bims-camemi Biomed News
on Mitochondrial metabolism in cancer
Issue of 2021‒01‒17
thirty-nine papers selected by
Christian Frezza
University of Cambridge, MRC Cancer Unit

  1. Nat Commun. 2021 01 14. 12(1): 366
      Many tumour cells show dependence on exogenous serine and dietary serine and glycine starvation can inhibit the growth of these cancers and extend survival in mice. However, numerous mechanisms promote resistance to this therapeutic approach, including enhanced expression of the de novo serine synthesis pathway (SSP) enzymes or activation of oncogenes that drive enhanced serine synthesis. Here we show that inhibition of PHGDH, the first step in the SSP, cooperates with serine and glycine depletion to inhibit one-carbon metabolism and cancer growth. In vitro, inhibition of PHGDH combined with serine starvation leads to a defect in global protein synthesis, which blocks the activation of an ATF-4 response and more broadly impacts the protective stress response to amino acid depletion. In vivo, the combination of diet and inhibitor shows therapeutic efficacy against tumours that are resistant to diet or drug alone, with evidence of reduced one-carbon availability. However, the defect in ATF4-response seen in vitro following complete depletion of available serine is not seen in mice, where dietary serine and glycine depletion and treatment with the PHGDH inhibitor lower but do not eliminate serine. Our results indicate that inhibition of PHGDH will augment the therapeutic efficacy of a serine depleted diet.
  2. Trends Cell Biol. 2021 Jan 11. pii: S0962-8924(20)30251-8. [Epub ahead of print]
      Mitochondria are dynamic organelles that undergo cycles of fission and fusion events depending on cellular requirements. During mitochondrial division, the GTPase dynamin-related protein-1 is recruited to endoplasmic reticulum (ER)-induced mitochondrial constriction sites where it drives fission. However, the events required to complete scission of mitochondrial membranes are not well understood. Here, we emphasize the recently described roles for Golgi-derived phosphatidylinositol 4-phosphate (PI4P)-containing vesicles in the last steps of mitochondrial division. We then propose how trans-Golgi network vesicles at mitochondria-ER contact sites and PI4P generation could mechanistically execute mitochondrial division, by recruiting PI4P effectors and/or the actin nucleation machinery. Finally, we speculate on mechanisms to explain why such a complex dance of different organelles is required to facilitate the remodelling of mitochondrial membranes.
    Keywords:  Drp1; PI4P; TGN vesicles; membrane contact sites; mitochondrial division
  3. Trends Cell Biol. 2021 Jan 06. pii: S0962-8924(20)30255-5. [Epub ahead of print]
      Mammalian cells, with the exception of erythrocytes, harbor mitochondria, which are organelles that provide energy, intermediate metabolites, and additional activities to sustain cell viability, replication, and function. Mitochondria contain multiple copies of a circular genome called mitochondrial DNA (mtDNA), whose individual sequences are rarely identical (homoplasmy) because of inherited or sporadic mutations that result in multiple mtDNA genotypes (heteroplasmy). Here, we examine potential mechanisms for maintenance or shifts in heteroplasmy that occur in induced pluripotent stem cells (iPSCs) generated by cellular reprogramming, and further discuss manipulations that can alter heteroplasmy to impact stem and differentiated cell performance. This additional insight will assist in developing more robust iPSC-based models of disease and differentiated cell therapies.
    Keywords:  heteroplasmy; induced pluripotent stem cell; mitochondrial DNA; pluripotency; reprogramming
  4. Mol Clin Oncol. 2021 Feb;14(2): 42
      Endometrial cancer (EC) is one of the ten most common gynecological cancers. As in most cancers, EC tumour progression involves alterations in cellular metabolism and can be associated with, for instance, altered levels of glycolytic enzymes. Mitochondrial functions and proteins are known to serve key roles in tumour metabolism and progression. The transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator 1 (PGC1α) is a major regulator of mitochondrial biogenesis and function, albeit of varying prognostic value in different cancers. The voltage-dependent anion channel type 1 (VDAC1) regulates apoptosis as well as metabolite import and export over the mitochondrial outer membrane, and is often used for comparative quantification of mitochondrial content. Using immunohistochemistry, the present study examined protein expression levels of PGC1α and VDAC1 in tumour and paired benign tissue samples from 148 patients with EC, in order to examine associations with clinical data, such as stage and grade, Ki-67, p53 status, clinical resistance and overall survival. The expression levels of both PGC1α and VDAC1, as well as a PGC1α downstream effector, were significantly lower in tumor tissues than in benign tissues, suggesting altered mitochondrial function in EC. However, Kaplan-Meier, log rank and Spearman's rank correlation tests revealed that their expression was not correlated with survival and clinical data. Therefore, PGC1α and VDAC1 are not of major prognostic value in EC.
    Keywords:  coactivator 1; endometrial cancer; mitochondria; peroxisome proliferator-activated receptor γ; prognosis; voltage-dependent anion channel type 1
  5. Free Radic Biol Med. 2021 Jan 12. pii: S0891-5849(21)00035-6. [Epub ahead of print]
      Mitochondria are the powerhouses of the cell. They produce a significant amount of the energy we need to grow, survive and reproduce. The same system that generates energy in the form of ATP also produces Reactive Oxygen Species (ROS). Mitochondrial Reactive Oxygen Species (mtROS) were considered for many years toxic by-products of metabolism, responsible for ageing and many degenerative diseases. Today, we know that mtROS are essential redox messengers required to determine cell fate and maintain cellular homeostasis. Most mtROS are produced by respiratory complex I (CI) and complex III (CIII). How and when CI and CIII produce ROS is determined by the redox state of the Coenzyme Q (CoQ) pool and the proton motive force (pmf) generated during respiration. During ageing, there is an accumulation of defective mitochondria that generate high levels of mtROS. This causes oxidative stress and disrupts redox signalling. Here, we review how mtROS are generated in young and old mitochondria and how CI and CIII derived ROS control physiological and pathological processes. Finally, we discuss why damaged mitochondria amass during ageing as well as methods to preserve mitochondrial redox signalling with age.
    Keywords:  Coenzyme Q; ROS; ageing; complex I; complex III; mitochondria; redox signalling
  6. J Clin Invest. 2021 Jan 14. pii: 141380. [Epub ahead of print]
      Mutations affecting mitochondrial coenzyme Q (CoQ) biosynthesis lead to kidney failure due to selective loss of podocytes, essential cells of the kidney filter. Curiously, neighboring tubular epithelial cells are spared early in disease, despite higher mitochondrial content. We sought to illuminate non-canonical, cell-specific roles for CoQ, independent of the electron transport chain (ETC). Here we demonstrate that CoQ depletion caused by Pdss2 enzyme deficiency in podocytes results in perturbations in polyunsaturated fatty acid (PUFA) metabolism and the Braf/Mapk pathway, rather than ETC dysfunction. Single nucleus RNA sequencing from kidneys of Pdss2kd/kd mice with nephrotic syndrome and global CoQ-deficiency identified a podocyte-specific perturbation of the Braf/Mapk pathway. Treatment with GDC-0879, a Braf/Mapk-targeting compound ameliorated kidney disease in Pdss2kd/kd mice. Mechanistic studies in Pdss2-depleted podocytes revealed a previously unknown perturbation in PUFA metabolism that was confirmed in vivo. Gpx4, an enzyme that protects against PUFA-mediated lipid peroxidation, was elevated in disease and restored after GDC-0879 treatment. We demonstrate broader human disease relevance by uncovering patterns of GPX4 and Braf/Mapk pathway gene expression in tissue from patients with kidney diseases. Our studies reveal ETC-independent roles for CoQ in podocytes and point to Braf/Mapk as a candidate pathway for the treatment of kidney diseases.
    Keywords:  Cell Biology; Chronic kidney disease; Genetic diseases; Mitochondria; Nephrology
  7. Nat Commun. 2021 01 12. 12(1): 290
      The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes hepatic toxicity associated with prominent lipid accumulation in humans. Here, the authors report that the lysosomal copper transporter SLC46A3 is induced by TCDD and underlies the hepatic lipid accumulation in mice, potentially via effects on mitochondrial function. SLC46A3 was localized to the lysosome where it modulated intracellular copper levels. Forced expression of hepatic SLC46A3 resulted in decreased mitochondrial membrane potential and abnormal mitochondria morphology consistent with lower copper levels. SLC46A3 expression increased hepatic lipid accumulation similar to the known effects of TCDD exposure in mice and humans. The TCDD-induced hepatic triglyceride accumulation was significantly decreased in Slc46a3-/- mice and was more pronounced when these mice were fed a high-fat diet, as compared to wild-type mice. These data are consistent with a model where lysosomal SLC46A3 induction by TCDD leads to cytosolic copper deficiency resulting in mitochondrial dysfunction leading to lower lipid catabolism, thus linking copper status to mitochondrial function, lipid metabolism and TCDD-induced liver toxicity.
  8. Mol Cell Proteomics. 2020 Jan;pii: S1535-9476(20)30006-2. [Epub ahead of print]19(1): 65-77
      NDUFAB1 is the mitochondrial acyl carrier protein (ACP) essential for cell viability. Through its pantetheine-4'-phosphate post-translational modification, NDUFAB1 interacts with members of the leucine-tyrosine-arginine motif (LYRM) protein family. Although several LYRM proteins have been described to participate in a variety of defined processes, the functions of others remain either partially or entirely unknown. We profiled the interaction network of NDUFAB1 to reveal associations with 9 known LYRM proteins as well as more than 20 other proteins involved in mitochondrial respiratory chain complex and mitochondrial ribosome assembly. Subsequent knockout and interaction network studies in human cells revealed the LYRM member AltMiD51 to be important for optimal assembly of the large mitoribosome subunit, consistent with recent structural studies. In addition, we used proteomics coupled with topographical heat-mapping to reveal that knockout of LYRM2 impairs assembly of the NADH-dehydrogenase module of complex I, leading to defects in cellular respiration. Together, this work adds to the catalogue of functions executed by LYRM family of proteins in building mitochondrial complexes and emphasizes the common and essential role of NDUFAB1 as a protagonist in mitochondrial metabolism.
    Keywords:  Mitochondria function or biology; acyl-carrier protein; affinity proteomics; blue native polyacrylamide gel electrophoresis; complex I; protein complex analysis; protein structure; protein-protein interactions
  9. Mol Cell Proteomics. 2020 Jul;pii: S1535-9476(20)34978-1. [Epub ahead of print]19(7): 1145-1160
      Assembly factors play a critical role in the biogenesis of mitochondrial respiratory chain complexes I-IV where they assist in the membrane insertion of subunits, attachment of co-factors, and stabilization of assembly intermediates. The major fraction of complexes I, III and IV are present together in large molecular structures known as respiratory chain supercomplexes. Several assembly factors have been proposed as required for supercomplex assembly, including the hypoxia inducible gene 1 domain family member HIGD2A. Using gene-edited human cell lines and extensive steady state, translation and affinity enrichment proteomics techniques we show that loss of HIGD2A leads to defects in the de novo biogenesis of mtDNA-encoded COX3, subsequent accumulation of complex IV intermediates and turnover of COX3 partner proteins. Deletion of HIGD2A also leads to defective complex IV activity. The impact of HIGD2A loss on complex IV was not altered by growth under hypoxic conditions, consistent with its role being in basal complex IV assembly. Although in the absence of HIGD2A we show that mitochondria do contain an altered supercomplex assembly, we demonstrate it to harbor a crippled complex IV lacking COX3. Our results redefine HIGD2A as a classical assembly factor required for building the COX3 module of complex IV.
    Keywords:  Mitochondria function or biology; OXPHOS; cytochrome c oxidase; energy metabolism; knockouts; mitochondria; mitochondrial disease; mtDNA translation; respirasome; translation
  10. Int J Mol Sci. 2021 Jan 08. pii: E586. [Epub ahead of print]22(2):
      Mitochondria are ubiquitous intracellular organelles found in almost all eukaryotes and involved in various aspects of cellular life, with a primary role in energy production. The interest in this organelle has grown stronger with the discovery of their link to various pathologies, including cancer, aging and neurodegenerative diseases. Indeed, dysfunctional mitochondria cannot provide the required energy to tissues with a high-energy demand, such as heart, brain and muscles, leading to a large spectrum of clinical phenotypes. Mitochondrial defects are at the origin of a group of clinically heterogeneous pathologies, called mitochondrial diseases, with an incidence of 1 in 5000 live births. Primary mitochondrial diseases are associated with genetic mutations both in nuclear and mitochondrial DNA (mtDNA), affecting genes involved in every aspect of the organelle function. As a consequence, it is difficult to find a common cause for mitochondrial diseases and, subsequently, to offer a precise clinical definition of the pathology. Moreover, the complexity of this condition makes it challenging to identify possible therapies or drug targets.
    Keywords:  ATP production; biogenesis of the respiratory chain; mi-tochondrial electrochemical gradient; mitochondrial disease; mitochondrial potential; mitochondrial proton pumping; mitochondrial respiratory chain; oxidative phosphorylation; respiratory complex; respiratory supercomplex
  11. Front Oncol. 2020 ;10 596197
      The metabolism of cancer cells is an issue of dealing with fluctuating and limiting levels of nutrients in a precarious microenvironment to ensure their vitality and propagation. Glucose and glutamine are central metabolites for catabolic and anabolic metabolism, which is in the limelight of numerous diagnostic methods and therapeutic targeting. Understanding tumor metabolism in conditions of nutrient depletion is important for such applications and for interpreting the readouts. To exemplify the metabolic network of tumor cells in a model system, the fate 13C6-glucose was tracked in a breast cancer cell line growing in variable low glucose/low glutamine conditions. 13C-glucose-derived metabolites allowed to deduce the engagement of metabolic pathways, namely glycolysis, the TCA-cycle including glutamine and pyruvate anaplerosis, amino acid synthesis (serine, glycine, aspartate, glutamate), gluconeogenesis, and pyruvate replenishment. While the metabolic program did not change, limiting glucose and glutamine supply reduced cellular metabolite levels and enhanced pyruvate recycling as well as pyruvate carboxylation for entry into the TCA-cycle. Otherwise, the same metabolic pathways, including gluconeogenesis, were similarly engaged with physiologically saturating as with limiting glucose and glutamine. Therefore, the metabolic plasticity in precarious nutritional microenvironment does not require metabolic reprogramming, but is based on dynamic changes in metabolite quantity, reaction rates, and directions of the existing metabolic network.
    Keywords:  13C-glucose tracing; TCA-cycle; anaplerosis; glutamine; glycolysis; metabolic network; nutrient deprivation; pyruvate replenishment
  12. Nat Commun. 2021 Jan 15. 12(1): 377
      Circadian clocks coordinate mammalian behavior and physiology enabling organisms to anticipate 24-hour cycles. Transcription-translation feedback loops are thought to drive these clocks in most of mammalian cells. However, red blood cells (RBCs), which do not contain a nucleus, and cannot perform transcription or translation, nonetheless exhibit circadian redox rhythms. Here we show human RBCs display circadian regulation of glucose metabolism, which is required to sustain daily redox oscillations. We found daily rhythms of metabolite levels and flux through glycolysis and the pentose phosphate pathway (PPP). We show that inhibition of critical enzymes in either pathway abolished 24-hour rhythms in metabolic flux and redox oscillations, and determined that metabolic oscillations are necessary for redox rhythmicity. Furthermore, metabolic flux rhythms also occur in nucleated cells, and persist when the core transcriptional circadian clockwork is absent in Bmal1 knockouts. Thus, we propose that rhythmic glucose metabolism is an integral process in circadian rhythms.
  13. Cell Calcium. 2021 Jan 12. pii: S0143-4160(20)30175-5. [Epub ahead of print]94 102333
      Anti-apoptotic Bcl-2 critically controls cell death by neutralizing pro-apoptotic Bcl-2-family members at the mitochondria. Bcl-2 proteins also act at the endoplasmic reticulum, the main intracellular Ca2+-storage organelle, where they inhibit IP3 receptors (IP3R) and prevent pro-apoptotic Ca2+-signaling events. IP3R channels are targeted by the BH4 domain of Bcl-2. Some cancer types rely on the IP3R-Bcl-2 interaction for survival. We previously developed a cell-permeable, BH4-domain-targeting peptide that can abrogate Bcl-2's inhibitory action on IP3Rs, named Bcl-2 IP3 receptor disrupter-2 (BIRD-2). This peptide kills several Bcl-2-dependent cancer cell types, including diffuse large B-cell lymphoma (DLBCL) and chronic lymphocytic leukaemia (CLL) cells, by eliciting intracellular Ca2+ signalling. However, the exact mechanisms by which these excessive Ca2+ signals triggered by BIRD-2 provoke cancer cell death remain elusive. Here, we demonstrate in DLBCL that although BIRD-2 activates caspase 3/7 and provokes cell death in a caspase-dependent manner, the cell death is independent of pro-apoptotic Bcl-2-family members, Bim, Bax and Bak. Instead, BIRD-2 provokes mitochondrial Ca2+ overload that is rapidly followed by opening of the mitochondrial permeability transition pore (mPTP). Inhibiting mitochondrial Ca2+ overload using Ru265, an inhibitor of the mitochondrial Ca2+ uniporter complex counteracts BIRD-2-induced cancer cell death. Finally, we validated our findings in primary CLL patient samples where BIRD-2 provoked mitochondrial Ca2+ overload and Ru265 counteracted BIRD-2-induced cell death. Overall, this work reveals the mechanisms by which BIRD-2 provokes cell death, which occurs via mitochondrial Ca2+ overload but acts independently of pro-apoptotic Bcl-2-family members.
    Keywords:  Apoptosis; B-cell lymphoma 2; Calcium signaling; Mitochondrial permeability transition pore; Targeted therapy
  14. Mol Cell Proteomics. 2019 Mar;pii: S1535-9476(20)31854-5. [Epub ahead of print]18(3): 504-519
      The fumarate ester dimethyl fumarate (DMF) has been introduced recently as a treatment for relapsing remitting multiple sclerosis (RRMS), a chronic inflammatory condition that results in neuronal demyelination and axonal loss. DMF is known to act by depleting intracellular glutathione and modifying thiols on Keap1 protein, resulting in the stabilization of the transcription factor Nrf2, which in turn induces the expression of antioxidant response element genes. We have previously shown that DMF reacts with a wide range of protein thiols, suggesting that the complete mechanisms of action of DMF are unknown. Here, we investigated other intracellular thiol residues that may also be irreversibly modified by DMF in neurons and astrocytes. Using mass spectrometry, we identified 24 novel proteins that were modified by DMF in neurons and astrocytes, including cofilin-1, tubulin and collapsin response mediator protein 2 (CRMP2). Using an in vitro functional assay, we demonstrated that DMF-modified cofilin-1 loses its activity and generates less monomeric actin, potentially inhibiting its cytoskeletal remodeling activity, which could be beneficial in the modulation of myelination during RRMS. DMF modification of tubulin did not significantly impact axonal lysosomal trafficking. We found that the oxygen consumption rate of N1E-115 neurons and the levels of proteins related to mitochondrial energy production were only slightly affected by the highest doses of DMF, confirming that DMF treatment does not impair cellular respiratory function. In summary, our work provides new insights into the mechanisms supporting the neuroprotective and remyelination benefits associated with DMF treatment in addition to the antioxidant response by Nrf2.
    Keywords:  Chemical biology; Dimethyl fumarate; Drug targets*; Mechanism of action; Post-translational modifications*; Protein adducts; Succination
  15. Mol Cell Proteomics. 2019 Feb;pii: S1535-9476(20)31868-5. [Epub ahead of print]18(2): 231-244
      Cancer cells are known to reprogram their metabolism to adapt to adverse conditions dictated by tumor growth and microenvironment. A subtype of cancer cells with stem-like properties, known as cancer stem cells (CSC), is thought to be responsible for tumor recurrence. In this study, we demonstrated that CSC and chemoresistant cells derived from triple negative breast cancer cells display an enrichment of up- and downregulated proteins from metabolic pathways that suggests their dependence on mitochondria for survival. Here, we selected antibiotics, in particular - linezolid, inhibiting translation of mitoribosomes and inducing mitochondrial dysfunction. We provided the first in vivo evidence demonstrating that linezolid suppressed tumor growth rate, accompanied by increased autophagy. In addition, our results revealed that bactericidal antibiotics used in combination with autophagy blocker decrease tumor growth. This study puts mitochondria in a spotlight for cancer therapy and places antibiotics as effective agents for eliminating CSC and resistant cells.
    Keywords:  Antibiotics; Autophagy; Breast Cancer; Cancer Biology; Cancer Stem Cells; Chemoresistance; Clinical Proteomics; Mitochondria; Mitochondria Function or Biology; NMR; NMR-metabolomic; Stem Cells
  16. Elife. 2021 Jan 13. pii: e63102. [Epub ahead of print]10
      Generating mammalian cells with specific mtDNA-nDNA combinations is desirable but difficult to achieve and would be enabling for studies of mitochondrial-nuclear communication and coordination in controlling cell fates and functions. We developed 'MitoPunch', a pressure-driven mitochondrial transfer device, to deliver isolated mitochondria into numerous target mammalian cells simultaneously. MitoPunch and MitoCeption, a previously described force-based mitochondrial transfer approach, both yield stable isolated mitochondrial recipient (SIMR) cells that permanently retain exogenous mtDNA, whereas coincubation of mitochondria with cells does not yield SIMR cells. Although a typical MitoPunch or MitoCeption delivery results in dozens of immortalized SIMR clones with restored oxidative phosphorylation, only MitoPunch can produce replication-limited, non-immortal human SIMR clones. The MitoPunch device is versatile, inexpensive to assemble, and easy to use for engineering mtDNA-nDNA combinations to enable fundamental studies and potential translational applications.
    Keywords:  cell biology; human
  17. Mol Cell Proteomics. 2019 Oct;pii: S1535-9476(20)31532-2. [Epub ahead of print]18(10): 2078-2088
      Aging is characterized by a gradual deterioration in proteome. However, how protein dynamics that changes with normal aging and in disease is less well understood. Here, we profiled the snapshots of aging proteome in Drosophila, from head and muscle tissues of post-mitotic somatic cells, and the testis of mitotically-active cells. Our data demonstrated that dysregulation of proteome homeostasis, or proteostasis, might be a common feature associated with age. We further used pulsed metabolic stable isotope labeling analysis to characterize protein synthesis. Interestingly, this study determined an age-modulated decline in protein synthesis with age, particularly in the pathways related to mitochondria, neurotransmission, and proteostasis. Importantly, this decline became dramatically accelerated in Pink1 mutants, a Drosophila model of human age-related Parkinson's disease. Taken together, our multidimensional proteomic study revealed tissue-specific protein dynamics with age, highlighting mitochondrial and proteostasis-related proteins. We suggest that declines in proteostasis and mitochondria early in life are critical signals prior to the onset of aging and aging-associated diseases.
    Keywords:  Drosophila melanogaster; aging; mitochondria function or biology; protein synthesis; proteome homeostasis; quantification
  18. EMBO J. 2021 Jan 13. e104705
      Degradation of mitochondria via a selective form of autophagy, named mitophagy, is a fundamental mechanism conserved from yeast to humans that regulates mitochondrial quality and quantity control. Mitophagy is promoted via specific mitochondrial outer membrane receptors, or ubiquitin molecules conjugated to proteins on the mitochondrial surface leading to the formation of autophagosomes surrounding mitochondria. Mitophagy-mediated elimination of mitochondria plays an important role in many processes including early embryonic development, cell differentiation, inflammation, and apoptosis. Recent advances in analyzing mitophagy in vivo also reveal high rates of steady-state mitochondrial turnover in diverse cell types, highlighting the intracellular housekeeping role of mitophagy. Defects in mitophagy are associated with various pathological conditions such as neurodegeneration, heart failure, cancer, and aging, further underscoring the biological relevance. Here, we review our current molecular understanding of mitophagy, and its physiological implications, and discuss how multiple mitophagy pathways coordinately modulate mitochondrial fitness and populations.
    Keywords:  autophagy; mitochondria; phosphorylation; quality and quantity control; ubiquitin
  19. Nat Commun. 2021 01 12. 12(1): 339
      Tuberous sclerosis complex (TSC) integrates upstream stimuli and regulates cell growth by controlling the activity of mTORC1. TSC complex functions as a GTPase-activating protein (GAP) towards small GTPase Rheb and inhibits Rheb-mediated activation of mTORC1. Mutations in TSC genes cause tuberous sclerosis. In this study, the near-atomic resolution structure of human TSC complex reveals an arch-shaped architecture, with a 2:2:1 stoichiometry of TSC1, TSC2, and TBC1D7. This asymmetric complex consists of two interweaved TSC1 coiled-coil and one TBC1D7 that spans over the tail-to-tail TSC2 dimer. The two TSC2 GAP domains are symmetrically cradled within the core module formed by TSC2 dimerization domain and central coiled-coil of TSC1. Structural and biochemical analyses reveal TSC2 GAP-Rheb complimentary interactions and suggest a catalytic mechanism, by which an asparagine thumb (N1643) stabilizes γ-phosphate of GTP and accelerate GTP hydrolysis of Rheb. Our study reveals mechanisms of TSC complex assembly and GAP activity.
  20. Cancer Res. 2021 Jan 14. pii: canres.3430.2020. [Epub ahead of print]
      BRCA1-associated protein 1 (BAP1) is emerging as an intensively studied cancer-associated gene. Germline mutations in BAP1 lead to a cancer syndrome, and somatic loss is found in several cancer types. BAP1 encodes a deubiquitinase enzyme, which plays key roles in cell cycle regulation, cell death, and differentiation. Recent studies have demonstrated that BAP1 is also involved in several aspects of cellular metabolism, including metabolic homeostasis, glucose utilization, control of ferroptosis, and stress response. A better knowledge of the metabolic roles of cancer-associated genes is important to understanding tumor initiation and progression, as well as highlighting potential therapeutic avenues. With this review, we summarize the current knowledge regarding BAP1-mediated regulation of metabolic activities that may support new strategies to treat BAP1-mutated cancers.
  21. J Biol Chem. 2021 Jan 12. pii: S0021-9258(21)00051-X. [Epub ahead of print] 100283
      Metabolic reprogramming provides transformed cells with proliferative and/or survival advantages. Capitalizing on this therapeutically, however, has been only moderately successful due to the relatively small magnitude of these differences and because cancers may further adapt their metabolism to evade metabolic pathway inhibition. Mice lacking the peroxisomal bi-functional enzyme enoyl-CoA hydratase/3-hydroxyacyl CoA dehydrogenase (Ehhadh) and supplemented with the 12-carbon fatty acid lauric acid (C12) accumulate the toxic metabolite dodecanedioic acid (DDDA), which causes acute hepatocyte necrosis and liver failure. We noted that, in a murine model of pediatric hepatoblastoma (HB) and in primary human HBs, down-regulation of Ehhadh occurs in association with the suppression of mitochondrial β- and endosomal/peroxisomal ω-fatty acid oxidation (FAO) pathways. This suggested that HBs might be more susceptible than normal liver tissue to C12 dietary intervention. Indeed, HB-bearing mice provided with C12- and/or DDDA-supplemented diets survived significantly longer than those on standard diets. Additionally, larger tumors developed massive necrosis following short-term DDDA administration. In some HBs, the eventual development of DDDA resistance was associated with 129 transcript differences, ∼90% of which were down-regulated and ∼two-thirds of which correlated with survival in numerous human cancers. These transcripts often encoded extracellular matrix components, suggesting that DDDA resistance arises from reduced Ehhadh uptake. Lower Ehhadh expression was also noted in murine hepatocellular carcinomas and in subsets of certain human cancers, supporting the likely generality of these results. Our results demonstrate the feasibility of C12 or DDDA dietary supplementation that is non-toxic, inexpensive and likely compatible with more standard chemotherapies.
    Keywords:  Ehhadh; Warburg effect; cancer metabolism; fatty acid oxidation; hepatocellular carcinoma; metabolic re-programming; oxidative phosphorylation; peroxisome
  22. Proc Natl Acad Sci U S A. 2021 Jan 19. pii: e2008890118. [Epub ahead of print]118(3):
      OCT4 is a fundamental component of the molecular circuitry governing pluripotency in vivo and in vitro. To determine how OCT4 establishes and protects the pluripotent lineage in the embryo, we used comparative single-cell transcriptomics and quantitative immunofluorescence on control and OCT4 null blastocyst inner cell masses at two developmental stages. Surprisingly, activation of most pluripotency-associated transcription factors in the early mouse embryo occurs independently of OCT4, with the exception of the JAK/STAT signaling machinery. Concurrently, OCT4 null inner cell masses ectopically activate a subset of trophectoderm-associated genes. Inspection of metabolic pathways implicates the regulation of rate-limiting glycolytic enzymes by OCT4, consistent with a role in sustaining glycolysis. Furthermore, up-regulation of the lysosomal pathway was specifically detected in OCT4 null embryos. This finding implicates a requirement for OCT4 in the production of normal trophectoderm. Collectively, our findings uncover regulation of cellular metabolism and biophysical properties as mechanisms by which OCT4 instructs pluripotency.
    Keywords:  OCT4; STAT3 pathway; developmental biology; metabolism; single-cell profiling
  23. Mol Cell. 2021 Jan 06. pii: S1097-2765(20)30947-3. [Epub ahead of print]
      R-2-hydroxyglutarate (R-2HG), a metabolite produced by mutant isocitrate dehydrogenases (IDHs), was recently reported to exhibit anti-tumor activity. However, its effect on cancer metabolism remains largely elusive. Here we show that R-2HG effectively attenuates aerobic glycolysis, a hallmark of cancer metabolism, in (R-2HG-sensitive) leukemia cells. Mechanistically, R-2HG abrogates fat-mass- and obesity-associated protein (FTO)/N6-methyladenosine (m6A)/YTH N6-methyladenosine RNA binding protein 2 (YTHDF2)-mediated post-transcriptional upregulation of phosphofructokinase platelet (PFKP) and lactate dehydrogenase B (LDHB) (two critical glycolytic genes) expression and thereby suppresses aerobic glycolysis. Knockdown of FTO, PFKP, or LDHB recapitulates R-2HG-induced glycolytic inhibition in (R-2HG-sensitive) leukemia cells, but not in normal CD34+ hematopoietic stem/progenitor cells, and inhibits leukemogenesis in vivo; conversely, their overexpression reverses R-2HG-induced effects. R-2HG also suppresses glycolysis and downregulates FTO/PFKP/LDHB expression in human primary IDH-wild-type acute myeloid leukemia (AML) cells, demonstrating the clinical relevance. Collectively, our study reveals previously unrecognized effects of R-2HG and RNA modification on aerobic glycolysis in leukemia, highlighting the therapeutic potential of targeting cancer epitranscriptomics and metabolism.
    Keywords:  FTO; LDHB; N(6)-methyladenosine (m(6)A) modification; PFKP; R-2HG; RNA stability; YTHDF2; cancer metabolism; glycolysis; leukemia
  24. FASEB J. 2021 Feb;35(2): e21319
      The tumor suppressor p53 is known as a critical mediator of many cellular processes, including cellular senescence, but its role in mitochondrial dynamics is not fully understood. We have previously shown that p53 regulates mitochondrial dynamics via the PKA-Drp1 pathway to induce cellular senescence. In this study, to further understand the role of p53-dependent regulation of mitochondrial dynamics, the effect of p53 expression on mitochondrial morphology was examined in various cancer cell lines and normal human cells. We found that p53 induced remarkable mitochondrial elongation and cellular senescence in various cancer cells regardless of their p53 status. p53 also induced mitochondrial elongation in various human primary normal cells, suggesting that p53-mediated mitochondrial elongation is a general phenomenon. Moreover, we found that p53 plays an essential role in mitochondrial elongation in H-Ras-induced cellular senescence and in the replicative senescence of normal human cells. Treatment with the MDM-2 antagonist Nutlin-3a also induced mitochondrial elongation through the PKA-Drp1 pathway in IMR90 normal human cells. Furthermore, the inhibition of PKA activity in late-passage normal cells significantly reduced both mitochondrial elongation and cellular senescence, suggesting that the p53-PKA pathway is essential for maintaining the senescence phenotype in normal cells. Together, these results further confirm the direct regulation of mitochondrial dynamics by p53 and the important role of p53-mediated mitochondrial elongation in cellular senescence.
    Keywords:  Drp1; PKA; cellular senescence; mitochondrial dynamics; p53
  25. EMBO Rep. 2021 Jan 11. e51519
      The MYC oncoprotein activates and represses gene expression in a transcription-dependent or transcription-independent manner. Modification of mRNA emerges as a key gene expression regulatory nexus. We sought to determine whether MYC alters mRNA modifications and report here that MYC promotes cancer progression by down-regulating N6-methyladenosine (m6 A) preferentially in transcripts of a subset of MYC-repressed genes (MRGs). We find that MYC activates the expression of ALKBH5 and reduces m6 A levels in the mRNA of the selected MRGs SPI1 and PHF12. We also show that MYC-regulated m6 A controls the translation of MRG mRNA via the specific m6 A reader YTHDF3. Finally, we find that inhibition of ALKBH5, or overexpression of SPI1 or PHF12, effectively suppresses the growth of MYC-deregulated B-cell lymphomas, both in vitro and in vivo. Our findings uncover a novel mechanism by which MYC suppresses gene expression by altering m6 A modifications in selected MRG transcripts promotes cancer progression.
    Keywords:  ALKBH5; MYC; MYC-repressed genes; m6A; oncogenesis
  26. Methods Mol Biol. 2021 ;2240 207-230
      Depletion of oxygen (O2) levels and reduction in the ATP synthesis (or even its complete blockage) are important characteristics of mitochondrial dysfunction; features that are often correlated with neurodegeneration. The measurement of oxygen consumption rate (OCR) is thus essential to evaluate cellular metabolism, survival, and neuroprotective strategies. In the present chapter, we describe the oxygen consumption assay using a Clark-type oxygen electrode in different types of samples named cells suspension (from primary and established cell culture), brain slices (ex vivo), and fresh brain tissues. In addition, we demonstrate herein how the program Oxygraph can be used in order to analyze the data and different approaches to normalize it.
    Keywords:  Brain slice; Cell suspension; Cellular function; Metabolism; Mitochondria; Neural deregulation
  27. Nature. 2021 Jan 13.
      The activation of mostly quiescent haematopoietic stem cells (HSCs) is a prerequisite for life-long production of blood cells1. This process requires major molecular adaptations to allow HSCs to meet the regulatory and metabolic requirements for cell division2-4. The mechanisms that govern cellular reprograming upon stem-cell activation, and the subsequent return of stem cells to quiescence, have not been fully characterized. Here we show that chaperone-mediated autophagy (CMA)5, a selective form of lysosomal protein degradation, is involved in sustaining HSC function in adult mice. CMA is required for protein quality control in stem cells and for the upregulation of fatty acid metabolism upon HSC activation. We find that CMA activity in HSCs decreases with age and show that genetic or pharmacological activation of CMA can restore the functionality of old mouse and human HSCs. Together, our findings provide mechanistic insights into a role for CMA in sustaining quality control, appropriate energetics and overall long-term HSC function. Our work suggests that CMA may be a promising therapeutic target for enhancing HSC function in conditions such as ageing or stem-cell transplantation.
  28. Nat Metab. 2021 Jan 11.
      The mammalian liver is a central hub for systemic metabolic homeostasis. Liver tissue is spatially structured, with hepatocytes operating in repeating lobules, and sub-lobule zones performing distinct functions. The liver is also subject to extensive temporal regulation, orchestrated by the interplay of the circadian clock, systemic signals and feeding rhythms. However, liver zonation has previously been analysed as a static phenomenon, and liver chronobiology has been analysed at tissue-level resolution. Here, we use single-cell RNA-seq to investigate the interplay between gene regulation in space and time. Using mixed-effect models of messenger RNA expression and smFISH validations, we find that many genes in the liver are both zonated and rhythmic, and most of them show multiplicative space-time effects. Such dually regulated genes cover not only key hepatic functions such as lipid, carbohydrate and amino acid metabolism, but also previously unassociated processes involving protein chaperones. Our data also suggest that rhythmic and localized expression of Wnt targets could be explained by rhythmically expressed Wnt ligands from non-parenchymal cells near the central vein. Core circadian clock genes are expressed in a non-zonated manner, indicating that the liver clock is robust to zonation. Together, our scRNA-seq analysis reveals how liver function is compartmentalized spatio-temporally at the sub-lobular scale.
  29. Biochim Biophys Acta Bioenerg. 2021 Jan 07. pii: S0005-2728(21)00001-3. [Epub ahead of print] 148368
      This review provides a retrospective on the role of osmotic regulation in the process of eukaryogenesis. Specifically, it focuses on the adjustments which must have been made by the original colonizing α-proteobacteria that led to the evolution of modern mitochondria. We focus on the cations that are fundamentally involved in volume determination and cellular metabolism and define the transporter landscape in relation to these ions in mitochondria as we know today. We provide analysis on how the cations interplay and together maintain osmotic balance that allows for effective ATP synthesis in the organelle.
    Keywords:  cation transporters; evolution; metabolism; mitochondria; osmotic regulation
  30. Proc Natl Acad Sci U S A. 2021 Jan 05. pii: e2006476118. [Epub ahead of print]118(1):
      Inositol-1,4,5-triphosphate (IP3) kinase B (ITPKB) is a ubiquitously expressed lipid kinase that inactivates IP3, a secondary messenger that stimulates calcium release from the endoplasmic reticulum (ER). Genome-wide association studies have identified common variants in the ITPKB gene locus associated with reduced risk of sporadic Parkinson's disease (PD). Here, we investigate whether ITPKB activity or expression level impacts PD phenotypes in cellular and animal models. In primary neurons, knockdown or pharmacological inhibition of ITPKB increased levels of phosphorylated, insoluble α-synuclein pathology following treatment with α-synuclein preformed fibrils (PFFs). Conversely, ITPKB overexpression reduced PFF-induced α-synuclein aggregation. We also demonstrate that ITPKB inhibition or knockdown increases intracellular calcium levels in neurons, leading to an accumulation of calcium in mitochondria that increases respiration and inhibits the initiation of autophagy, suggesting that ITPKB regulates α-synuclein pathology by inhibiting ER-to-mitochondria calcium transport. Furthermore, the effects of ITPKB on mitochondrial calcium and respiration were prevented by pretreatment with pharmacological inhibitors of the mitochondrial calcium uniporter complex, which was also sufficient to reduce α-synuclein pathology in PFF-treated neurons. Taken together, these results identify ITPKB as a negative regulator of α-synuclein aggregation and highlight modulation of ER-to-mitochondria calcium flux as a therapeutic strategy for the treatment of sporadic PD.
    Keywords:  Parkinson’s disease; calcium signaling; genetics; mitochondria; α-synuclein
  31. Am J Physiol Cell Physiol. 2021 Jan 13.
      Tumor cell proliferation requires sufficient metabolic flux through the pentose phosphate pathway to meet the demand for biosynthetic precursors and to increase protection against oxidative stress which in turn requires an upregulation of substrate flow through glycolysis. This metabolic poise is often coupled with a shift in ATP production from mitochondrial OXPHOS to substrate-level phosphorylation. Despite major advances that were facilitated by using tumor-derived cell lines in research areas spanning from membrane to cytoskeletal biology, this distorted metabolic profile limits their impact as a model in physiology and toxicology. Substitution of glucose with galactose in the cell culture medium has been demonstrated to shift ATP production from substrate-level phosphorylation to mitochondrial OXPHOS. This increase in oxygen utilization is coupled to a global metabolic reorganization with potential impacts on macromolecule biosynthesis and cellular redox homeostasis, but a comprehensive analysis on the effects of sugar substitution in tumor-derived cells is still missing. To address this gap in knowledge we performed transcriptomic and metabolomic analyses on human hepatocellular carcinoma (HepG2) cells adapted to either glucose or galactose as the aldohexose source. We observed a shift towards oxidative metabolism in all primary metabolic pathways at both transcriptomic and metabolomic levels. We also observed a decrease in nicotinamide dinucleotide (NAD(P)) levels and subcellular NAD+-to-NADH ratios in cells cultured with galactose compared to glucose control cells. Our results suggest that galactose reduces both glycolytic and biosynthetic flux and restores a metabolic poise in HepG2 cells that closely reflects the metabolic state observed in primary hepatocytes.
    Keywords:  Cancer; Galactose; Mitochondria; NAD; Redox state
  32. Free Radic Biol Med. 2021 Jan 06. pii: S0891-5849(20)32120-1. [Epub ahead of print]
      Superoxide produced by mitochondria has been implicated in numerous physiologies and pathologies. Eleven different mitochondrial sites that can produce superoxide and/or hydrogen peroxide (O2.-/H2O2) have been identified in vitro, but little is known about their contributions in vivo. We introduce novel variants of S1QELs and S3QELs (small molecules that suppress O2.-/H2O2 production specifically from mitochondrial sites IQ and IIIQo, respectively, without compromising bioenergetics), that are suitable for use in vivo. When administered by intraperitoneal injection, they achieve total tissue concentrations exceeding those that are effective in vitro. We use them to study the engagement of sites IQ and IIIQo in mice lacking functional manganese-superoxide dismutase (SOD2). Lack of SOD2 is expected to elevate superoxide levels in the mitochondrial matrix, and leads to severe pathologies and death about 8 days after birth. Compared to littermate wild-type mice, 6-day-old Sod2-/- mice had significantly lower body weight, lower heart succinate dehydrogenase activity, and greater hepatic lipid accumulation. These pathologies were ameliorated by treatment with a SOD/catalase mimetic, EUK189, confirming previous observations. A 3-day treatment with S1QEL352 decreased the inactivation of cardiac succinate dehydrogenase and hepatic steatosis in Sod2-/- mice. S1QEL712, which has a distinct chemical structure, also decreased hepatic steatosis, confirming that O2.- derived specifically from mitochondrial site IQ is a significant driver of hepatic steatosis in Sod2-/- mice. These findings also demonstrate the ability of these new S1QELs to suppress O2.- production in the mitochondrial matrix in vivo. In contrast, suppressing site IIIQo using S3QEL941 did not protect, suggesting that site IIIQo does not contribute significantly to mitochondrial O2.- production in the hearts or livers of Sod2-/- mice. We conclude that the novel S1QELs are effective in vivo, and that site IQ runs in vivo and is a significant driver of pathology in Sod2-/- mice.
    Keywords:  Complex I; Complex II; Complex III; Mitochondria; S1QEL; S3QEL; hepatosteatosis; reactive oxygen species; succinate dehydrogenase; superoxide dismutase
  33. Cell Rep. 2021 Jan 12. pii: S2211-1247(20)31602-8. [Epub ahead of print]34(2): 108613
      Individuals with malaria exhibit increased morbidity and mortality when infected with Gram-negative (Gr-) bacteria. To explore this experimentally, we performed co-infection of mice with Plasmodium chabaudi and Citrobacter rodentium, an extracellular Gr- bacterial pathogen that infects the large intestine. While single infections are controlled effectively, co-infection results in enhanced virulence that is characterized by prolonged systemic bacterial persistence and high mortality. Mortality in co-infected mice is associated with disrupted iron metabolism, elevated levels of plasma heme, and increased mitochondrial reactive oxygen species (ROS) production by phagocytes. In addition, iron acquisition by the bacterium plays a key role in pathogenesis because co-infection with a mutant C. rodentium strain lacking a critical iron acquisition pathway does not cause mortality. These results indicate that disrupted iron metabolism may drive mortality during co-infection with C. rodentium and P. chabaudi by both altering host immune responses and facilitating bacterial persistence.
    Keywords:  Citrobacter rodentium; Gram-negative bacteria; Plasmodium; co-infection; heme; hemolysis; iron; malaria; mortality
  34. Sci Rep. 2021 Jan 14. 11(1): 1458
      T cell activation is intimately linked to metabolism, as distinct metabolic requirements support the functional and phenotypical differences between quiescent and activated T cells. Metabolic transition from mitochondrial oxidative phosphorylation to aerobic glycolysis is crucial for a proper T cell activation. However, the role of tricarboxylic acid cycle (TCA), and in particular succinate dehydrogenase (SDH) in activated T cells needs further elucidation. Here we show that inhibition of SDH during activation of T cells results in strong impairment of proliferation, expression of activation markers, and production of key inflammatory cytokines, despite a concomitant increase in glycolytic metabolic activity. Similar effect of SDH inhibition were demonstrated in pre-activated T cell. Interestingly, itaconic acid, an endogenous SDH inhibitor released from activated macrophages and dendritic cells, had no immunomodulator effect. Taken together, our findings demonstrate that SDH enzyme fitness is critical for mounting and maintaining appropriate activation and function of human T cells.
  35. Nat Commun. 2021 01 12. 12(1): 326
      Adipose tissue-resident T cells have been recognized as a critical regulator of thermogenesis and energy expenditure, yet the underlying mechanisms remain unclear. Here, we show that high-fat diet (HFD) feeding greatly suppresses the expression of disulfide-bond A oxidoreductase-like protein (DsbA-L), a mitochondria-localized chaperone protein, in adipose-resident T cells, which correlates with reduced T cell mitochondrial function. T cell-specific knockout of DsbA-L enhances diet-induced thermogenesis in brown adipose tissue (BAT) and protects mice from HFD-induced obesity, hepatosteatosis, and insulin resistance. Mechanistically, DsbA-L deficiency in T cells reduces IFN-γ production and activates protein kinase A by reducing phosphodiesterase-4D expression, leading to increased BAT thermogenesis. Taken together, our study uncovers a mechanism by which T cells communicate with brown adipocytes to regulate BAT thermogenesis and whole-body energy homeostasis. Our findings highlight a therapeutic potential of targeting T cells for the treatment of over nutrition-induced obesity and its associated metabolic diseases.
  36. Cell Rep. 2021 Jan 12. pii: S2211-1247(20)31613-2. [Epub ahead of print]34(2): 108624
      Thermoneutral conditions typical for standard human living environments result in brown adipose tissue (BAT) involution, characterized by decreased mitochondrial mass and increased lipid deposition. Low BAT activity is associated with poor metabolic health, and BAT reactivation may confer therapeutic potential. However, the molecular drivers of this BAT adaptive process in response to thermoneutrality remain enigmatic. Using metabolic and lipidomic approaches, we show that endogenous fatty acid synthesis, regulated by carbohydrate-response element-binding protein (ChREBP), is the central regulator of BAT involution. By transcriptional control of lipogenesis-related enzymes, ChREBP determines the abundance and composition of both storage and membrane lipids known to regulate organelle turnover and function. Notably, ChREBP deficiency and pharmacological inhibition of lipogenesis during thermoneutral adaptation preserved mitochondrial mass and thermogenic capacity of BAT independently of mitochondrial biogenesis. In conclusion, we establish lipogenesis as a potential therapeutic target to prevent loss of BAT thermogenic capacity as seen in adult humans.
    Keywords:  ChREBP; brown adipose tissue; cardiolipins; de novo lipogenesis; energy expenditure; fatty acid synthesis; fatty acids; lipidome; mitochondria; mitophagy; non-shivering thermogenesis; phospholipids; thermoneutrality; triacylglycerols; whitening
  37. Trends Ecol Evol. 2021 Jan 09. pii: S0169-5347(20)30354-2. [Epub ahead of print]
      Biologists have long appreciated the critical role that energy turnover plays in understanding variation in performance and fitness among individuals. Whole-organism metabolic studies have provided key insights into fundamental ecological and evolutionary processes. However, constraints operating at subcellular levels, such as those operating within the mitochondria, can also play important roles in optimizing metabolism over different energetic demands and time scales. Herein, we explore how mitochondrial aerobic metabolism influences different aspects of organismal performance, such as through changing adenosine triphosphate (ATP) and reactive oxygen species (ROS) production. We consider how such insights have advanced our understanding of the mechanisms underpinning key ecological and evolutionary processes, from variation in life-history traits to adaptation to changing thermal conditions, and we highlight key areas for future research.
    Keywords:  bioenergetics; life-history trade-off; metabolic rate; mitochondrial efficiency; mitochondrial uncoupling; reactive oxygen species
  38. Nat Commun. 2021 Jan 15. 12(1): 401
      Mechanisms regulating DNA repair processes remain incompletely defined. Here, the circadian factor CRY1, an evolutionally conserved transcriptional coregulator, is identified as a tumor specific regulator of DNA repair. Key findings demonstrate that CRY1 expression is androgen-responsive and associates with poor outcome in prostate cancer. Functional studies and first-in-field mapping of the CRY1 cistrome and transcriptome reveal that CRY1 regulates DNA repair and the G2/M transition. DNA damage stabilizes CRY1 in cancer (in vitro, in vivo, and human tumors ex vivo), which proves critical for efficient DNA repair. Further mechanistic investigation shows that stabilized CRY1 temporally regulates expression of genes required for homologous recombination. Collectively, these findings reveal that CRY1 is hormone-induced in tumors, is further stabilized by genomic insult, and promotes DNA repair and cell survival through temporal transcriptional regulation. These studies identify the circadian factor CRY1 as pro-tumorigenic and nominate CRY1 as a new therapeutic target.