function were obtained. A negative correlation between age and respiratory parameters of PBMCs from control individuals was observed. Surprisingly, respiratory parameters of PBMCs normalized by cell number were similar in patients and young controls. Considering possible compensatory mechanisms, mtDNA copy number in PBMCs was quantified and an increase was found in patients with respect to controls. Hence, respiratory parameters normalized by mtDNA copy number were determined, and in these conditions a decrease in maximum respiration rate and spare respiratory capacity was observed in patients relative to control individuals. In platelets no decay was seen in mitochondrial function with age, while a reduction in basal, ATP-independent and ATP-dependent respiration normalized by cell number was detected in patients compared to control subjects. In summary, our results offer promising perspectives regarding the assessment of mitochondrial function in blood cells for the diagnosis mitochondrial disease, minimizing the need for invasive procedures such as muscle biopsies, and for following disease progression and response to treatments.
    Keywords:  PBMC; aging; bioenergetics; mitochondrial disease; mtDNA; platelets
    DOI:  https://doi.org/10.1016/j.mito.2021.09.004
  18. Mol Cell. 2021 Sep 16. pii: S1097-2765(21)00712-7. [Epub ahead of print]81(18): 3670-3671
      Schuler et al. (2021) demonstrate that mitochondrial-derived compartments protect cells from amino acid toxicity by activation of amino acid catabolism through the Ehrlich pathway, thus highlighting the incredible plasticity of mitochondria in rewiring cellular metabolism.
    DOI:  https://doi.org/10.1016/j.molcel.2021.08.032
  19. Cancer Discov. 2021 Jun 10. pii: candisc.0276.2021. [Epub ahead of print]
      While cancers evolve during disease progression and in response to therapy, temporal dynamics remain difficult to study in humans due to the lack of consistent barcodes marking individual clones in vivo. We employ mitochondrial single-cell assay for transposase-accessible chromatin with sequencing to profile 163,279 cells from 9 patients with chronic lymphocytic leukemia (CLL) collected across disease course and utilize mitochondrial DNA (mtDNA) mutations as natural genetic markers of cancer clones. We observe stable propagation of mtDNA mutations over years in the absence of strong selective pressure indicating clonal persistence, but dramatic changes following tight bottlenecks including disease transformation and relapse post-therapy, paralleled by acquisition of copy number variants, changes in chromatin accessibility and gene expression. Furthermore, we link CLL subclones to distinct chromatin states, providing insight into non-genetic sources of relapse. mtDNA mutations thus mirror disease history and provide naturally-occurring genetic barcodes to enable patient-specific study of cancer subclonal dynamics.
    DOI:  https://doi.org/10.1158/2159-8290.CD-21-0276
  20. Mol Genet Metab. 2021 Sep 14. pii: S1096-7192(21)00778-2. [Epub ahead of print]
      Acyl CoA Dehydrogenase 9 (ACAD9) is a member of the family of flavoenzymes that catalyze the dehydrogenation of acyl-CoAs to 2,3 enoyl-CoAs in mitochondrial fatty acid oxidation (FAO). Inborn errors of metabolism of all family members, including ACAD9, have been described in humans, and represent significant causes of morbidity and mortality particularly in children. ACAD9 deficiency leads to a combined defect in fatty acid oxidation and oxidative phosphorylation (OXPHOS) due to a dual role in the pathways. In addition to its function in mitochondrial FAO, ACAD9 has a second function as one of 14 factors responsible for assembly of complex I of the electron transport chain (ETC). Considerable controversy remains over the relative role of these two functions in normal physiology and the disparate clinical findings described in patients with ACAD9 deficiency. To better understand the normal function of ACAD9 and the pathophysiology of its deficiency, several knock out mouse models were developed. Homozygous total body knock out appeared to be lethal as no ACAD9 animals were obtained. Cre-lox technology was then used to generate tissue-specific deletion of the gene. Cardiac-specific ACAD9 deficient animals had severe neonatal cardiomyopathy and died by 17 days of age. They had severe mitochondrial dysfunction in vitro. Muscle-specific mutants were viable but exhibited muscle weakness. Additional studies of heart muscle from the cardiac specific deficient animals were used to examine the evolutionarily conserved signaling Intermediate in toll pathway (ECSIT) protein, a known binding partner of ACAD9 in the electron chain complex I assembly pathway. As expected, ECSIT levels were significantly reduced in the absence of ACAD9 protein, consistent with the demonstrated impairment of the complex I assembly. The various ACAD9 deficient animals should serve as useful models for development of novel therapeutics for this disorder.
    Keywords:  Acyl-CoA dehydrogenase 9; Cardiomyopathy; Fatty acid oxidation; Mouse models; Myopathy; Respiratory chain; Supercomplexes
    DOI:  https://doi.org/10.1016/j.ymgme.2021.09.002
  21. Mol Cell. 2021 Sep 16. pii: S1097-2765(21)00688-2. [Epub ahead of print]81(18): 3786-3802.e13
      Amino acids are essential building blocks of life. However, increasing evidence suggests that elevated amino acids cause cellular toxicity associated with numerous metabolic disorders. How cells cope with elevated amino acids remains poorly understood. Here, we show that a previously identified cellular structure, the mitochondrial-derived compartment (MDC), functions to protect cells from amino acid stress. In response to amino acid elevation, MDCs are generated from mitochondria, where they selectively sequester and deplete SLC25A nutrient carriers and their associated import receptor Tom70 from the organelle. Generation of MDCs promotes amino acid catabolism, and their formation occurs simultaneously with transporter removal at the plasma membrane via the multivesicular body (MVB) pathway. The combined loss of vacuolar amino acid storage, MVBs, and MDCs renders cells sensitive to high amino acid stress. Thus, we propose that MDCs operate as part of a coordinated cell network that facilitates amino acid homeostasis through post-translational nutrient transporter remodeling.
    Keywords:  MDC; Tom70; amino acid; lysosome; mitochondria; nutrient carrier; vacuole
    DOI:  https://doi.org/10.1016/j.molcel.2021.08.021
  22. Free Radic Biol Med. 2021 Sep 18. pii: S0891-5849(21)00725-5. [Epub ahead of print]
      The CISD2 (NAF-1) protein plays a key role in regulating cellular homeostasis, aging, cancer and neurodegenerative diseases. It was found to control different calcium, reactive oxygen species (ROS), and iron signaling mechanisms. However, since most studies of CISD2 to date were conducted with cells that constitutively lack, overexpress, or contain mutations in CISD2, the relationships between these different signaling processes are unclear. To address the hierarchy of signaling events occurring in cells upon CISD2 disruption, we developed an inducible system to express CISD2, or the dominant-negative H114C inhibitor of CISD2, in human breast cancer cells. Here, we report that inducible disruption of CISD2 function causes an immediate disruption in mitochondrial labile iron (mLI), and that this disruption results in enhanced mitochondrial ROS (mROS) levels. We further show that alterations in cytosolic and ER calcium levels occur only after the changes in mLI and mROS levels happen and are unrelated to them. Interestingly, disrupting CISD2 function resulted in the enhanced expression of the tumor suppressor thioredoxin-interacting protein (TXNIP) that was dependent on the accumulation of mLI and associated with ferroptosis activation. CISD2 could therefore regulate the expression of TXNIP in cancer cells, and this regulation is dependent on alterations in mLI levels.
    Keywords:  CISD2; Cancer; Ferroptosis; Iron homeostasis; Iron-sulfur cluster [Fe–S]; Mitochondria; NAF-1; Oxidative stress; Reactive oxygen species (ROS); TXNIP
    DOI:  https://doi.org/10.1016/j.freeradbiomed.2021.09.013
  23. Sci Rep. 2021 Sep 21. 11(1): 18770
      Mitochondrial malate dehydrogenase (MDH)-citrate synthase (CS) multi-enzyme complex is a part of the Krebs tricarboxylic acid (TCA) cycle 'metabolon' which is enzyme machinery catalyzing sequential reactions without diffusion of reaction intermediates into a bulk matrix. This complex is assumed to be a dynamic structure involved in the regulation of the cycle by enhancing metabolic flux. Microscale Thermophoresis analysis of the porcine heart MDH-CS complex revealed that substrates of the MDH and CS reactions, NAD+ and acetyl-CoA, enhance complex association while products of the reactions, NADH and citrate, weaken the affinity of the complex. Oxaloacetate enhanced the interaction only when it was present together with acetyl-CoA. Structural modeling using published CS structures suggested that the binding of these substrates can stabilize the closed format of CS which favors the MDH-CS association. Two other TCA cycle intermediates, ATP, and low pH also enhanced the association of the complex. These results suggest that dynamic formation of the MDH-CS multi-enzyme complex is modulated by metabolic factors responding to respiratory metabolism, and it may function in the feedback regulation of the cycle and adjacent metabolic pathways.
    DOI:  https://doi.org/10.1038/s41598-021-98314-z
  24. Mol Cancer Ther. 2021 Sep 22. pii: molcanther.0033.2021. [Epub ahead of print]
      Heme is an essential nutritional, metabolic, and signaling molecule in living organisms. Pathogenic microbes extract heme from hosts to obtain metallonutrient, while heme fuels mitochondrial respiration and ATP generation in lung tumor cells. Here, we generated small heme-sequestering proteins (HeSPs) based on bacterial hemophores. These HeSPs contain neutral mutations in the heme-binding pocket and hybrid sequences from hemophores of different bacteria. We showed that HeSPs bound to heme and effectively extracted heme from hemoglobin. They strongly inhibited heme uptake and cell proliferation and induced apoptosis in non-small lung cancer (NSCLC) cells, while their effects on non-tumorigenic cell lines representing normal lung cells were not significant. HeSPs strongly suppressed the growth of human NSCLC tumor xenografts in mice. HeSPs decreased oxygen consumption rates and ATP levels in tumor cells isolated from treated mice, while they did not affect liver and blood cell functions. Immunohistochemistry, along with data from Western blotting and functional assays, revealed that HeSPs reduced the levels of key proteins involved in heme uptake, as well as the consumption of major fuels for tumor cells, glucose and glutamine. Further, we found that HeSPs reduced the levels of angiogenic and vascular markers, as well as vessel density in tumor tissues. Together, these results demonstrate that HeSPs act via multiple mechanisms, including the inhibition of oxidative phosphorylation, to suppress tumor growth and progression. Evidently, heme sequestration can be a powerful strategy for suppressing lung tumors and likely drug-resistant tumors that rely on oxidative phosphorylation for survival.
    DOI:  https://doi.org/10.1158/1535-7163.MCT-21-0033
  25. Methods Mol Biol. 2022 ;2363 111-119
      Blue native electrophoresis (BN-PAGE) is a highly resolutive method suited to the study of high molecular weight protein complexes between 100 and >3000 kDa. One of the drawbacks of this method is that it is very time-consuming and requires high quantities of purified organelles. Here we describe a high throughput BN-PAGE method allowing to screen libraries of plants potentially altered in respiratory metabolism.
    Keywords:  Blue Native PAGE; Immunoblots; Mitochondria; Respiratory complexes
    DOI:  https://doi.org/10.1007/978-1-0716-1653-6_10
  26. Cell Syst. 2021 Sep 16. pii: S2405-4712(21)00338-0. [Epub ahead of print]
      NAD+ is an essential coenzyme for all living cells. NAD+ concentrations decline with age, but whether this reflects impaired production or accelerated consumption remains unclear. We employed isotope tracing and mass spectrometry to probe age-related changes in NAD+ metabolism across tissues. In aged mice, we observed modest tissue NAD+ depletion (median decrease ∼30%). Circulating NAD+ precursors were not significantly changed, and isotope tracing showed the unimpaired synthesis of nicotinamide from tryptophan. In most tissues of aged mice, turnover of the smaller tissue NAD+ pool was modestly faster such that absolute NAD+ biosynthetic flux was maintained, consistent with more active NAD+-consuming enzymes. Calorie restriction partially mitigated age-associated NAD+ decline by decreasing consumption. Acute inflammatory stress induced by LPS decreased NAD+ by impairing synthesis in both young and aged mice. Thus, the decline in NAD+ with normal aging is relatively subtle and occurs despite maintained NAD+ production, likely due to increased consumption.
    Keywords:  CD38; NAD; NADH; PARP; PARP1; SIRT1; aging; flux; mononucleotide; niacin; nicotinamide; redox; riboside; sirtuins
    DOI:  https://doi.org/10.1016/j.cels.2021.09.001
  27. J Cachexia Sarcopenia Muscle. 2021 Sep 24.
       BACKGROUND: Oxidative stress and damage are associated with a number of ageing phenotypes, including age-related loss of muscle mass and reduced contractile function (sarcopenia). Our group and others have reported loss of neuromuscular junction (NMJ) integrity and increased denervation as initiating factors in sarcopenia, leading to mitochondrial dysfunction, generation of reactive oxygen species and peroxides, and loss of muscle mass and weakness. Previous studies from our laboratory show that denervation-induced skeletal muscle mitochondrial peroxide generation is highly correlated to muscle atrophy. Here, we directly test the impact of scavenging muscle mitochondrial hydrogen peroxide on the structure and function of the NMJ and muscle mass and function in a mouse model of denervation-induced muscle atrophy CuZnSOD (Sod1-/- mice, Sod1KO).
    METHODS: Whole-body Sod1KO mice were crossed to mice with increased expression of human catalase (MCAT) targeted specifically to mitochondria in skeletal muscle (mMCAT mice) to determine the impact of reduced hydrogen peroxide levels on key targets of sarcopenia, including mitochondrial function, NMJ structure and function, and indices of muscle mass and function.
    RESULTS: Female adult (~12-month-old) Sod1KO mice show a number of sarcopenia-related phenotypes in skeletal muscle including reduced mitochondrial oxygen consumption and elevated reactive oxygen species generation, fragmentation, and loss of innervated NMJs (P < 0.05), a 30% reduction in muscle mass (P < 0.05), a 36% loss of force generation (P < 0.05), and a loss of exercise capacity (305 vs. 709 m in wild-type mice, P < 0.05). Muscle from Sod1KO mice also shows a 35% reduction in sarco(endo)plasmic reticulum ATPase activity (P < 0.05), changes in the amount of calcium-regulating proteins, and altered fibre-type composition. In contrast, increased catalase expression in the mMCAT × Sod1KO mice completely prevents the mitochondrial and NMJ-related phenotypes and maintains muscle mass and force generation. The reduction in exercise capacity is also partially inhibited (~35%, P < 0.05), and the loss of fibre cross-sectional area is inhibited by ~50% (P < 0.05).
    CONCLUSIONS: Together, these striking findings suggest that scavenging of mitochondrial peroxide generation by mMCAT expression efficiently prevents mitochondrial dysfunction and NMJ disruption associated with denervation-induced atrophy and weakness, supporting mitochondrial H2 O2 as an important effector of NMJ alterations that lead to phenotypes associated with sarcopenia.
    Keywords:  Catalase; Mitochondria; Neuromuscular junction; Oxidative stress; ROS; Skeletal muscle; Sod
    DOI:  https://doi.org/10.1002/jcsm.12768
  28. Cell Death Differ. 2021 Sep 23.
      Developing methods to improve the regenerative capacity of somatic stem cells (SSCs) is a major challenge in regenerative medicine. Here, we propose the forced expression of LIN28A as a method to modulate cellular metabolism, which in turn enhances self-renewal, differentiation capacities, and engraftment after transplantation of various human SSCs. Mechanistically, in undifferentiated/proliferating SSCs, LIN28A induced metabolic reprogramming from oxidative phosphorylation (OxPhos) to glycolysis by activating PDK1-mediated glycolysis-TCA/OxPhos uncoupling. Mitochondria were also reprogrammed into healthy/fused mitochondria with improved functional capacity. The reprogramming allows SSCs to undergo cell proliferation more extensively with low levels of oxidative and mitochondrial stress. When the PDK1-mediated uncoupling was untethered upon differentiation, LIN28A-SSCs differentiated more efficiently with an increase of OxPhos by utilizing the reprogrammed mitochondria. This study provides mechanistic and practical approaches of utilizing LIN28A and metabolic reprogramming in order to improve SSCs utility in regenerative medicine.
    DOI:  https://doi.org/10.1038/s41418-021-00873-1
  29. Oxid Med Cell Longev. 2021 ;2021 7569168
      Due to high energy and material metabolism requirements, mitochondria are frequently active in tumor cells. Our study found that the high energy metabolism status is positively correlated with the poor prognosis of patients with lung adenocarcinoma. We constructed a scoring system (mitoRiskscore) based on the gene expression of specific mitochondrial localized proteins through univariate and LASSO cox regression. It has been shown that high mitoRiskscore was correlated with a shorter survival time after surgery in patients with lung adenocarcinoma. Compared with the typical TNM grading system, the mitoRiskscore gene panel had higher prediction accuracy. A vast number of external verification results ensured its universality. Additionally, the mitoRiskscore could evaluate the metabolic pattern and chemotherapy sensitivity of the tumor samples. Lung adenocarcinoma with higher mitoRiskscore was more active in glycolysis, and oxidative phosphorylation expression of proliferation-related pathway genes was also significantly upregulated. In contrast, patients with low mitoRiskscore had similar metabolic patterns to normal tissues. In order to improve the accuracy of prediction ability and promote clinical usage, we developed a nomogram that combined mitoRiskscore and clinical prognostic factors to predict the 3-year, 5-year, and 10-year survival rates of patients. We also performed in vitro experiments to verify the function of the key genes in the mitoRiskscore panel. In conclusion, the mitoRiskscore scoring system may assist clinicians to judge the postoperative survival rate and chemotherapy of patients with lung adenocarcinoma.
    DOI:  https://doi.org/10.1155/2021/7569168
  30. Mitochondrion. 2021 Sep 15. pii: S1567-7249(21)00121-5. [Epub ahead of print]
      Mitochondria are dynamic, interactive organelles that connect cellular signaling and whole-cell homeostasis. This "mitochatting" allows the cell to receive information about the mitochondria's condition before accommodating energy demands. Mitofusin 2 (Mfn2), an outer mitochondrial membrane fusion protein specializes in mediating mitochondrial homeostasis. Early studies defined the biological significance of Mfn2, latter studies highlighted its role in substrate metabolism. However, determining Mfn2 potential to contribute to energy homeostasis needs study. This review summarizes current literature on mitochondrial metabolic processes, dynamics, and evidence of interactions among Mfn2 and regulatory processes that may link Mfn2's role in maintaining mitochondrial function and substrate metabolism.
    Keywords:  fatty acid oxidation; fission; fusion; glycolysis; mitochondrial dynamics; mitophagy
    DOI:  https://doi.org/10.1016/j.mito.2021.09.003
  31. Mol Cell. 2021 Sep 16. pii: S1097-2765(21)00692-4. [Epub ahead of print]81(18): 3803-3819.e7
      Mitochondrial dynamics regulated by mitochondrial fusion and fission maintain mitochondrial functions, whose alterations underline various human diseases. Here, we show that inositol is a critical metabolite directly restricting AMPK-dependent mitochondrial fission independently of its classical mode as a precursor for phosphoinositide generation. Inositol decline by IMPA1/2 deficiency elicits AMPK activation and mitochondrial fission without affecting ATP level, whereas inositol accumulation prevents AMPK-dependent mitochondrial fission. Metabolic stress or mitochondrial damage causes inositol decline in cells and mice to elicit AMPK-dependent mitochondrial fission. Inositol directly binds to AMPKγ and competes with AMP for AMPKγ binding, leading to restriction of AMPK activation and mitochondrial fission. Our study suggests that the AMP/inositol ratio is a critical determinant for AMPK activation and establishes a model in which AMPK activation requires inositol decline to release AMPKγ for AMP binding. Hence, AMPK is an inositol sensor, whose inactivation by inositol serves as a mechanism to restrict mitochondrial fission.
    Keywords:  AMP; AMPK; IMPA1; energy stress; glucose deprivation; inosiotl sensor; inositol; inositol/AMP ratio; mitochondrial fission; mitocondrial dynamics
    DOI:  https://doi.org/10.1016/j.molcel.2021.08.025
  32. Elife. 2021 Sep 21. pii: e68394. [Epub ahead of print]10
      Gene knockout of the master regulator of mitochondrial fission, Drp1, prevents neoplastic transformation. Also, mitochondrial fission and its opposing process of mitochondrial fusion are emerging as crucial regulators of stemness. Intriguingly, stem/progenitor cells maintaining repressed mitochondrial fission are primed for self-renewal and proliferation. Using our newly derived carcinogen transformed human cell model we demonstrate that fine-tuned Drp1 repression primes a slow cycling 'stem/progenitor-like state', which is characterized by small networks of fused mitochondria and a gene-expression profile with elevated functional stem/progenitor markers (Krt15, Sox2 etc) and their regulators (Cyclin E). Fine tuning Drp1 protein by reducing its activating phosphorylation sustains the neoplastic stem cell markers. Whereas, fine-tuned reduction of Drp1 protein maintains the characteristic mitochondrial shape and gene-expression of the primed 'stem/progenitor-like state' to accelerate neoplastic transformation, and more complete reduction of Drp1 protein prevents it. Therefore, our data highlights a 'goldilocks'; level of Drp1 repression supporting stem/progenitor state dependent neoplastic transformation.
    Keywords:  cancer biology; cell biology; mouse
    DOI:  https://doi.org/10.7554/eLife.68394
  33. Cancer Metab. 2021 Sep 23. 9(1): 33
       INTRODUCTION: The transcription factor MYC is overexpressed in 30% of small cell lung cancer (SCLC) tumors and is known to modulate the balance between two major pathways of metabolism: glycolysis and mitochondrial respiration. This duality of MYC underscores the importance of further investigation into its role in SCLC metabolism and could lead to insights into metabolic targeting approaches.
    METHODS: We investigated differences in metabolic pathways in transcriptional and metabolomics datasets based on cMYC expression in patient and cell line samples. Metabolic pathway utilization was evaluated by flow cytometry and Seahorse extracellular flux methodology. Glycolysis inhibition was evaluated in vitro and in vivo using PFK158, a small molecular inhibitor of PFKFB3.
    RESULTS: MYC-overexpressing SCLC patient samples and cell lines exhibited increased glycolysis gene expression directly mediated by MYC. Further, MYC-overexpressing cell lines displayed enhanced glycolysis consistent with the Warburg effect, while cell lines with low MYC expression appeared more reliant on oxidative metabolism. Inhibition of glycolysis with PFK158 preferentially attenuated glucose uptake, ATP production, and lactate in MYC-overexpressing cell lines. Treatment with PFK158 in xenografts delayed tumor growth and decreased glycolysis gene expression.
    CONCLUSIONS: Our study highlights an in-depth characterization of SCLC metabolic programming and presents glycolysis as a targetable mechanism downstream of MYC that could offer therapeutic benefit in a subset of SCLC patients.
    Keywords:  Glycolysis; MYC; Metabolism; PFK158; Small cell lung cancer
    DOI:  https://doi.org/10.1186/s40170-021-00270-9
  34. Exp Ther Med. 2021 Nov;22(5): 1246
      Glioma is a common malignant tumor of the central nervous system, accounting for ~50% of intracranial tumors. The current standard therapy for glioma is surgical resection followed by postoperative adjuvant radiotherapy and temozolomide (TMZ) chemotherapy. However, resistance to TMZ is one of the factors affecting prognosis. It has been reported that TNF receptor-associated protein 1 (TRAP1) is overexpressed in numerous types of tumor and that interfering with its function may abrogate chemotherapy resistance. TRAP1 inhibitor Gamitrinib triphenylphosphonium (G-TPP) and shRNA were used in the present study to suppress the function of this molecule in glioblastoma multiforme (GBM) cell lines. MTT assay was performed to evaluate the combined effect of G-TPP and TMZ treatment. To investigate the underlying mechanism responsible for this combined effect, the mitochondrial unfolded protein response (mtUPR), mitophagy, mitochondrial fusion and reactive oxygen species (ROS) were quantified using western blotting and immunofluorescence techniques. TMZ treatment induced apoptosis in GBM cells by activating the p53 pathway, whilst simultaneously downregulating mitophagy and enhancing mitochondrial fusion. The latter may occur in order to compensate for the defect caused by downregulated mitophagy. Suppressing the function of TRAP1 disturbed this compensatory mechanism by inducing mtUPR, which resulted in a burst of ROS formation and sensitized the GBM cells to the effects of TMZ treatment. Thus, suppressing the function of TRAP1 sensitized GBM cells to TMZ lysis by inducing mtUPR and the subsequent ROS burst. TRAP1 is therefore considered to be a promising target for GBM therapy.
    Keywords:  TNF receptor-associated protein 1; glioblastoma multiforme; mitochondrial unfolded protein response; reactive oxygen species; temozolomide
    DOI:  https://doi.org/10.3892/etm.2021.10681
  35. Cell Metab. 2021 Sep 17. pii: S1550-4131(21)00421-6. [Epub ahead of print]
      One of the defining characteristics of a pre-metastatic niche, a fundamental requirement for primary tumor metastasis, is infiltration of immunosuppressive macrophages. How these macrophages acquire their phenotype remains largely unexplored. Here, we demonstrate that tumor-derived exosomes (TDEs) polarize macrophages toward an immunosuppressive phenotype characterized by increased PD-L1 expression through NF-kB-dependent, glycolytic-dominant metabolic reprogramming. TDE signaling through TLR2 and NF-κB leads to increased glucose uptake. TDEs also stimulate elevated NOS2, which inhibits mitochondrial oxidative phosphorylation resulting in increased conversion of pyruvate to lactate. Lactate feeds back on NF-κB, further increasing PD-L1. Analysis of metastasis-negative lymph nodes of non-small-cell lung cancer patients revealed that macrophage PD-L1 positively correlates with levels of GLUT-1 and vesicle release gene YKT6 from primary tumors. Collectively, our study provides a novel mechanism by which macrophages within a pre-metastatic niche acquire their immunosuppressive phenotype and identifies an important link among exosomes, metabolism, and metastasis.
    Keywords:  NF-kB; PD-L1; exosomes; glycolysis; immunosuppression; lactate; metastasis
    DOI:  https://doi.org/10.1016/j.cmet.2021.09.002
  36. iScience. 2021 Sep 24. 24(9): 103038
      Mitochondrial biogenesis is a cell response to external stimuli which is generally believed to suppress apoptosis. However, during the process of apoptosis, whether mitochondrial biogenesis occurs in the early stage of the apoptotic cells remains unclear. To address this question, we constructed the COX8-EGFP-ACTIN-mCherry HeLa cells with recombinant fluorescent proteins respectively tagged on the nucleus and mitochondria and monitored the mitochondrial changes in the living cells exposed to gamma-ray radiation. Besides in situ detection of mitochondrial fluorescence changes, we also examined the cell viability, nuclear DNA damage, reactive oxygen species (ROS), mitochondrial superoxide, citrate synthase activity, ATP, cytoplasmic and mitochondrial calcium, mitochondrial mass, mitochondrial morphology, and protein expression related to mitochondrial biogenesis, as well as the apoptosis biomarkers. As a result, we confirmed that significant mitochondrial biogenesis took place preceding the radiation-induced apoptosis, and it was closely correlated with the apoptotic cells at late stage. The involved mechanism was also discussed.
    Keywords:  Biochemistry methods; Biomolecular engineering; Cell biology
    DOI:  https://doi.org/10.1016/j.isci.2021.103038
  37. Mol Cell. 2021 Sep 16. pii: S1097-2765(21)00685-7. [Epub ahead of print]81(18): 3691-3707
      Redox reactions are intrinsically linked to energy metabolism. Therefore, redox processes are indispensable for organismal physiology and life itself. The term reactive oxygen species (ROS) describes a set of distinct molecular oxygen derivatives produced during normal aerobic metabolism. Multiple ROS-generating and ROS-eliminating systems actively maintain the intracellular redox state, which serves to mediate redox signaling and regulate cellular functions. ROS, in particular hydrogen peroxide (H2O2), are able to reversibly oxidize critical, redox-sensitive cysteine residues on target proteins. These oxidative post-translational modifications (PTMs) can control the biological activity of numerous enzymes and transcription factors (TFs), as well as their cellular localization or interactions with binding partners. In this review, we describe the diverse roles of redox regulation in the context of physiological cellular metabolism and provide insights into the pathophysiology of diseases when redox homeostasis is dysregulated.
    Keywords:  ROS; cysteine oxidation; hydrogen peroxide; post-translational modification; reactive oxygen species; redox metabolism; redox signaling
    DOI:  https://doi.org/10.1016/j.molcel.2021.08.018
  38. Proc Natl Acad Sci U S A. 2021 09 28. pii: e2106947118. [Epub ahead of print]118(39):
      Reduced succinate dehydrogenase (SDH) activity resulting in adverse succinate accumulation was previously considered relevant only in 0.05 to 0.5% of kidney cancers associated with germline SDH mutations. Here, we sought to examine a broader role for SDH loss in kidney cancer pathogenesis/progression. We report that underexpression of SDH subunits resulting in accumulation of oncogenic succinate is a common feature in clear cell renal cell carcinoma (ccRCC) (∼80% of all kidney cancers), with a marked adverse impact on survival in ccRCC patients (n = 516). We show that SDH down-regulation is a critical brake in the TCA cycle during ccRCC pathogenesis and progression. In exploring mechanisms of SDH down-regulation in ccRCC, we report that Von Hippel-Lindau loss-induced hypoxia-inducible factor-dependent up-regulation of miR-210 causes direct inhibition of the SDHD transcript. Moreover, shallow deletion of SDHB occurs in ∼20% of ccRCC. We then demonstrate that SDH loss-induced succinate accumulation contributes to adverse loss of 5-hydroxymethylcytosine, gain of 5-methylcytosine, and enhanced invasiveness in ccRCC via inhibition of ten-eleven translocation (TET)-2 activity. Intriguingly, binding affinity between the catalytic domain of recombinant TET-2 and succinate was found to be very low, suggesting that the mechanism of succinate-induced attenuation of TET-2 activity is likely via product inhibition rather than competitive inhibition. Finally, exogenous ascorbic acid, a TET-activating demethylating agent, led to reversal of the above oncogenic effects of succinate in ccRCC cells. Collectively, our study demonstrates that functional SDH deficiency is a common adverse feature of ccRCC and not just limited to the kidney cancers associated with germline SDH mutations.
    Keywords:  TET-2; kidney cancer; succinate; succinate dehydrogenase
    DOI:  https://doi.org/10.1073/pnas.2106947118
  39. Bioengineered. 2021 Dec;12(1): 7143-7155
      Cancer stem cell (CSC) has been confirmed to trigger tumor occurrence and progression and CSC can develop strategies to maintain a lower reactive oxygen species (ROS) level compared to cancer cells. However, the mechanisms contributing to ROS homeostasis in CSC are still lacking key elements. In the current study, we found that reductive redox states and ROS levels were suppressed in non-adherent spheres formed by non-small cell lung cancer (NSCLC) cells, which were confirmed to hold CSC-like traits. However, mitochondria DNA content and cellular oxygen consumption rate analyses revealed fewer numbers of mitochondria in NSCLC spheres. Further exploration attributed this result to decreased mitochondrial biogenesis, likely resulted from the accelerated degradation of peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α). Mechanistic studies indicated that Ubiquilin 1 (UBQLN1) increased PGC1α protein stability via reducing the ubiquitination of PGC1α protein. Moreover, UBQLN1 was lowly expressed in NSCLC spheres compared to that in parental NSCLC cells and UBQLN1 overexpression suppressed the CSC-like traits of NSCLC cells, which was characterized as the decrease of ALDH1 activity, sphere-formation ability, and CSC marker expression. Finally, clinical investigations further demonstrated that UBQLN1 level was positively correlated with patient's survival of lung adenocarcinoma, but not squamous cell carcinoma of lung. Taken together, our results revealed a novel mechanism involving ROS homeostasis and mitochondrial biogenesis in non-small cell lung CSCs, which may provide novel potential targets and methods for NSCLC patients.
    Keywords:  PGC1α; UBQLN1; cancer stem cell; mitochondrial biogenesis; reactive oxygen species
    DOI:  https://doi.org/10.1080/21655979.2021.1979353
  40. Nat Metab. 2021 Sep;3(9): 1202-1216
      Excess nutrient uptake and altered hormone secretion in the gut contribute to a systemic energy imbalance, which causes obesity and an increased risk of type 2 diabetes and colorectal cancer. This functional maladaptation is thought to emerge at the level of the intestinal stem cells (ISCs). However, it is not clear how an obesogenic diet affects ISC identity and fate. Here we show that an obesogenic diet induces ISC and progenitor hyperproliferation, enhances ISC differentiation and cell turnover and changes the regional identities of ISCs and enterocytes in mice. Single-cell resolution of the enteroendocrine lineage reveals an increase in progenitors and peptidergic enteroendocrine cell types and a decrease in serotonergic enteroendocrine cell types. Mechanistically, we link increased fatty acid synthesis, Ppar signaling and the Insr-Igf1r-Akt pathway to mucosal changes. This study describes molecular mechanisms of diet-induced intestinal maladaptation that promote obesity and therefore underlie the pathogenesis of the metabolic syndrome and associated complications.
    DOI:  https://doi.org/10.1038/s42255-021-00458-9
  41. Front Cell Dev Biol. 2021 ;9 738932
      Autophagy, an essential biological process that affects immunity, is a powerful tool that host cells can use to defend against infections caused by pathogenic microorganisms. Autophagy can not only initiate innate immune responses but also degrade the cellular components that provide the conditions for removing the invaders. However, hyperactivated or inhibited autophagy leads to mitochondrial dysfunction, which is harmful to the host itself and is involved in many types of diseases. Mitochondria perform the functions of biological oxidation and energy exchange. In addition, mitochondrial functions are closely related to cell death, oxygen radical formation, and disease. Accumulation of mitochondrial metabolites affects survival of intracellular pathogens. In this mini-review, we focus on the crosstalk between autophagy and mitochondrial homeostasis during infection.
    Keywords:  autophagy; dual role; homeostasis; mitochondria; pathogen infection
    DOI:  https://doi.org/10.3389/fcell.2021.738932
  42. Biochem Biophys Res Commun. 2021 Sep 14. pii: S0006-291X(21)01298-5. [Epub ahead of print]578 70-76
      Lung cancer is one of the most malignant and prevalent tumors and accounts for the vast majority of cancer death worldwide. However, the molecular mechanisms underlying lung cancer progression are poorly understood. Here, we reveal that both transcription and protein expression levels of Cox15 were increased in lung cancer. Nrf2 specifically binds to the Cox15 promoter and triggers Cox15 expression at the transcriptional level. Cox15 functions as a novel oncogene that facilitates lung cancer cell proliferation. Additionally, Aripiprazole, a potent inhibitor of Cox15, executives profoundly suppressive effects on lung cancers cells growth and tumor progression in vivo and in vitro through exerting therapeutic effects. Taken together, our results unravel that Cox15 holds great potential to act as a prognostic molecule for lung cancer patients' prognosis in the future.
    Keywords:  Aripiprazole; Biomarker; Cox15; Lung cancer; Targeted therapy
    DOI:  https://doi.org/10.1016/j.bbrc.2021.09.010
  43. Stem Cell Reports. 2021 Sep 14. pii: S2213-6711(21)00434-3. [Epub ahead of print]
      Therapeutic application of induced pluripotent stem cell (iPSC) derivatives requires comprehensive assessment of the integrity of their nuclear and mitochondrial DNA (mtDNA) to exclude oncogenic potential and functional deficits. It is unknown, to which extent mtDNA variants originate from their parental cells or from de novo mutagenesis, and whether dynamics in heteroplasmy levels are caused by inter- and intracellular selection or genetic drift. Sequencing of mtDNA of 26 iPSC clones did not reveal evidence for de novo mutagenesis, or for any selection processes during reprogramming or differentiation. Culture expansion, however, selected against putatively actionable mtDNA mutations. Altogether, our findings point toward a scenario in which intracellular selection of mtDNA variants during culture expansion shapes the mutational landscape of the mitochondrial genome. Our results suggest that intercellular selection and genetic drift exert minor impact and that the bottleneck effect in context of the mtDNA genetic pool might have been overestimated.
    Keywords:  genomic integrity; induced pluripotent stem cells; mitochondrial genome; prolonged expansion culture; reprogramming; selection; small-scale mutations
    DOI:  https://doi.org/10.1016/j.stemcr.2021.08.016
  44. Leukemia. 2021 Sep 24.
      While the understanding of the genomic aberrations that underpin chronic and acute myeloid leukaemia (CML and AML) has allowed the development of therapies for these diseases, limitations remain. These become apparent when looking at the frequency of treatment resistance leading to disease relapse in leukaemia patients. Key questions regarding the fundamental biology of the leukaemic cells, such as their metabolic dependencies, are still unresolved. Even though a majority of leukaemic cells are killed during initial treatment, persistent leukaemic stem cells (LSCs) and therapy-resistant cells are still not eradicated with current treatments, due to various mechanisms that may contribute to therapy resistance, including cellular metabolic adaptations. In fact, recent studies have shown that LSCs and treatment-resistant cells are dependent on mitochondrial metabolism, hence rendering them sensitive to inhibition of mitochondrial oxidative phosphorylation (OXPHOS). As a result, rewired energy metabolism in leukaemic cells is now considered an attractive therapeutic target and the significance of this process is increasingly being recognised in various haematological malignancies. Therefore, identifying and targeting aberrant metabolism in drug-resistant leukaemic cells is an imperative and a relevant strategy for the development of new therapeutic options in leukaemia. In this review, we present a detailed overview of the most recent studies that present experimental evidence on how leukaemic cells can metabolically rewire, more specifically the importance of OXPHOS in LSCs and treatment-resistant cells, and the current drugs available to target this process. We highlight that uncovering specific energy metabolism dependencies will guide the identification of new and more targeted therapeutic strategies for myeloid leukaemia.
    DOI:  https://doi.org/10.1038/s41375-021-01416-w
  45. Mol Cell. 2021 Sep 16. pii: S1097-2765(21)00690-0. [Epub ahead of print]81(18): 3672-3674
      Igelmann et al. report a novel metabolic cycle, which they name HTC, that converts NADH into the key antioxidant factor NADPH. The HTC is repressed by the tumor suppressors p53 and RB, and this determines whether oncogene-expressing cells undergo senescence (HTCoff) or malignant transformation (HTCon).
    DOI:  https://doi.org/10.1016/j.molcel.2021.08.023