bims-camemi 2019-03-17 papers

bims-camemi

Biomed News

on Mitochondrial metabolism in cancer

Issue of 2019‒03‒17
33 papers selected by
Christian Frezza,

ELMOD2 regulates mitochondrial fusion in a mitofusin-dependent manner, downstream of ARL2.
AMPK-mediated activation of MCU stimulates mitochondrial Ca2+ entry to promote mitotic progression.
UVB-induced inactivation of manganese-containing superoxide dismutase promotes mitophagy via ROS-mediated mTORC2 pathway activation.
The E3 ubiquitin ligase parkin is dispensable for metabolic homeostasis in murine pancreatic β cells and adipocytes.
Perm1 regulates CaMKII activation and shapes skeletal muscle responses to endurance exercise training.
Acute increases in O-GlcNAc indirectly impairs mitochondrial bioenergetics through dysregulation of LonP1-mediated mitochondrial protein complex turnover.
A KLF6-driven transcriptional network links lipid homeostasis and tumour growth in renal carcinoma.
Spatiotemporal Control of ULK1 Activation by NDP52 and TBK1 during Selective Autophagy.
Substrate-dependent and cyclophilin D-independent regulation of mitochondrial flashes in skeletal and cardiac muscle.
p53 Promotes Cancer Cell Adaptation to Glutamine Deprivation by Upregulating Slc7a3 to Increase Arginine Uptake.
Molecular evolution of proteins mediating mitochondrial fission-fusion dynamics.
Myoglobin induces mitochondrial fusion thereby inhibiting breast cancer cell proliferation.
Mitophagy regulates mitochondrial network signaling, oxidative stress, and apoptosis during myoblast differentiation.
ATP-citrate lyase multimerization is required for coenzyme-A substrate binding and catalysis.
Increased calpain-1 in mitochondria induces dilated heart failure in mice: role of mitochondrial superoxide anion.
The 'Achilles Heel' of Metabolism in Renal Cell Carcinoma: Glutaminase Inhibition as a Rational Treatment Strategy.
Energetic Trade-Offs and Hypometabolic States Promote Disease Tolerance.
Bim escapes displacement by BH3-mimetic anti-cancer drugs by double-bolt locking both Bcl-XL and Bcl-2.
p53 β-hydroxybutyrylation attenuates p53 activity.
Sigma-1 receptor ablation impairs autophagosome clearance.
PGC-1α overexpression partially rescues impaired oxidative and contractile pathophysiology following volumetric muscle loss injury.
Mutant and wild-type isocitrate dehydrogenase 1 share enhancing mechanisms involving distinct tyrosine kinase cascades in cancer.
TFEB-driven lysosomal biogenesis is pivotal for PGC1α-dependent renal stress resistance.
ER-mitochondria contacts are required for pexophagy in Saccharomyces cerevisiae.
Metabolic engineering of Chinese hamster ovary cells towards reduced biosynthesis and accumulation of novel growth inhibitors in fed-batch cultures.
Pivotal role of inter-organ aspartate metabolism for treatment of mitochondrial aspartate-glutamate carrier 2 (citrin) deficiency, based on the mouse model.
The 5-formyltetrahydrofolate futile cycle reduces pathway stochasticity in an extended hybrid-stochastic model of folate-mediated one-carbon metabolism.
Organelle Interplay - Peroxisome Interactions in Health and Disease.
High Glucose Triggers Nucleotide Imbalance through O-GlcNAcylation of Key Enzymes and Induces KRAS Mutation in Pancreatic Cells.
RIT1 oncoproteins escape LZTR1-mediated proteolysis.
Glycolysis Is an Intrinsic Factor for Optimal Replication of a Norovirus.
A GPR17-cAMP-Lactate Signaling Axis in Oligodendrocytes Regulates Whole-Body Metabolism.
Inflammatory macrophage dependence on NAD+ salvage is a consequence of reactive oxygen species-mediated DNA damage.

Mol Biol Cell. 2019 Mar 13. mbcE18120804

ELMOD2 regulates mitochondrial fusion in a mitofusin-dependent manner, downstream of ARL2.

Cara R Schiavon, Rachel E Turn, Laura E Newman, Richard A Kahn.

Mitochondria are essential and dynamic organelles, undergoing constant fission and fusion. The primary players in mitochondrial morphology (MFN1/2, OPA1, DRP1) have been identified, but their mechanism(s) of regulation are still being elucidated. ARL2 is a regulatory GTPase which has previously been shown to play a role in the regulation of mitochondrial morphology. Here we demonstrate that ELMOD2, an ARL2 GTPase activating protein (GAP), is necessary for ARL2 to promote mitochondrial elongation. We show that loss of ELMOD2 causes mitochondrial fragmentation and a lower rate of mitochondrial fusion, while ELMOD2 overexpression promotes mitochondrial tubulation and increases the rate of fusion in a mitofusin-dependent fashion. We also show that a mutant of ELMOD2 lacking GAP activity is capable of promoting fusion, suggesting that ELMOD2 does not require GAP activity to influence mitochondrial morphology. Finally, we show that ELMOD2, ARL2, Mitofusins 1 and 2, Miros 1 and 2, and mitochondrial phospholipase D (mitoPLD) all localize to discrete, regularly-spaced puncta along mitochondria. These results suggest that ELMOD2 is functioning as an effector downstream of ARL2 and upstream of the mitofusins to promote mitochondrial fusion. Our data provide insights into the pathway by which mitochondrial fusion is regulated in the cell.

DOI: https://doi.org/10.1091/mbc.E18-12-0804
Nat Cell Biol. 2019 Mar 11.

AMPK-mediated activation of MCU stimulates mitochondrial Ca2+ entry to promote mitotic progression.

Haixin Zhao, Teng Li, Kai Wang, Fei Zhao, Jiayi Chen, Guang Xu, Jie Zhao, Ting Li, Liang Chen, Lin Li, Qing Xia, Tao Zhou, Hui-Yan Li, Ai-Ling Li, Toren Finkel, Xue-Min Zhang, Xin Pan.

The capacity of cells to alter bioenergetics in response to the demands of various biological processes is essential for normal physiology. The coordination of energy sensing and production with highly energy-demanding cellular processes, such as cell division, is poorly understood. Here, we show that a cell cycle-dependent mitochondrial Ca2+ transient connects energy sensing to mitochondrial activity for mitotic progression. The mitochondrial Ca2+ uniporter (MCU) mediates a rapid mitochondrial Ca2+ transient during mitosis. Inhibition of mitochondrial Ca2+ transients via MCU depletion causes spindle checkpoint-dependent mitotic delay. Cellular ATP levels drop during early mitosis, and the mitochondrial Ca2+ transients boost mitochondrial respiration to restore energy homeostasis. This is achieved through mitosis-specific MCU phosphorylation and activation by the mitochondrial translocation of energy sensor AMP-activated protein kinase (AMPK). Our results establish a critical role for AMPK- and MCU-dependent mitochondrial Ca2+ signalling in mitosis and reveal a mechanism of mitochondrial metabolic adaptation to acute cellular energy stress.

DOI: https://doi.org/10.1038/s41556-019-0296-3
J Biol Chem. 2019 Mar 11. pii: jbc.RA118.006595. [Epub ahead of print]

UVB-induced inactivation of manganese-containing superoxide dismutase promotes mitophagy via ROS-mediated mTORC2 pathway activation.

Sanjit K Dhar, Ines Batinic-Haberle, Daret K St Clair.

  Mitochondria are major sites of energy metabolism that influence numerous cellular events including immunity and cancer development. Previously, we reported that the mitochondrion specific antioxidant enzyme, manganese containing superoxide dismutase (MnSOD), has dual roles in early- and late- carcinogenesis stages. However, how defective MnSOD impacts the chain of events that leads to cell transformation in pathologically normal epidermal cells that have been exposed to carcinogens is unknown. Here, we show that UVB radiation causes nitration and inactivation of MnSOD leading to mitochondrial injury and mitophagy. In keratinocytes, exposure to UVB radiation decreased mitochondrial oxidative phosphorylation, increased glycolysis and the expression of autophagy-related genes, and enhanced AKT Ser/Thr kinase (AKT) phosphorylation and cell growth. Interestingly, UVB initiated a prosurvival mitophagy response by mitochondria-mediated reactive oxygen species (ROS) signaling via the mammalian target of the mTOR complex 2 (mTORC2) pathway. Knock-down of rictor but not raptor abrogated UVB-induced mitophagy responses. Furthermore, fractionation and proximity-ligation assays reveal that ROS-mediated mTOC2 activation in mitochondria is necessary for UVB-induced mitophagy. Importantly, pretreatment with the MnSOD mimic MnTnBuOE-2-PyP5+ (MnP) attenuates mTORC2 activation and suppresses UVB-induced mitophagy. UVB radiation exposure also increased cell growth as assessed by soft-agar colony survival and cell growth assays, and pretreatment with MnP or the known autophagy inhibitor 3-MA, abrogated UVB-induced cell growth. These results indicat that MnSOD is a major redox regulator that maintains mitochondrial health and show that UVB-mediated MnSOD inactivation promotes mitophagy and thereby prevents accumulation of damaged mitochondria.

Keywords:  MnSOD; autophagy; cancer; mTOR; mTOR complex 2 (mTOR C2); metabolism; mitophagy; oxidative stress; reactive oxygen species (ROS); rictor

DOI:  https://doi.org/10.1074/jbc.RA118.006595
J Biol Chem. 2019 Mar 15. pii: jbc.RA118.006763. [Epub ahead of print]

The E3 ubiquitin ligase parkin is dispensable for metabolic homeostasis in murine pancreatic β cells and adipocytes.

Callie A S Corsa, Gemma L Pearson, Aaron Renberg, Matthew M Askar, Tracy Vozheiko, Ormond A MacDougald, Scott A Soleimanpour.

  The E3 ubiquitin ligase parkin is a critical regulator of mitophagy and has been identified as a susceptibility gene for type 2 diabetes (T2D). However, its role in metabolically active tissues that precipitate T2D development is unknown. Specifically, pancreatic β cells and adipocytes both rely heavily on mitochondrial function in the regulation of optimal glycemic control to prevent T2D, but parkin's role in preserving quality control of β-cell or adipocyte mitochondria is unclear. Although parkin has been previously reported to control mitophagy, here we show that parkin surprisingly is dispensable for glucose homeostasis in both β cells and adipocytes during diet-induced insulin resistance in mice. We observed that insulin secretion, β-cell formation, and islet architecture were preserved in parkin-deficient β cells and islets, suggesting that parkin is not necessary for control of β-cell function and islet compensation for diet-induced obesity. Although transient parkin deficiency mildly impaired mitochondrial turnover in β-cell lines, parkin deletion in primary β cells yielded no deficits in mitochondrial clearance. In adipocyte-specific deletion models, lipid uptake and β-oxidation were increased in cultured cells, whereas adipose tissue morphology, glucose homeostasis, and the beige-to-white adipocyte transition were unaffected in vivo. In key metabolic tissues where mitochondrial dysfunction has been implicated in T2D development, our experiments unexpectedly revealed that parkin is not an essential regulator of glucose tolerance, whole-body energy metabolism, or mitochondrial quality control. These findings highlight that parkin-independent processes maintain β-cell and adipocyte mitochondrial quality control in diet-induced obesity.

Keywords:  E3 ubiquitin ligase; adipocyte; adipose tissue; autophagy; beta cell (B-cell); diabetes; islet; metabolism; mitochondria; mitophagy

DOI:  https://doi.org/10.1074/jbc.RA118.006763
Mol Metab. 2019 Feb 27. pii: S2212-8778(18)31063-9. [Epub ahead of print]

Perm1 regulates CaMKII activation and shapes skeletal muscle responses to endurance exercise training.

Yoshitake Cho, Shizuko Tachibana, Bethany C Hazen, James J Moresco, John R Yates, Bernard Kok, Enrique Saez, Robert S Ross, Aaron P Russell, Anastasia Kralli.

  OBJECTIVE: Endurance exercise training remodels skeletal muscle, leading to increased mitochondrial content and oxidative capacity. How exercise entrains skeletal muscle signaling pathways to induce adaptive responses remains unclear. In past studies, we identified Perm1 (PGC-1 and ERR induced regulator, muscle 1) as an exercise-induced gene and showed that Perm1 overexpression elicits similar muscle adaptations as endurance exercise training. The mechanism of action and the role of Perm1 in exercise-induced responses are not known. In this study, we aimed to determine the pathway by which Perm1 acts as well as the importance of Perm1 for acute and long-term responses to exercise.METHODS: We performed immunoprecipitation and mass spectrometry to identify Perm1 associated proteins, and validated Perm1 interactions with the Ca2+/calmodulin-dependent protein kinase II (CaMKII). We also knocked down Perm1 expression in gastrocnemius muscles of mice via AAV-mediated delivery of shRNA and assessed the impact of reduced Perm1 expression on both acute molecular responses to a single treadmill exercise bout and long-term adaptive responses to four weeks of voluntary wheel running training. Finally, we asked whether Perm1 levels are modulated by diet or diseases affecting skeletal muscle function.
RESULTS: We show that Perm1 associates with skeletal muscle CaMKII and promotes CaMKII activation. In response to an acute exercise bout, muscles with a knock down of Perm1 showed defects in the activation of CaMKII and p38 MAPK and blunted induction of regulators of oxidative metabolism. Following four weeks of voluntary training, Perm1 knockdown muscles had attenuated mitochondrial biogenesis. Finally, we found that Perm1 expression is reduced in diet-induced obese mice and in muscular dystrophy patients and mouse models.
CONCLUSIONS: Our findings identify Perm1 as a muscle-specific regulator of exercise-induced signaling and Perm1 levels as tuners of the skeletal muscle response to exercise. The decreased Perm1 levels in states of obesity or muscle disease suggest that Perm1 may link pathological states to inefficient exercise responses.

Keywords:  CaMKII signaling; Endurance exercise training; Mitochondrial biogenesis; Skeletal muscle; p38 MAPK regulation

DOI:  https://doi.org/10.1016/j.molmet.2019.02.009
Am J Physiol Cell Physiol. 2019 Mar 13.

Acute increases in O-GlcNAc indirectly impairs mitochondrial bioenergetics through dysregulation of LonP1-mediated mitochondrial protein complex turnover.

JaLessa N Wright, Gloria A Benavides, Michelle S Johnson, Willayat Wani, Xiaosen Ouyang, Luyun Zou, Helen E Collins, Jianhua Zhang, Victor Darley-Usmar, John C Chatham.

  The attachment of O-linked β-N-acetylglucosamine (O-GlcNAc) to the serine and threonine residues of proteins in distinct cellular compartments is increasingly recognized as an important mechanism regulating cellular function. Importantly, the O-GlcNAc modification of mitochondrial proteins has been identified as a potential mechanism to modulate metabolism under stress with both potentially beneficial and detrimental effects. This suggests that temporal and dose-dependent changes in O-GlcNAcylation may have different effects on mitochondrial function. In the current study we found that acutely augmenting O-GlcNAc levels by inhibiting O-GlcNAcase with Thiamet-G for up to 6 hours resulted in a time-dependent decrease in cellular bioenergetics and decreased mitochondrial complex I, II and IV activities. Under these conditions, mitochondrial number was unchanged, whereas an increase in the protein levels of the subunits of several electron transport complex proteins was observed. However, the observed bioenergetic changes appeared not to be due direct increased O-GlcNAc modification of complex subunit proteins. Increases in O-GlcNAc were also associated with an accumulation of mitochondrial ubiquitinated proteins; PINK1 and p62 protein levels were also significantly increased. Interestingly, the increase in O-GlcNAc levels were associated with a decrease in the protein levels of the mitochondrial protease LonP1, which is known to target complex IV subunits and PINK1, in addition to other mitochondrial proteins. These data suggest that impaired bioenergetics associated with short-term increases in O-GlcNAc levels could be due to impaired, LonP1-dependent, mitochondrial complex protein turnover.

Keywords:  Bioenergetics; Mitochondria; O-GlcNAc

DOI:  https://doi.org/10.1152/ajpcell.00491.2018
Nat Commun. 2019 Mar 11. 10(1): 1152

A KLF6-driven transcriptional network links lipid homeostasis and tumour growth in renal carcinoma.

Saiful E Syafruddin, Paulo Rodrigues, Erika Vojtasova, Saroor A Patel, M Nazhif Zaini, Johanna Burge, Anne Y Warren, Grant D Stewart, Tim Eisen, Dóra Bihary, Shamith A Samarajiwa, Sakari Vanharanta.

Transcriptional networks are critical for the establishment of tissue-specific cellular states in health and disease, including cancer. Yet, the transcriptional circuits that control carcinogenesis remain poorly understood. Here we report that Kruppel like factor 6 (KLF6), a transcription factor of the zinc finger family, regulates lipid homeostasis in clear cell renal cell carcinoma (ccRCC). We show that KLF6 supports the expression of lipid metabolism genes and promotes the expression of PDGFB, which activates mTOR signalling and the downstream lipid metabolism regulators SREBF1 and SREBF2. KLF6 expression is driven by a robust super enhancer that integrates signals from multiple pathways, including the ccRCC-initiating VHL-HIF2A pathway. These results suggest an underlying mechanism for high mTOR activity in ccRCC cells. More generally, the link between super enhancer-driven transcriptional networks and essential metabolic pathways may provide clues to the mechanisms that maintain the stability of cell identity-defining transcriptional programmes in cancer.

DOI: https://doi.org/10.1038/s41467-019-09116-x
Mol Cell. 2019 Mar 06. pii: S1097-2765(19)30100-5. [Epub ahead of print]

Spatiotemporal Control of ULK1 Activation by NDP52 and TBK1 during Selective Autophagy.

Jose Norberto S Vargas, Chunxin Wang, Eric Bunker, Ling Hao, Dragan Maric, Giampietro Schiavo, Felix Randow, Richard J Youle.

  Selective autophagy recycles damaged organelles and clears intracellular pathogens to prevent their aberrant accumulation. How ULK1 kinase is targeted and activated during selective autophagic events remains to be elucidated. In this study, we used chemically inducible dimerization (CID) assays in tandem with CRISPR KO lines to systematically analyze the molecular basis of selective autophagosome biogenesis. We demonstrate that ectopic placement of NDP52 on mitochondria or peroxisomes is sufficient to initiate selective autophagy by focally localizing and activating the ULK1 complex. The capability of NDP52 to induce mitophagy is dependent on its interaction with the FIP200/ULK1 complex, which is facilitated by TBK1. Ectopically tethering ULK1 to cargo bypasses the requirement for autophagy receptors and TBK1. Focal activation of ULK1 occurs independently of AMPK and mTOR. Our findings provide a parsimonious model of selective autophagy, which highlights the coordination of ULK1 complex localization by autophagy receptors and TBK1 as principal drivers of targeted autophagosome biogenesis.

Keywords:  ATG13; FIP200; P62; PINK1; Parkin; TAX1BP1; lysosome; mitochondria; mitophagy; optineurin

DOI:  https://doi.org/10.1016/j.molcel.2019.02.010
Arch Biochem Biophys. 2019 Mar 11. pii: S0003-9861(18)30802-6. [Epub ahead of print]

Substrate-dependent and cyclophilin D-independent regulation of mitochondrial flashes in skeletal and cardiac muscle.

Lan Wei-LaPierre, Alina Ainbinder, Kevin M Tylock, Robert T Dirksen.

  Mitochondrial flashes (mitoflashes) are stochastic events in the mitochondrial matrix detected by mitochondrial-targeted cpYFP (mt-cpYFP). Mitoflashes are quantal bursts of reactive oxygen species (ROS) production accompanied by modest matrix alkalinization and depolarization of the mitochondrial membrane potential. Mitoflashes are fundamental events present in a wide range of cell types. To date, the precise mechanisms for mitoflash generation and termination remain elusive. Transient opening of the mitochondrial membrane permeability transition pore (mPTP) during a mitoflash is proposed to account for the mitochondrial membrane potential depolarization. Here, we set out to compare the tissue-specific effects of cyclophilin D (CypD)-deficiency and mitochondrial substrates on mitoflash activity in skeletal and cardiac muscle. In contrast to previous reports, we found that CypD knockout did not alter the mitoflash frequency or other mitoflash properties in acutely isolated cardiac myocytes, skeletal muscle fibers, or isolated mitochondria from skeletal muscle and the heart. However, in skeletal muscle fibers, CypD deficiency resulted in a parallel increase in both activity-dependent mitochondrial Ca2+ uptake and activity-dependent mitoflash activity. Increases in both mitochondrial Ca2+ uptake and mitoflash activity following electrical stimulation were abolished by inhibition of mitochondrial Ca2+ uptake. We also found that mitoflash frequency and amplitude differ greatly between intact skeletal muscle fibers and cardiac myocytes, but that this difference is absent in isolated mitochondria. We propose that this difference may be due, in part, to differences in substrate availability in intact skeletal muscle fibers (primarily glycolytic) and cardiac myocytes (largely oxidative). Overall, we find that CypD does not contribute significantly in mitoflash biogenesis under basal conditions in skeletal and cardiac muscle, but does regulate mitoflash events during muscle activity. In addition, tissue-dependent differences in mitoflash frequency are strongly regulated by mitochondrial substrate availability.

Keywords:  CypD; Mt-cpYFP; Substrates; mitoflashes

DOI:  https://doi.org/10.1016/j.abb.2019.03.003
Cell Rep. 2019 Mar 12. pii: S2211-1247(19)30208-6. [Epub ahead of print]26(11): 3051-3060.e4

p53 Promotes Cancer Cell Adaptation to Glutamine Deprivation by Upregulating Slc7a3 to Increase Arginine Uptake.

Xazmin H Lowman, Eric A Hanse, Ying Yang, Mari B Ishak Gabra, Thai Q Tran, Haiqing Li, Mei Kong.

  Cancer cells heavily depend on the amino acid glutamine to meet the demands associated with growth and proliferation. Due to the rapid consumption of glutamine, cancer cells frequently undergo glutamine starvation in vivo. We and others have shown that p53 is a critical regulator in metabolic stress resistance. To better understand the molecular mechanisms by which p53 activation promotes cancer cell adaptation to glutamine deprivation, we identified p53-dependent genes that are induced upon glutamine deprivation by using RNA-seq analysis. We show that Slc7a3, an arginine transporter, is significantly induced by p53. We also show that increased intracellular arginine levels following glutamine deprivation are dependent on p53. The influx of arginine has minimal effects on known metabolic pathways upon glutamine deprivation. Instead, we found arginine serves as an effector for mTORC1 activation to promote cell growth in response to glutamine starvation. Therefore, we identify a p53-inducible gene that contributes to the metabolic stress response.

Keywords:  Slc7a3; arginine; glutamine deprivation; p53 activation

DOI:  https://doi.org/10.1016/j.celrep.2019.02.037
FEBS Lett. 2019 Mar 12.

Molecular evolution of proteins mediating mitochondrial fission-fusion dynamics.

Sansrity Sinha, Narayanan Manoj.

  Eukaryotes employ a subset of dynamins to mediate mitochondrial fusion and fission dynamics. Here we report the molecular evolution and diversification of the dynamin-related mitochondrial proteins that drive the fission (Drp1) and the fusion processes (mitofusin and OPA1). We demonstrate that the three paralogs emerged concurrently in an early mitochondriate eukaryotic ancestor. Furthermore, multiple independent duplication events from an ancestral bifunctional fission protein gave rise to specialized fission proteins. The evolutionary history of these proteins is marked by transformations that include independent gain and loss events occurring at the levels of entire genes, specific functional domains, and intronic regions. The domain level variations primarily comprise loss-gain of lineage specific domains that are present in the terminal regions of the sequences. This article is protected by copyright. All rights reserved.

Keywords:  Drp1; Dynamin related proteins; Mitochondrial fission and fusion; Mitofusin; Molecular evolution; OPA1

DOI:  https://doi.org/10.1002/1873-3468.13356
J Biol Chem. 2019 Mar 14. pii: jbc.RA118.006673. [Epub ahead of print]

Myoglobin induces mitochondrial fusion thereby inhibiting breast cancer cell proliferation.

Andrea Braganza, Kelly Quesnelle, Janelle Bickta, Christopher Reyes, Yinna Wang, Morgan Jessup, Claudette St Croix, Julie Scott, Shivendra V Singh, Sruti Shiva.

  Myoglobin is a monomeric heme protein expressed ubiquitously in skeletal and cardiac muscle and is traditionally considered to function as an oxygen reservoir for mitochondria during hypoxia. It is now well established that low concentrations of myoglobin are aberrantly expressed in a significant proportion of breast cancer tumors. Despite being expressed only at low levels in these tumors, myoglobin is associated with attenuated tumor growth and a better prognosis in breast cancer patients, but the mechanism of this myoglobin-mediated protection against further cancer growth remains unclear. Herein, we report a signaling pathway by which myoglobin regulates mitochondrial dynamics and thereby decreases cell proliferation. We demonstrate in vitro that expression of human myoglobin in MDA-MB-231, MDA-MB-468, and MCF7 breast cancer cells induces mitochondrial hyperfusion by up-regulating mitofusins 1 and 2, the predominant catalysts of mitochondrial fusion. This hyperfusion causes cell cycle arrest and subsequent inhibition of cell proliferation. Mechanistically, increased mitofusin expression was due to myoglobin-dependent free-radical production, leading to the oxidation and degradation of the E3 ubiquitin ligase parkin. We recapitulated this pathway in a murine model in which myoglobin-expressing xenografts exhibited decreased tumor volume with increased mitofusin, markers of cell cycle arrest, and decreased parkin expression. Further, in human triple-negative breast tumor tissues, mitofusin and myoglobin levels were positively correlated. Collectively, these results elucidate a new function for myoglobin as a modulator of mitochondrial dynamics and reveal a novel pathway by which myoglobin decreases breast cancer cell proliferation and tumor growth by up-regulating mitofusin levels.

Keywords:  breast cancer; cell cycle; heme protein; mitochondrial fusion; myoglobin; oxidative stress; parkin; proliferation; reactive oxygen species (ROS); tumor growth

DOI:  https://doi.org/10.1074/jbc.RA118.006673
Autophagy. 2019 Mar 12.

Mitophagy regulates mitochondrial network signaling, oxidative stress, and apoptosis during myoblast differentiation.

Brittany L Baechler, Darin Bloemberg, Joe Quadrilatero.

  Macroautophagy/autophagy is a degradative process essential for various cellular processes. We previously demonstrated that autophagy-deficiency causes myoblast apoptosis and impairs myotube formation. In this study, we continued this work with particular emphasis on mitochondrial remodelling and stress/apoptotic signaling. We found increased (p<0.05) autophagic (e.g., altered LC3B levels, increased ATG7, decreased SQSTM1) and mitophagic (e.g., BNIP3 upregulation, mitochondrial localized GFP-LC3 puncta, and elevated mitochondrial LC3B-II) signaling during myoblast differentiation. shRNA-mediated knockdown of ATG7 (shAtg7) decreased these autophagic and mitophagic responses, while increasing CASP3 activity and ANXA5/annexin V staining in differentiating myoblasts; ultimately resulting in dramatically impaired myogenesis. Further confirming the importance of mitophagy in these responses, CRISPR-Cas9-mediated knockout of Bnip3 (bnip3-/-) resulted in increased CASP3 activity and DNA fragmentation as well as impaired myoblast differentiation. In addition, shAtg7 myoblasts displayed greater endoplasmic reticulum (e.g., increased CAPN activity and HSPA) and mitochondrial (e.g., mPTP formation, reduced mitochondrial membrane potential, elevated mitochondrial 4-HNE) stress. shAtg7 and bnip3-/- myoblasts also displayed altered mitochondria-associated signaling (e.g., PPARGC1A, DNM1L, OPA1) and protein content (e.g., SLC25A4, VDAC1, CYCS). Moreover, shAtg7 myoblasts displayed CYCS and AIFM1 release from mitochondria, and CASP9 activation. Similarly, bnip3-/- myoblasts had significantly higher CASP9 activation during differentiation. Importantly, administration of a chemical inhibitor of CASP9 (Ac-LEHD-CHO) or dominant-negative CASP9 (ad-DNCASP9) partially recovered differentiation and myogenesis in shAtg7 myoblasts. Together, these data demonstrate an essential role for autophagy in protecting myoblasts from mitochondrial oxidative stress and apoptotic signaling during differentiation, as well as in the regulation of mitochondrial network remodelling and myogenesis.

Keywords:  apoptosis; autophagy; caspase 9; differentiation; mitochondria; mitophagy; myogenesis; oxidative stress; skeletal muscle

DOI:  https://doi.org/10.1080/15548627.2019.1591672
J Biol Chem. 2019 Mar 15. pii: jbc.RA118.006685. [Epub ahead of print]

ATP-citrate lyase multimerization is required for coenzyme-A substrate binding and catalysis.

Gleb A Bazilevsky, Hayley C Affronti, Xuepeng Wei, Sydney L Campbell, Kathryn E Wellen, Ronen Marmorstein.

  ATP-citrate lyase (ACLY) is the predominant source of nucleocytosolic acetyl-CoA, a fundamental building block of carbon metabolism in eukaryotes. ACLY is aberrantly regulated in many cancers, cardiovascular disease, and metabolic disorders. However, the molecular mechanisms determining ACLY activity and function are unclear. To this end, we investigated the role of the uncharacterized ACLY C-terminal citrate synthase homology domain (CSHD) in the mechanism of acetyl-CoA formation. Using recombinant and purified ACLY and a suite of biochemical and biophysical approaches, including analytical ultracentrifugation, dynamic light scattering, and thermal stability assays, we demonstrate that the C-terminus maintains ACLY tetramerization, a conserved and essential quaternary structure in vitro and likely also in vivo. Furthermore, we show that the C-terminus, only in the context of the full-length enzyme, is necessary for full ACLY binding to CoA, stability and catalysis. Together, we demonstrate that ACLY forms a homotetramer through the C-terminus to facilitate CoA binding and acetyl-CoA production. Our findings highlight a novel and unique role of the C-terminal citrate synthase homology domain in ACLY function and catalysis, adding to the understanding of the molecular basis for acetyl-CoA synthesis by ACLY. This newly discovered means of ACLY regulation has implications for the development of novel ACLY modulators to target acetyl-CoA dependent cellular processes for potential therapeutic use.

Keywords:  ATP-citrate lyase; acetyl coenzyme A (acetyl-CoA); citrate synthase; coenzyme A (CoA); enzyme mechanism; metabolism; protein assembly

DOI:  https://doi.org/10.1074/jbc.RA118.006685
Basic Res Cardiol. 2019 Mar 15. 114(3): 17

Increased calpain-1 in mitochondria induces dilated heart failure in mice: role of mitochondrial superoxide anion.

Ting Cao, Shuai Fan, Dong Zheng, Grace Wang, Yong Yu, Ruizhen Chen, Long-Sheng Song, Guo-Chang Fan, Zhuxu Zhang, Tianqing Peng.

  We and others have reported that calpain-1 was increased in myocardial mitochondria from various animal models of heart disease. This study investigated whether constitutive up-regulation of calpain-1 restricted to mitochondria induced myocardial injury and heart failure and, if so, whether these phenotypes could be rescued by selective inhibition of mitochondrial superoxide production. Transgenic mice with human CAPN1 up-regulation restricted to mitochondria in cardiomyocytes (Tg-mtCapn1/tTA) were generated and characterized with low and high over-expression of transgenic human CAPN1 restricted to mitochondria, respectively. Transgenic up-regulation of mitochondria-targeted CAPN1 dose-dependently induced cardiac cell death, adverse myocardial remodeling, heart failure, and early death in mice, the changes of which were associated with mitochondrial dysfunction and mitochondrial superoxide generation. Importantly, a daily injection of mitochondria-targeted superoxide dismutase mimetics mito-TEMPO for 1 month starting from age 2 months attenuated cardiac cell death, adverse myocardial remodeling and heart failure, and reduced mortality in Tg-mtCapn1/tTA mice. In contrast, administration of TEMPO did not achieve similar cardiac protection in transgenic mice. Furthermore, transgenic up-regulation of mitochondria-targeted CAPN1 induced a reduction of ATP5A1 protein and ATP synthase activity in hearts. In cultured cardiomyocytes, increased calpain-1 in mitochondria promoted mitochondrial permeability transition pore (mPTP) opening and induced cell death, which were prevented by over-expression of ATP5A1, mito-TEMPO or cyclosporin A, an inhibitor of mPTP opening. In conclusion, this study has provided direct evidence demonstrating that increased mitochondrial calpain-1 is an important mechanism contributing to myocardial injury and heart failure by disrupting ATP synthase, and promoting mitochondrial superoxide generation and mPTP opening.

Keywords:  ATP synthase; Calpain; Heart failure; Mitochondria; Superoxide onion

DOI:  https://doi.org/10.1007/s00395-019-0726-1
Kidney Cancer. 2019 Feb 05. 3(1): 15-29

The 'Achilles Heel' of Metabolism in Renal Cell Carcinoma: Glutaminase Inhibition as a Rational Treatment Strategy.

Christian R Hoerner, Viola J Chen, Alice C Fan.

  An important hallmark of cancer is 'metabolic reprogramming' or the rewiring of cellular metabolism to support rapid cell proliferation [1-5]. Metabolic reprogramming through oncometabolite-mediated transformation or activation of oncogenes in renal cell carcinoma (RCC) globally impacts energy production as well as glucose and glutamine utilization in RCC cells, which can promote dependence on glutamine supply to support cell growth and proliferation [6, 7]. Novel inhibitors of glutaminase, a key enzyme in glutamine metabolism, target glutamine addiction as a viable treatment strategy in metastatic RCC (mRCC). Here, we review glutamine metabolic pathways and how changes in cellular glutamine utilization enable the progression of RCC. This overview provides scientific rationale for targeting this pathway in patients with mRCC. We will summarize the current understanding of cellular and molecular mechanisms underlying anti-tumor efficacy of glutaminase inhibitors in RCC, provide an overview of clinical efforts targeting glutaminase in mRCC, and review approaches for identifying biomarkers for patient stratification and detecting therapeutic response early on in patients treated with this novel class of anti-cancer drug. Ultimately, results of ongoing clinical trials will demonstrate whether glutaminase inhibition can be a worthy addition to the current armamentarium of drugs used for patients with mRCC.

Keywords:  Kidney cancer; biomarkers; glutaminase; glutaminase inhibitor; glutamine; metabolic reprogramming; metabolism; renal cell carcinoma; treatment

DOI:  https://doi.org/10.3233/KCA-180043
Cell. 2019 Feb 26. pii: S0092-8674(19)30113-8. [Epub ahead of print]

Energetic Trade-Offs and Hypometabolic States Promote Disease Tolerance.

Kirthana Ganeshan, Joni Nikkanen, Kevin Man, Yew Ann Leong, Yoshitaka Sogawa, J Alan Maschek, Tyler Van Ry, D Nyasha Chagwedera, James E Cox, Ajay Chawla.

  Host defenses against pathogens are energetically expensive, leading ecological immunologists to postulate that they might participate in energetic trade-offs with other maintenance programs. However, the metabolic costs of immunity and the nature of physiologic trade-offs it engages are largely unknown. We report here that activation of immunity causes an energetic trade-off with the homeothermy (the stable maintenance of core temperature), resulting in hypometabolism and hypothermia. This immunity-induced physiologic trade-off was independent of sickness behaviors but required hematopoietic sensing of lipopolysaccharide (LPS) via the toll-like receptor 4 (TLR4). Metabolomics and genome-wide expression profiling revealed that distinct metabolic programs supported entry and recovery from the energy-conserving hypometabolic state. During bacterial infections, hypometabolic states, which could be elicited by competition for energy between maintenance programs or energy restriction, promoted disease tolerance. Together, our findings suggest that energy-conserving hypometabolic states, such as dormancy, might have evolved as a mechanism of tissue tolerance.

Keywords:  caloric restriction; dormancy; hibernation; innate immunity; ketones; metabolism; resistance; thermoneutrality; torpor; triglycerides

DOI:  https://doi.org/10.1016/j.cell.2019.01.050
Elife. 2019 Mar 12. pii: e37689. [Epub ahead of print]8

Bim escapes displacement by BH3-mimetic anti-cancer drugs by double-bolt locking both Bcl-XL and Bcl-2.

Qian Liu, Elizabeth J Oesterlund, Xiaoke Chi, Justin Pogmore, Brian Leber, David William Andrews.

  Tumor initiation, progression and resistance to chemotherapy rely on cancer cells bypassing programmed cell death by apoptosis. We report that unlike other pro-apoptotic proteins, Bim contains two distinct binding sites for the anti-apoptotic proteins Bcl-XL and Bcl-2. These include the BH3 sequence shared with other pro-apoptotic proteins and an unexpected sequence located near the Bim carboxyl-terminus (residues 181-192). Using automated Fluorescence Lifetime Imaging Microscopy - Fluorescence Resonance Energy Transfer (FLIM-FRET) we show that the two binding interfaces enable Bim to double-bolt lock Bcl-XL and Bcl-2 in complexes resistant to displacement by BH3-mimetic drugs currently in use or being evaluated for cancer therapy. Quantifying in live cells the contributions of individual amino acids revealed that residue L185 previously thought involved in binding Bim to membranes, instead contributes to binding to anti-apoptotic proteins. This double-bolt lock mechanism has profound implications for the utility of BH3-mimetics as drugs. .

Keywords:  BH3-mimetic drugs; Bcl-2 family proteins; Forster resonance energy transfer; apoptosis; cancer biology; cell biology; fluorescence lifetime imaging; none; tissue culture cells

DOI:  https://doi.org/10.7554/eLife.37689
Cell Death Dis. 2019 Mar 11. 10(3): 243

p53 β-hydroxybutyrylation attenuates p53 activity.

Kun Liu, Fangzhou Li, Qianqian Sun, Ning Lin, Haichao Han, Kaiqiang You, Feng Tian, Zebin Mao, Tingting Li, Tanjun Tong, Meiyu Geng, Yingming Zhao, Wei Gu, Wenhui Zhao.

p53 is an essential tumor suppressor, whose activity is finely tuned by the posttranslational modifications. Previous research has reported that β-hydroxybutyrate (BHB) induces β-hydroxybutyrylation (Kbhb), which is a novel histone posttranslational modification. Here we report that p53 is modified by kbhb and that this modification occurs at lysines 120, 319, and 370 of p53. We demonstrate that the level of p53 kbhb is dramatically increased in cultured cells treated with BHB and in thymus tissues of fasted mice, and that CBP catalyze p53 kbhb. We show that p53 kbhb results in lower levels of p53 acetylation and reduced expression of the p53 downstream genes p21 and PUMA, as well as reduced cell growth arrest and apoptosis in cultured cells under p53-activating conditions. Similar results were observed in mouse thymus tissue under starvation conditions, which result in increased concentrations of serum BHB, and in response to genotoxic stress caused by γ-irradiation to activate p53. Our findings thus show that BHB-mediated p53 kbhb is a novel mechanism of p53 activity regulation, which may explain the link between ketone bodies and tumor, and which may provide promising therapeutic target for cancer treatment.

DOI: https://doi.org/10.1038/s41419-019-1463-y
Autophagy. 2019 Mar 14. 1-19

Sigma-1 receptor ablation impairs autophagosome clearance.

Huan Yang, Hongtao Shen, Jing Li, Lian-Wang Guo.

  Autophagosome-lysosome fusion is a common critical step in various forms of macroautophagy including mitophagy, the selective degradation of mitochondria. Regulations of this fusion process remain poorly defined. Here we have determined the role of the sigma-1 receptor (Sig1R), a unique endoplasmic reticulum membrane protein. Knockout of Sig1R impaired mitochondrial clearance without altering the PINK1/Parkin signaling, in mouse retinal explants and cultured cells treated with carbonyl cyanide m-chlorophenyl hydrazone (CCCP) for induction of mitophagy. Sig1R depletion also caused accumulation of autophagosome markers LC3-II and SQSTM1, but did not change the levels of Beclin1 and ATG7, proteins associated with autophagosome biogenesis. Lysosomal pH and protease activities were not negatively affected. However, Sig1R knockout partially compromised autophagosome-lysosome fusion in CCCP-treated NSC34 cells, as revealed by reduced GFP fluorescence quenching of GFP-RFP-LC3-II puncta and co-localization of lysosomes with mitochondria. Furthermore, Sig1R co-immunoprecipitated with ATG14, STX17, and VAMP8 (but not SNAP29), proteins key to autophagosome-lysosome membrane fusion. Re-expressing Sig1R in the null background rescued clearance of mitochondria and autophagosomes. In summary, we started out finding that Sig1R knockout impaired the clearance of mitochondria and autophagosomes, and then narrowed down the Sig1R modulation to the autophagosome-lysosome fusion step. This study may shed new light on understanding autophagy-associated cyto-protection and disease mechanisms. Abbreviations: APEX2, a genetically engineered peroxidase; BiFC, bimolecule fluorescence complementation; CCCP, a mitophagy inducing compound; CRISPR, clustered regularly interspaced short palindromic repeats; EM, electron microscopy; ER, endoplasmic reticulum; LC3, Microtubule-associated protein 1A/1B-light chain 3; Sig1R, sigma-1 receptor.

Keywords:  CRISPR knockout and knockdown; Sigma-1 receptor; autophagosome-lysosome fusion; autophagy; mitophagy

DOI:  https://doi.org/10.1080/15548627.2019.1586248
Sci Rep. 2019 Mar 11. 9(1): 4079

PGC-1α overexpression partially rescues impaired oxidative and contractile pathophysiology following volumetric muscle loss injury.

William M Southern, Anna S Nichenko, Kayvan F Tehrani, Melissa J McGranahan, Laxminarayanan Krishnan, Anita E Qualls, Nathan T Jenkins, Luke J Mortensen, Hang Yin, Amelia Yin, Robert E Guldberg, Sarah M Greising, Jarrod A Call.

Volumetric muscle loss (VML) injury is characterized by a non-recoverable loss of muscle fibers due to ablative surgery or severe orthopaedic trauma, that results in chronic functional impairments of the soft tissue. Currently, the effects of VML on the oxidative capacity and adaptability of the remaining injured muscle are unclear. A better understanding of this pathophysiology could significantly shape how VML-injured patients and clinicians approach regenerative medicine and rehabilitation following injury. Herein, the data indicated that VML-injured muscle has diminished mitochondrial content and function (i.e., oxidative capacity), loss of mitochondrial network organization, and attenuated oxidative adaptations to exercise. However, forced PGC-1α over-expression rescued the deficits in oxidative capacity and muscle strength. This implicates physiological activation of PGC1-α as a limiting factor in VML-injured muscle's adaptive capacity to exercise and provides a mechanistic target for regenerative rehabilitation approaches to address the skeletal muscle dysfunction.

DOI: https://doi.org/10.1038/s41598-019-40606-6
Cancer Discov. 2019 Mar 12. pii: CD-18-1040. [Epub ahead of print]

Mutant and wild-type isocitrate dehydrogenase 1 share enhancing mechanisms involving distinct tyrosine kinase cascades in cancer.

Dong Chen, Siyuan Xia, Mei Wang, Ruiting Lin, Yuancheng Li, Hui Mao, Mike Aguiar, Christopher A Famulare, Alan H Shih, Cameron W Brennan, Xue Gao, Yaozhu Pan, Shuangping Liu, Jun Fan, Lingtao Jin, Lina Song, An Zhou, Joydeep Mukherjee, Russell O Pieper, Ashutosh Mishra, Junmin Peng, Martha Arellano, William G Blum, Sagar Lonial, Titus J Boggon, Ross L Levine, Jing Chen.

Isocitrate dehydrogenase 1 (IDH1) is important for reductive carboxylation in cancer cells, and IDH1 R132H mutant plays a pathogenic role in cancers including acute myeloid leukemia (AML). However, the regulatory mechanisms modulating IDH1 mutant and/or wild-type (WT) function remain unknown. Here we show that two groups of tyrosine kinases (TKs) enhance the activation of IDH1 mutant and WT through preferential Y42 or Y391 phosphorylation. Mechanistically, Y42 phosphorylation occurs in IDH1 monomers, which promotes dimer formation with enhanced substrate (isocitrate or a-ketoglutarate) binding, while Y42-phosphorylated dimers show attenuated disruption to monomers. Y391 phosphorylation occurs in both monomeric and dimeric IDH1, which enhances cofactor (NADP+ or NADPH) binding. Diverse oncogenic TKs activate Src to achieve Y42 and Y391 phosphorylation of IDH1, respectively, which contributes to reductive carboxylation and tumor growth, while FLT3 or FLT3-ITD mutant activate JAK2 to enhance IDH1 mutant activity through phosphorylation of Y391 and Y42, respectively, in AML cells.

DOI: https://doi.org/10.1158/2159-8290.CD-18-1040
JCI Insight. 2019 Mar 14. pii: 126749. [Epub ahead of print]5

TFEB-driven lysosomal biogenesis is pivotal for PGC1α-dependent renal stress resistance.

Matthew R Lynch, Mei T Tran, Kenneth M Ralto, Zsuzsanna K Zsengeller, Vinod Raman, Swati S Bhasin, Nuo Sun, Xiuying Chen, Daniel Brown, Ilsa I Rovira, Kensei Taguchi, Craig R Brooks, Isaac E Stillman, Manoj K Bhasin, Toren Finkel, Samir M Parikh.

  Because injured mitochondria can accelerate cell death through the elaboration of oxidative free radicals and other mediators, it is striking that proliferator gamma coactivator 1-alpha (PGC1α), a stimulator of increased mitochondrial abundance, protects stressed renal cells instead of potentiating injury. Here we report that PGC1α's induction of lysosomes via transcription factor EB (TFEB) may be pivotal for kidney protection. CRISPR and stable gene transfer showed that PGC1α knockout tubular cells were sensitized to the genotoxic stressor cisplatin whereas transgenic cells were protected. The biosensor mtKeima unexpectedly revealed that cisplatin blunts mitophagy both in cells and mice. PGC1α not only counteracted this effect but also raised basal mitophagy, as did the downstream mediator nicotinamide adenine dinucleotide (NAD+). PGC1α did not consistently affect known autophagy pathways modulated by cisplatin. Instead RNA sequencing identified coordinated regulation of lysosomal biogenesis via TFEB. This effector pathway was sufficiently important that inhibition of TFEB or lysosomes unveiled a striking harmful effect of excess PGC1α in cells and conditional mice. These results uncover an unexpected effect of cisplatin on mitophagy and PGC1α's exquisite reliance on lysosomes for kidney protection. Finally, the data illuminate TFEB as a novel target for renal tubular stress resistance.

Keywords:  Mitochondria; Nephrology

DOI:  https://doi.org/10.1172/jci.insight.126749
Contact (Thousand Oaks). 2018 Jan-Dec;2:2

ER-mitochondria contacts are required for pexophagy in Saccharomyces cerevisiae.

Xu Liu, Xin Wen, Daniel J Klionsky.

  Peroxisomes play important roles in lipid metabolism. Surplus or damaged peroxisomes can be selectively targeted for autophagic degradation, a process termed pexophagy. Maintaining a proper level of pexophagy is critical for cellular homeostasis. Here we found that endoplasmic reticulum (ER)-mitochondria contact sites are necessary for efficient pexophagy. During pexophagy, the peroxisomes destined for degradation are adjacent to the ER-mitochondria encounter structure (ERMES) that mediates formation of ER- mitochondria contacts; disruption of the ERMES results in a severe defect in pexophagy. We show that a mutant form of Mdm34, a component of the ERMES, which impairs ERMES formation and diminishes its association with the peroxisomal membrane protein Pex11, also leads to defects in pexophagy. The dynamin-related GTPase Vps1, which is specific for peroxisomal fission, is recruited to the peroxisomes at ER-mitochondria contacts by the selective autophagy scaffold Atg11 and the pexophagy receptor Atg36, facilitating peroxisome degradation.

Keywords:  ER-mitochondria contact sites; peroxisomes; pexophagy; stress; yeast

DOI:  https://doi.org/10.1177/2515256418821584
Metab Eng. 2019 Mar 06. pii: S1096-7176(18)30397-5. [Epub ahead of print]

Metabolic engineering of Chinese hamster ovary cells towards reduced biosynthesis and accumulation of novel growth inhibitors in fed-batch cultures.

Bhanu Chandra Mulukutla, Pauline Geoffroy, Jeffrey Mitchell, Cameron Harrington, Manisha Krishnan, Taylor Kalomeris, Caitlin Morris, Lin Zhang, Pamela Pegman, Gregory W Hiller.

Chinese hamster ovary (CHO) cells in fed-batch cultures are known to consume large amounts of nutrients and divert significant portion of them towards the formation of byproducts, some of which, including lactate and ammonia, are known to be growth inhibitory in nature. A major fraction of these inhibitory metabolites are byproducts or intermediates of amino acid catabolism. Limiting the supply of amino acids has been shown to curtail the production of corresponding inhibitory byproducts resulting in enhanced growth and productivities in CHO fed-batch cultures (Mulukutla et al., 2017). In the current study, metabolic engineering of CHO cells was undertaken in order to reduce the biosynthesis of these novel growth inhibitors. Phenylalanine-tyrosine (Phe-Tyr) and branched chain amino acid (BCAA) pathways were engineered as part of this effort. Four genes that encode enzymes in the Phe-Tyr pathway, which were observed to be minimally expressed in CHO cells, were in turn overexpressed. Metabolically engineered cells were prototrophic to tyrosine and had reduced production of the inhibitory byproducts from Phe-Tyr pathway including 3-phenyllactate and 4-hydroxyphenyllactate. In case of BCAA catabolic pathway, branched chain aminotransferase 1 (BCAT1) gene, which encodes the enzyme that catalyzes the first step in the catabolism of BCAAs, was knocked out in CHO cells. Knockout (KO) of BCAT1 function completely eradicated production of inhibitory byproducts from BCAA catabolic pathway, including isovalerate, isobutyrate and 2-methylbutyrate, resulting in significantly enhanced cell growth and productivities in fed-batch cultures. This study is first of its kind to demonstrate that metabolic engineering of essential amino acid metabolism of CHO cells can significantly improve cell culture process performance.

DOI: https://doi.org/10.1016/j.ymben.2019.03.001
Sci Rep. 2019 Mar 12. 9(1): 4179

Pivotal role of inter-organ aspartate metabolism for treatment of mitochondrial aspartate-glutamate carrier 2 (citrin) deficiency, based on the mouse model.

Takeyori Saheki, Mitsuaki Moriyama, Eishi Kuroda, Aki Funahashi, Izumi Yasuda, Yoshiko Setogawa, Qinghua Gao, Miharu Ushikai, Sumie Furuie, Ken-Ichi Yamamura, Katsura Takano, Yoichi Nakamura, Kazuhiro Eto, Takashi Kadowaki, David S Sinasac, Tatsuhiko Furukawa, Masahisa Horiuchi, Yen How Tai.

Previous studies using citrin/mitochondrial glycerol-3-phosphate (G3P) dehydrogenase (mGPD) double-knockout mice have demonstrated that increased dietary protein reduces the extent of carbohydrate-induced hyperammonemia observed in these mice. This study aimed to further elucidate the mechanisms of this effect. Specific amino acids were initially found to decrease hepatic G3P, or increase aspartate or citrulline levels, in mGPD-knockout mice administered ethanol. Unexpectedly, oral glycine increased ammonia in addition to lowering G3P and increasing citrulline. Subsequently, simultaneous glycine-plus-sucrose (Gly + Suc) administration led to a more severe hyperammonemic state in double-KO mice compared to sucrose alone. Oral arginine, ornithine, aspartate, alanine, glutamate and medium-chain triglycerides all lowered blood ammonia following Gly + Suc administration, with combinations of ornithine-plus-aspartate (Orn + Asp) or ornithine-plus-alanine (Orn + Ala) suppressing levels similar to wild-type. Liver perfusion and portal vein-arterial amino acid differences suggest that oral aspartate, similar to alanine, likely activated ureagenesis from ammonia and lowered the cytosolic NADH/NAD+ ratio through conversion to alanine in the small intestine. In conclusion, Gly + Suc administration induces a more severe hyperammonemic state in double-KO mice that Orn + Asp or Orn + Ala both effectively suppress. Aspartate-to-alanine conversion in the small intestine allows for effective oral administration of either, demonstrating a pivotal role of inter-organ aspartate metabolism for the treatment of citrin deficiency.

DOI: https://doi.org/10.1038/s41598-019-39627-y
Sci Rep. 2019 Mar 13. 9(1): 4322

The 5-formyltetrahydrofolate futile cycle reduces pathway stochasticity in an extended hybrid-stochastic model of folate-mediated one-carbon metabolism.

Karla Misselbeck, Luca Marchetti, Corrado Priami, Patrick J Stover, Martha S Field.

In folate-mediated one-carbon metabolism (FOCM), 5-formyltetrahydrofolate (5fTHF), a one-carbon substituted tetrahydrofolate (THF) vitamer, acts as an intracellular storage form of folate and as an inhibitor of the folate-dependent enzymes phosphoribosylaminoimidazolecarboxamide formyltransferase (AICARFT) and serine hydroxymethyltransferase (SHMT). Cellular levels of 5fTHF are regulated by a futile cycle comprising the enzymes SHMT and 5,10-methenyltetrahydrofolate synthetase (MTHFS). MTHFS is an essential gene in mice; however, the roles of both 5fTHF and MTHFS in mammalian FOCM remain to be fully elucidated. We present an extension of our previously published hybrid-stochastic model of FOCM by including the 5fTHF futile-cycle to explore its effect on the FOCM network. Model simulations indicate that MTHFS plays an essential role in preventing 5fTHF accumulation, which consequently averts inhibition of all other reactions in the metabolic network. Moreover, in silico experiments show that 10-formylTHF inhibition of MTHFS is critical for regulating purine synthesis. Model simulations also provide evidence that 5-methylTHF (and not 5fTHF) is the predominant physiological binder/inhibitor of SHMT. Finally, the model simulations indicate that the 5fTHF futile cycle dampens the stochastic noise in FOCM that results from both folate deficiency and a common variant in the methylenetetrahydrofolate reductase (MTHFR) gene.

DOI: https://doi.org/10.1038/s41598-019-40230-4
J Inherit Metab Dis. 2019 Mar 12.

Organelle Interplay - Peroxisome Interactions in Health and Disease.

Michael Schrader, Maki Kamoshita, Markus Islinger.

Peroxisomes are multifunctional, dynamic, membrane-bound organelles with important functions in cellular lipid metabolism, rendering them essential for human health and development. Important roles for peroxisomes in signalling and the fine-tuning of cellular processes are emerging, which integrate them in a complex network of interacting cellular compartments. Like many other organelles, peroxisomes communicate through membrane contact sites. For example, peroxisomal growth, positioning and lipid metabolism involves contacts with the endoplasmic reticulum (ER). Here we discuss the most recent findings on peroxisome-organelle interactions including peroxisome-ER interplay at membrane contacts sites, and functional interplay with mitochondria, lysosomes and lipid droplets in mammalian cells. We address tether proteins, metabolic cooperation and the impact of peroxisome interactions on human health and disease. This article is protected by copyright. All rights reserved.

DOI: https://doi.org/10.1002/jimd.12083
Cell Metab. 2019 Mar 04. pii: S1550-4131(19)30069-5. [Epub ahead of print]

High Glucose Triggers Nucleotide Imbalance through O-GlcNAcylation of Key Enzymes and Induces KRAS Mutation in Pancreatic Cells.

Chun-Mei Hu, Sui-Chih Tien, Ping-Kun Hsieh, Yung-Ming Jeng, Ming-Chu Chang, Yu-Ting Chang, Yi-Ju Chen, Yu-Ju Chen, Eva Y-H P Lee, Wen-Hwa Lee.

  KRAS mutations are the earliest events found in approximately 90% of pancreatic ductal adenocarcinomas (PDACs). However, little is known as to why KRAS mutations preferentially occur in PDACs and what processes/factors generate these mutations. While abnormal carbohydrate metabolism is associated with a high risk of pancreatic cancer, it remains elusive whether a direct relationship between KRAS mutations and sugar metabolism exists. Here, we show that under high-glucose conditions, cellular O-GlcNAcylation is significantly elevated in pancreatic cells that exhibit lower phosphofructokinase (PFK) activity than other cell types. This post-translational modification specifically compromises the ribonucleotide reductase (RNR) activity, leading to deficiency in dNTP pools, genomic DNA alterations with KRAS mutations, and cellular transformation. These results establish a mechanistic link between a perturbed sugar metabolism and genomic instability that induces de novo oncogenic KRAS mutations preferentially in pancreatic cells.

Keywords:  KRAS mutation; O-GlcNAcylation; PFK; RNR; RRM1; high glucose; nucleotide imbalance

DOI:  https://doi.org/10.1016/j.cmet.2019.02.005
Science. 2019 03 15. 363(6432): 1226-1230

RIT1 oncoproteins escape LZTR1-mediated proteolysis.

Pau Castel, Alice Cheng, Antonio Cuevas-Navarro, David B Everman, Alex G Papageorge, Dhirendra K Simanshu, Alexandra Tankka, Jacqueline Galeas, Anatoly Urisman, Frank McCormick.

RIT1 oncoproteins have emerged as an etiologic factor in Noonan syndrome and cancer. Despite the resemblance of RIT1 to other members of the Ras small guanosine triphosphatases (GTPases), mutations affecting RIT1 are not found in the classic hotspots but rather in a region near the switch II domain of the protein. We used an isogenic germline knock-in mouse model to study the effects of RIT1 mutation at the organismal level, which resulted in a phenotype resembling Noonan syndrome. By mass spectrometry, we detected a RIT1 interactor, leucine zipper-like transcription regulator 1 (LZTR1), that acts as an adaptor for protein degradation. Pathogenic mutations affecting either RIT1 or LZTR1 resulted in incomplete degradation of RIT1. This led to RIT1 accumulation and dysregulated growth factor signaling responses. Our results highlight a mechanism of pathogenesis that relies on impaired protein degradation of the Ras GTPase RIT1.

DOI: https://doi.org/10.1126/science.aav1444
MBio. 2019 Mar 12. pii: e02175-18. [Epub ahead of print]10(2):

Glycolysis Is an Intrinsic Factor for Optimal Replication of a Norovirus.

Karla D Passalacqua, Jia Lu, Ian Goodfellow, Abimbola O Kolawole, Jacob R Arche, Robert J Maddox, Kelly E Carnahan, Mary X D O'Riordan, Christiane E Wobus.

  The metabolic pathways of central carbon metabolism, glycolysis and oxidative phosphorylation (OXPHOS), are important host factors that determine the outcome of viral infections and can be manipulated by some viruses to favor infection. However, mechanisms of metabolic modulation and their effects on viral replication vary widely. Herein, we present the first metabolomics and energetic profiling of norovirus-infected cells, which revealed increases in glycolysis, OXPHOS, and the pentose phosphate pathway (PPP) during murine norovirus (MNV) infection. Inhibiting glycolysis with 2-deoxyglucose (2DG) in macrophages revealed that glycolysis is an important factor for optimal MNV infection, while inhibiting the PPP and OXPHOS showed a relatively minor impact of these pathways on MNV infection. 2DG affected an early stage in the viral life cycle after viral uptake and capsid uncoating, leading to decreased viral protein production and viral RNA. The requirement of glycolysis was specific for MNV (but not astrovirus) infection, independent of the type I interferon antiviral response, and unlikely to be due to a lack of host cell nucleotide synthesis. MNV infection increased activation of the protein kinase Akt, but not AMP-activated protein kinase (AMPK), two master regulators of cellular metabolism, implicating Akt signaling in upregulating host metabolism during norovirus infection. In conclusion, our findings suggest that the metabolic state of target cells is an intrinsic host factor that determines the extent of norovirus replication and implicates glycolysis as a virulence determinant. They further point to cellular metabolism as a novel therapeutic target for norovirus infections and improvements in current human norovirus culture systems.IMPORTANCE Viruses depend on the host cells they infect to provide the machinery and substrates for replication. Host cells are highly dynamic systems that can alter their intracellular environment and metabolic behavior, which may be helpful or inhibitory for an infecting virus. In this study, we show that macrophages, a target cell of murine norovirus (MNV), increase glycolysis upon viral infection, which is important for early steps in MNV infection. Human noroviruses (hNoV) are a major cause of gastroenteritis globally, causing enormous morbidity and economic burden. Currently, no effective antivirals or vaccines exist for hNoV, mainly due to the lack of high-efficiency in vitro culture models for their study. Thus, insights gained from the MNV model may reveal aspects of host cell metabolism that can be targeted for improving hNoV cell culture systems and for developing effective antiviral therapies.

Keywords:  calicivirus; carbon metabolism; glycolysis; noroviruses; oxidative phosphorylation; pentose phosphate pathway

DOI:  https://doi.org/10.1128/mBio.02175-18
Cell Rep. 2019 Mar 12. pii: S2211-1247(19)30239-6. [Epub ahead of print]26(11): 2984-2997.e4

A GPR17-cAMP-Lactate Signaling Axis in Oligodendrocytes Regulates Whole-Body Metabolism.

Zhimin Ou, Yanchen Ma, Yuxia Sun, Gege Zheng, Shiyun Wang, Rui Xing, Xiang Chen, Ying Han, Jiajia Wang, Q Richard Lu, Tong-Jin Zhao, Ying Chen.

  The CNS plays a pivotal role in energy homeostasis, but whether oligodendrocytes are involved has been largely unexplored. Here, we show that signaling through GPR17, a G-protein-coupled receptor predominantly expressed in the oligodendrocyte lineage, regulates food intake by modulating hypothalamic neuronal activities. GPR17-null mice and mice with an oligodendrocyte-specific knockout of GPR17 have lean phenotypes on a high-fat diet, suggesting that GPR17 regulates body weight by way of oligodendrocytes. Downregulation of GPR17 results in activation of cAMP-protein kinase A (PKA) signaling in oligodendrocytes and upregulated expression of pyruvate dehydrogenase kinase 1 (PDK1), which promotes lactate production. Elevation of lactate activates AKT and STAT3 signaling in the hypothalamic neurons, leading to increased expression of Pomc and suppression of Agrp. Our findings uncover a critical role of oligodendrocytes in metabolic homeostasis, where GPR17 modulates the production of lactate, which, in turn, acts as a metabolic signal to regulate neuronal activity.

Keywords:  GPR17; lactate; metabolic homeostasis; oligodendroyctes

DOI:  https://doi.org/10.1016/j.celrep.2019.02.060
Nat Immunol. 2019 Mar 11.

Inflammatory macrophage dependence on NAD+ salvage is a consequence of reactive oxygen species-mediated DNA damage.

Alanna M Cameron, Angela Castoldi, David E Sanin, Lea J Flachsmann, Cameron S Field, Daniel J Puleston, Ryan L Kyle, Annette E Patterson, Fabian Hässler, Joerg M Buescher, Beth Kelly, Erika L Pearce, Edward J Pearce.

The adoption of Warburg metabolism is critical for the activation of macrophages in response to lipopolysaccharide. Macrophages stimulated with lipopolysaccharide increase their expression of nicotinamide phosphoribosyltransferase (NAMPT), a key enzyme in NAD+ salvage, and loss of NAMPT activity alters their inflammatory potential. However, the events that lead to the cells' becoming dependent on NAD+ salvage remain poorly defined. We found that depletion of NAD+ and increased expression of NAMPT occurred rapidly after inflammatory activation and coincided with DNA damage caused by reactive oxygen species (ROS). ROS produced by complex III of the mitochondrial electron-transport chain were required for macrophage activation. DNA damage was associated with activation of poly(ADP-ribose) polymerase, which led to consumption of NAD+. In this setting, increased NAMPT expression allowed the maintenance of NAD+ pools sufficient for glyceraldehyde-3-phosphate dehydrogenase activity and Warburg metabolism. Our findings provide an integrated explanation for the dependence of inflammatory macrophages on the NAD+ salvage pathway.

DOI: https://doi.org/10.1038/s41590-019-0336-y