bims-camemi 2019-09-15 papers

bims-camemi

Biomed News

on Mitochondrial metabolism in cancer

Issue of 2019–09–15
23 papers selected by
Christian Frezza, , University of Cambridge, MRC Cancer Unit

Fascin Controls Metastatic Colonization and Mitochondrial Oxidative Phosphorylation by Remodeling Mitochondrial Actin Filaments.
Activation of BNIP3-mediated mitophagy protects against renal ischemia-reperfusion injury.
Methionine metabolism in health and cancer: a nexus of diet and precision medicine.
Glutamine independence is a selectable feature of pluripotent stem cells.
Mitochondrial supercomplex assembly promotes breast and endometrial tumorigenesis by metabolic alterations and enhanced hypoxia tolerance.
Vitamin C in combination with inhibition of mutant IDH1 synergistically activates TET enzymes and epigenetically modulates gene silencing in colon cancer cells.
The Good and the Bad of Mitochondrial Breakups.
PINK1 import regulation at a crossroad of mitochondrial fate.
Mechanism of mitochondrial complex I damage in brain ischemia/reperfusion injury. A hypothesis.
Mitochondria supply ATP to the ER through a mechanism antagonized by cytosolic Ca2.
Selective autophagy maintains centrosome integrity and accurate mitosis by turnover of centriolar satellites.
Mitochondrial fragmentation, elevated mitochondrial superoxide and respiratory supercomplexes disassembly is connected with the tamoxifen-resistant phenotype of breast cancer cells.
MCU Up-regulation contributes to myocardial ischemia-reperfusion Injury through calpain/OPA-1-mediated mitochondrial fusion/mitophagy Inhibition.
The Mitochondria-Endoplasmic Reticulum Contacts and Their Critical Role in Aging and Age-Associated Diseases.
SLC19A1 transports immunoreactive cyclic dinucleotides.
Molecular basis for the function of the αβ heterodimer of human NAD-dependent isocitrate dehydrogenase.
Proteomics of Melanoma Response to Immunotherapy Reveals Mitochondrial Dependence.
JAK2 mutant hematopoietic cells display metabolic alterations that can be targeted to treat myeloproliferative neoplasms.
Coupling of import and assembly pathways in mitochondrial protein biogenesis.
ATG2 regulation of phagophore expansion at mitochondria-associated ER membranes.
Tracing Tumor Evolution in Sarcoma Reveals Clonal Origin of Advanced Metastasis.
Kidney harvesting and metabolite extraction for metabolomics studies in rodents.
Isocitrate Lyase and Succinate Semialdehyde Dehydrogenase Mediate the Synthesis of α-Ketoglutarate in Pseudomonas fluorescens.

Cell Rep. 2019 Sep 10. pii: S2211-1247(19)31044-7. [Epub ahead of print]28(11): 2824-2836.e8

Fascin Controls Metastatic Colonization and Mitochondrial Oxidative Phosphorylation by Remodeling Mitochondrial Actin Filaments.

Shengchen Lin, Chongbiao Huang, Venugopal Gunda, Jianwei Sun, Srikumar P Chellappan, Zengxun Li, Victoria Izumi, Bin Fang, John Koomen, Pankaj K Singh, Jihui Hao, Shengyu Yang.

  The deregulation of the actin cytoskeleton has been extensively studied in metastatic dissemination. However, the post-dissemination role of the actin cytoskeleton dysregulation is poorly understood. Here, we report that fascin, an actin-bundling protein, promotes lung cancer metastatic colonization by augmenting metabolic stress resistance and mitochondrial oxidative phosphorylation (OXPHOS). Fascin is directly recruited to mitochondria under metabolic stress to stabilize mitochondrial actin filaments (mtF-actin). Using unbiased metabolomics and proteomics approaches, we discovered that fascin-mediated mtF-actin remodeling promotes mitochondrial OXPHOS by increasing the biogenesis of respiratory Complex I. Mechanistically, fascin and mtF-actin control the homeostasis of mtDNA to promote mitochondrial OXPHOS. The disruption of mtF-actin abrogates fascin-mediated lung cancer metastasis. Conversely, restoration of mitochondrial respiration by using yeast NDI1 in fascin-depleted cancer cells is able to rescue lung metastasis. Our findings indicate that the dysregulated actin cytoskeleton in metastatic lung cancer could be targeted to rewire mitochondrial metabolism and to prevent metastatic recurrence.

Keywords:  NSCLC; OXPHOS; actin; fascin; metastasis; metastatic colonization; mitochondria

DOI:  https://doi.org/10.1016/j.celrep.2019.08.011
Cell Death Dis. 2019 Sep 12. 10(9): 677

Activation of BNIP3-mediated mitophagy protects against renal ischemia-reperfusion injury.

Chengyuan Tang, Hailong Han, Zhiwen Liu, Yuxue Liu, Lijun Yin, Juan Cai, Liyu He, Yu Liu, Guochun Chen, Zhuohua Zhang, Xiao-Ming Yin, Zheng Dong.

Acute kidney injury (AKI) is a syndrome of abrupt loss of renal functions. The underlying pathological mechanisms of AKI remain largely unknown. BCL2-interacting protein 3 (BNIP3) has dual functions of regulating cell death and mitophagy, but its pathophysiological role in AKI remains unclear. Here, we demonstrated an increase of BNIP3 expression in cultured renal proximal tubular epithelial cells following oxygen-glucose deprivation-reperfusion (OGD-R) and in renal tubules after renal ischemia-reperfusion (IR)-induced injury in mice. Functionally, silencing Bnip3 by specific short hairpin RNAs in cultured renal tubular cells reduced OGD-R-induced mitophagy, and potentiated OGD-R-induced cell death. In vivo, Bnip3 knockout worsened renal IR injury, as manifested by more severe renal dysfunction and tissue injury. We further showed that Bnip3 knockout reduced mitophagy, which resulted in the accumulation of damaged mitochondria, increased production of reactive oxygen species, and enhanced cell death and inflammatory response in kidneys following renal IR. Taken together, these findings suggest that BNIP3-mediated mitophagy has a critical role in mitochondrial quality control and tubular cell survival during AKI.

DOI: https://doi.org/10.1038/s41419-019-1899-0
Nat Rev Cancer. 2019 Sep 12.

Methionine metabolism in health and cancer: a nexus of diet and precision medicine.

Sydney M Sanderson, Xia Gao, Ziwei Dai, Jason W Locasale.

Methionine uptake and metabolism is involved in a host of cellular functions including methylation reactions, redox maintenance, polyamine synthesis and coupling to folate metabolism, thus coordinating nucleotide and redox status. Each of these functions has been shown in many contexts to be relevant for cancer pathogenesis. Intriguingly, the levels of methionine obtained from the diet can have a large effect on cellular methionine metabolism. This establishes a link between nutrition and tumour cell metabolism that may allow for tumour-specific metabolic vulnerabilities that can be influenced by diet. Recently, a number of studies have begun to investigate the molecular and cellular mechanisms that underlie the interaction between nutrition, methionine metabolism and effects on health and cancer.

DOI: https://doi.org/10.1038/s41568-019-0187-8
Nat Metab. 2019 Jul;1(7): 676-687

Glutamine independence is a selectable feature of pluripotent stem cells.

Santosha A Vardhana, Paige K Arnold, Bess P Rosen, Yanyang Chen, Bryce W Carey, Danwei Huangfu, Carlos Carmona Fontaine, Craig B Thompson, Lydia W S Finley.

Most rapidly proliferating mammalian cells rely on the oxidation of exogenous glutamine to support cell proliferation. We previously found that culture of mouse embryonic stem cells (ESCs) in the presence of inhibitors against MEK and GSK3β to maintain pluripotency reduces cellular reliance on glutamine for tricarboxylic acid (TCA) cycle anaplerosis, enabling ESCs to proliferate in the absence of exogenous glutamine. Here we show that reduced dependence on exogenous glutamine is a generalizable feature of pluripotent stem cells. Enhancing self-renewal, through either overexpression of pluripotency-associated transcription factors or altered signal transduction, decreases the utilization of glutamine-derived carbons in the TCA cycle. As a result, cells with the highest potential for self-renewal can be enriched by transient culture in glutamine-deficient media. During pluripotent cell culture or reprogramming to pluripotency, transient glutamine withdrawal selectively leads to the elimination of non-pluripotent cells. These data reveal that reduced dependence on glutamine anaplerosis is an inherent feature of self-renewing pluripotent stem cells and reveal a simple, non-invasive mechanism to select for mouse and human pluripotent stem cells within a heterogeneous population during both ESC passage and induced pluripotent cell reprogramming.

DOI: https://doi.org/10.1038/s42255-019-0082-3
Nat Commun. 2019 Sep 11. 10(1): 4108

Mitochondrial supercomplex assembly promotes breast and endometrial tumorigenesis by metabolic alterations and enhanced hypoxia tolerance.

Kazuhiro Ikeda, Kuniko Horie-Inoue, Takashi Suzuki, Rutsuko Hobo, Norie Nakasato, Satoru Takeda, Satoshi Inoue.

Recent advance in cancer research sheds light on the contribution of mitochondrial respiration in tumorigenesis, as they efficiently produce ATP and oncogenic metabolites that will facilitate cancer cell growth. Here we show that a stabilizing factor for mitochondrial supercomplex assembly, COX7RP/COX7A2L/SCAF1, is abundantly expressed in clinical breast and endometrial cancers. Moreover, COX7RP overexpression associates with prognosis of breast cancer patients. We demonstrate that COX7RP overexpression in breast and endometrial cancer cells promotes in vitro and in vivo growth, stabilizes mitochondrial supercomplex assembly even in hypoxic states, and increases hypoxia tolerance. Metabolomic analyses reveal that COX7RP overexpression modulates the metabolic profile of cancer cells, particularly the steady-state levels of tricarboxylic acid cycle intermediates. Notably, silencing of each subunit of the 2-oxoglutarate dehydrogenase complex decreases the COX7RP-stimulated cancer cell growth. Our results indicate that COX7RP is a growth-regulatory factor for breast and endometrial cancer cells by regulating metabolic pathways and energy production.

DOI: https://doi.org/10.1038/s41467-019-12124-6
Epigenetics. 2019 Sep 11.

Vitamin C in combination with inhibition of mutant IDH1 synergistically activates TET enzymes and epigenetically modulates gene silencing in colon cancer cells.

Christian Gerecke, Fabian Schumacher, Alide Berndzen, Thomas Homann, Burkhard Kleuser.

  Mutations in the enzyme isocitrate dehydrogenase 1 (IDH1) lead to metabolic alterations and a sustained formation of 2-hydroxyglutarate (2-HG). 2-HG is an oncometabolite as it inhibits the activity of α-ketoglutarate-dependent dioxygenases such as ten-eleven translocation (TET) enzymes. Inhibitors of mutant IDH enzymes, like ML309, are currently tested in order to lower the levels of 2-HG. Vitamin C (VC) is an inducer of TET enzymes. To test a new therapeutic avenue of synergistic effects, the anti-neoplastic activity of inhibition of mutant IDH1 via ML309 in the presence of VC was investigated in the colon cancer cell line HCT116 IDH1R132H/+ (harboring a mutated IDH1 allele) and the parental cells HCT116 IDH1+/+ (wild type IDH1). Measurement of the oncometabolite indicated a 56-fold higher content of 2-HG in mutated cells compared to wild type cells. A significant reduction of 2-HG was observed in mutated cells after treatment with ML 309, whereas VC produced only minimally changes of the oncometabolite. However, combinatorial treatment with both, ML309 and VC, in mutated cells induced pronounced reduction of 2-HG leading to levels comparable to those in wild type cells. The decreased level of 2-HG in mutated cells after combinatorial treatment was accompanied by an enhanced global DNA hydroxymethylation and an increased gene expression of certain tumor suppressors. Moreover, mutated cells showed an increased percentage of apoptotic cells after treatment with non-cytotoxic concentrations of ML309 and VC. These results suggest that combinatorial therapy is of interest for further investigation to rescue TET activity and treatment of IDH1/2 mutated cancers.

Keywords:  IDH1; TET; cancer cells; epigenetics; vitamin C

DOI:  https://doi.org/10.1080/15592294.2019.1666652
Trends Cell Biol. 2019 Sep 05. pii: S0962-8924(19)30139-4. [Epub ahead of print]

The Good and the Bad of Mitochondrial Breakups.

Hans-Georg Sprenger, Thomas Langer.

  Mitochondrial morphology is a crucial determinant of mitochondrial and cellular function. Opposing fusion and fission events shape the tubular mitochondrial reticulum and ensure mitochondrial transport within cells. Cellular stress and pathophysiological conditions can lead to fragmentation of the mitochondrial network, which facilitates mitophagy and is associated with cell death. However, mitochondrial shape changes are also intertwined with the cellular metabolism, and metabolic switches can induce but also result from alterations in mitochondrial morphology. Here, we discuss recent advances in the field of mitochondrial dynamics, demonstrating cell- and tissue-specific effects of mitochondrial fragmentation on cellular metabolism, cell survival, and mitochondrial quality control.

Keywords:  metabolism; mitochondrial dynamics; mitochondrial fission; mitochondrial fragmentation; mitochondrial fusion; mitochondrial quality control

DOI:  https://doi.org/10.1016/j.tcb.2019.08.003
J Biochem. 2019 Sep 04. pii: mvz069. [Epub ahead of print]

PINK1 import regulation at a crossroad of mitochondrial fate.

Shiori Sekine.

  PTEN-induced kinase 1 (PINK1) is a mitochondrial kinase whose activity is tightly regulated by the mitochondrial health status. In response to mitochondrial damage, activated PINK1 can promote mitophagy, an autophagic elimination of damaged mitochondria, by cooperating with Parkin ubiquitin ligase. Loss-of-function of PINK1/Parkin-mediated mitophagy results in the accumulation of dysfunctional mitochondria, which could be one etiology of Parkinson's disease (PD). Within step-by-step signaling cascades of PINK1/Parkin-mediated mitophagy, mitochondrial damage-dependent PINK1 kinase activation is a critical step to trigger the mitophagy signal. Recent investigation of this process reveals that this stress-dependent PINK1 kinase activation is achieved by its regulated import into different mitochondrial compartments. Thus, PINK1 import regulation stands at an important crossroad to determine the mitochondrial fate - "keep" or "remove"? In this review, we will summarize how the PINK1 import is regulated in a mitochondrial health status-dependent manner and how this process could be pharmacologically modulated to activate the PINK1/Parkin pathway.

Keywords:  Mitochondrial import; Mitochondrial protease; Mitophagy; PINK1; Parkinson’s Disease

DOI:  https://doi.org/10.1093/jb/mvz069
Mol Cell Neurosci. 2019 Sep 05. pii: S1044-7431(19)30102-2. [Epub ahead of print] 103408

Mechanism of mitochondrial complex I damage in brain ischemia/reperfusion injury. A hypothesis.

Vadim Ten, Alexander Galkin.

  The purpose of this review is to integrate available data on the effect of brain ischemia/reperfusion (I/R) on mitochondrial complex I. Complex I is a key component of the mitochondrial respiratory chain and it is the only enzyme responsible for regenerating NAD+ for the maintenance of energy metabolism. The vulnerability of brain complex I to I/R injury has been observed in multiple animal models, but the mechanisms of enzyme damage have not been studied. This review summarizes old and new data on the effect of cerebral I/R on mitochondrial complex I, focusing on a recently discovered mechanism of the enzyme impairment. We found that the loss of the natural cofactor flavin mononucleotide (FMN) by complex I takes place after brain I/R. Reduced FMN dissociates from the enzyme if complex I is maintained under conditions of reverse electron transfer when mitochondria oxidize succinate accumulated during ischemia. The potential role of this process in the development of mitochondrial I/R damage in the brain is discussed.

Keywords:  Complex I; Flavin; Ischemia-reperfusion injury; Mitochondria; Riboflavin; Stroke

DOI:  https://doi.org/10.1016/j.mcn.2019.103408
Elife. 2019 Sep 09. pii: e49682. [Epub ahead of print]8

Mitochondria supply ATP to the ER through a mechanism antagonized by cytosolic Ca2.

Jing Yong, Helmut Bischof, Sandra Burgstaller, Marina Siirin, Anne Murphy, Roland Malli, Randal J Kaufman.

  The endoplasmic reticulum (ER) imports ATP and uses energy from ATP hydrolysis for protein folding and trafficking. However, little is known about how this vital ATP transport occurs across the ER membrane. Here, using three commonly used cell lines (CHO, INS1 and HeLa), we report that ATP enters the ER lumen through a cytosolic Ca2+-antagonized mechanism, or CaATiER (Ca2+-Antagonized Transport into ER). Significantly, we show that mitochondria supply ATP to the ER and a SERCA-dependent Ca2+ gradient across the ER membrane is necessary for ATP transport into the ER, through SLC35B1/AXER. We propose that under physiological conditions, increases in cytosolic Ca2+ inhibit ATP import into the ER lumen to limit ER ATP consumption. Furthermore, the ATP level in the ER is readily depleted by oxidative phosphorylation (OxPhos) inhibitors and that ER protein misfolding increases ATP uptake from mitochondria into the ER. These findings suggest that ATP usage in the ER may increase mitochondrial OxPhos while decreasing glycolysis, i.e., an 'anti-Warburg' effect.

Keywords:  biochemistry; cell biology; chemical biology

DOI:  https://doi.org/10.7554/eLife.49682
Nat Commun. 2019 Sep 13. 10(1): 4176

Selective autophagy maintains centrosome integrity and accurate mitosis by turnover of centriolar satellites.

Søs Grønbæk Holdgaard, Valentina Cianfanelli, Emanuela Pupo, Matteo Lambrughi, Michal Lubas, Julie C Nielsen, Susana Eibes, Emiliano Maiani, Lea M Harder, Nicole Wesch, Mads Møller Foged, Kenji Maeda, Francesca Nazio, Laura R de la Ballina, Volker Dötsch, Andreas Brech, Lisa B Frankel, Marja Jäättelä, Franco Locatelli, Marin Barisic, Jens S Andersen, Simon Bekker-Jensen, Anders H Lund, Vladimir V Rogov, Elena Papaleo, Letizia Lanzetti, Daniela De Zio, Francesco Cecconi.

The centrosome is the master orchestrator of mitotic spindle formation and chromosome segregation in animal cells. Centrosome abnormalities are frequently observed in cancer, but little is known of their origin and about pathways affecting centrosome homeostasis. Here we show that autophagy preserves centrosome organization and stability through selective turnover of centriolar satellite components, a process we termed doryphagy. Autophagy targets the satellite organizer PCM1 by interacting with GABARAPs via a C-terminal LIR motif. Accordingly, autophagy deficiency results in accumulation of large abnormal centriolar satellites and a resultant dysregulation of centrosome composition. These alterations have critical impact on centrosome stability and lead to mitotic centrosome fragmentation and unbalanced chromosome segregation. Our findings identify doryphagy as an important centrosome-regulating pathway and bring mechanistic insights to the link between autophagy dysfunction and chromosomal instability. In addition, we highlight the vital role of centriolar satellites in maintaining centrosome integrity.

DOI: https://doi.org/10.1038/s41467-019-12094-9
Free Radic Biol Med. 2019 Sep 05. pii: S0891-5849(19)31004-4. [Epub ahead of print]

Mitochondrial fragmentation, elevated mitochondrial superoxide and respiratory supercomplexes disassembly is connected with the tamoxifen-resistant phenotype of breast cancer cells.

Veronika Tomková, Cristian Sandoval-Acuña, Natalia Torrealba, Jaroslav Truksa.

  Tamoxifen resistance remains a clinical obstacle in the treatment of hormone sensitive breast cancer. It has been reported that tamoxifen is able to target respiratory complex I within mitochondria. Therefore, we established two tamoxifen-resistant cell lines, MCF7 Tam5R and T47D Tam5R resistant to 5 μM tamoxifen and investigated whether tamoxifen-resistant cells exhibit mitochondrial changes which could help them to survive the treatment. The function of mitochondria in this experimental model was evaluated in detail by studying i) the composition and activity of mitochondrial respiratory complexes; ii) respiration and glycolytic status; iii) mitochondrial distribution, dynamics and reactive oxygen species production. We show that Tam5R cells exhibit a significant decrease in mitochondrial respiration, low abundance of assembled mitochondrial respiratory supercomplexes, a more fragmented mitochondrial network connected with DRP1 Ser637 phosphorylation, higher glycolysis and sensitivity to 2-deoxyglucose. Tam5R cells also produce significantly higher levels of mitochondrial superoxide but at the same time increase their antioxidant defense (CAT, SOD2) through upregulation of SIRT3 and show phosphorylation of AMPK at Ser 485/491. Importantly, MCF7 ρ0 cells lacking functional mitochondria exhibit a markedly higher resistance to tamoxifen, supporting the role of mitochondria in tamoxifen resistance. We propose that reduced mitochondrial function and higher level of reactive oxygen species within mitochondria in concert with metabolic adaptations contribute to the phenotype of tamoxifen resistance.

Keywords:  Breast cancer; Mitochondria; Mitochondrial fragmentation; Reactive oxygen species; Tamoxifen resistance

DOI:  https://doi.org/10.1016/j.freeradbiomed.2019.09.004
J Cell Mol Med. 2019 Sep 09.

MCU Up-regulation contributes to myocardial ischemia-reperfusion Injury through calpain/OPA-1-mediated mitochondrial fusion/mitophagy Inhibition.

Lichun Guan, Zhimei Che, Xiangdong Meng, Yong Yu, Minghui Li, Ziqin Yu, Hui Shi, Dicheng Yang, Min Yu.

  Mitochondrial dynamic disorder is involved in myocardial ischemia/reperfusion (I/R) injury. To explore the effect of mitochondrial calcium uniporter (MCU) on mitochondrial dynamic imbalance under I/R and its related signal pathways, a mouse myocardial I/R model and hypoxia/reoxygenation model of mouse cardiomyocytes were established. The expression of MCU during I/R increased and related to myocardial injury, enhancement of mitochondrial fission, inhibition of mitochondrial fusion and mitophagy. Suppressing MCU functions by Ru360 during I/R could reduce myocardial infarction area and cardiomyocyte apoptosis, alleviate mitochondrial fission and restore mitochondrial fusion and mitophagy. However, spermine administration, which could enhance MCU function, deteriorated the above-mentioned myocardial cell injury and mitochondrial dynamic imbalanced. In addition, up-regulation of MCU promoted the expression and activation of calpain-1/2 and down-regulated the expression of Optic atrophy type 1 (OPA1). Meantime, in transgenic mice (overexpression calpastatin, the endogenous inhibitor of calpain) I/R model and OPA1 knock-down cultured cell. In I/R models of transgenic mice over-expressing calpastatin, which is the endogenous inhibitor of calpain, and in H/R models with siOPA1 transfection, inhibition of calpains could enhance mitochondrial fusion and mitophagy, and inhibit excessive mitochondrion fission and apoptosis through OPA1. Therefore, we conclude that during I/R, MCU up-regulation induces calpain activation, which down-regulates OPA1, consequently leading to mitochondrial dynamic imbalance.

Keywords:  calpain; ischemia/reperfusion (I/R); mitochondrial calcium uniporter (MCU); mitochondrial fission; mitophagy

DOI:  https://doi.org/10.1111/jcmm.14662
Front Cell Dev Biol. 2019 ;7 172

The Mitochondria-Endoplasmic Reticulum Contacts and Their Critical Role in Aging and Age-Associated Diseases.

Ornella Moltedo, Paolo Remondelli, Giuseppina Amodio.

  The recent discovery of interconnections between the endoplasmic reticulum (ER) membrane and those of almost all the cell compartments is providing novel perspectives for the understanding of the molecular events underlying cellular mechanisms in both physiological and pathological conditions. In particular, growing evidence strongly supports the idea that the molecular interactions occurring between ER and mitochondrial membranes, referred as the mitochondria (MT)-ER contacts (MERCs), may play a crucial role in aging and in the development of age-associated diseases. As emerged in the last decade, MERCs behave as signaling hubs composed by structural components that act as critical players in different age-associated disorders, such as neurodegenerative diseases and motor disorders, cancer, metabolic syndrome, as well as cardiovascular diseases. Age-associated disorders often derive from mitochondrial or ER dysfunction as consequences of oxidative stress, mitochondrial DNA mutations, accumulation of misfolded proteins, and defective organelle turnover. In this review, we discuss the recent advances associating MERCs to aging in the context of ER-MT crosstalk regulating redox signaling, ER-to MT lipid transfer, mitochondrial dynamics, and autophagy.

Keywords:  age-associated diseases; aging; endoplasmic reticulum; mitochondria; oxidative stress; senescence

DOI:  https://doi.org/10.3389/fcell.2019.00172
Nature. 2019 Sep 11.

SLC19A1 transports immunoreactive cyclic dinucleotides.

Rutger D Luteijn, Shivam A Zaver, Benjamin G Gowen, Stacia K Wyman, Nick E Garelis, Liberty Onia, Sarah M McWhirter, George E Katibah, Jacob E Corn, Joshua J Woodward, David H Raulet.

The accumulation of DNA in the cytosol serves as a key immunostimulatory signal associated with infections, cancer and genomic damage1,2. Cytosolic DNA triggers immune responses by activating the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway3. The binding of DNA to cGAS activates its enzymatic activity, leading to the synthesis of a second messenger, cyclic guanosine monophosphate-adenosine monophosphate (2'3'-cGAMP)4-7. This cyclic dinucleotide (CDN) activates STING8, which in turn activates the transcription factors interferon regulatory factor 3 (IRF3) and nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB), promoting the transcription of genes encoding type I interferons and other cytokines and mediators that stimulate a broader immune response. Exogenous 2'3'-cGAMP produced by malignant cells9 and other CDNs, including those produced by bacteria10-12 and synthetic CDNs used in cancer immunotherapy13,14, must traverse the cell membrane to activate STING in target cells. How these charged CDNs pass through the lipid bilayer is unknown. Here we used a genome-wide CRISPR-interference screen to identify the reduced folate carrier SLC19A1, a folate-organic phosphate antiporter, as the major transporter of CDNs. Depleting SLC19A1 in human cells inhibits CDN uptake and functional responses, and overexpressing SLC19A1 increases both uptake and functional responses. In human cell lines and primary cells ex vivo, CDN uptake is inhibited by folates as well as two medications approved for treatment of inflammatory diseases, sulfasalazine and the antifolate methotrexate. The identification of SLC19A1 as the major transporter of CDNs into cells has implications for the immunotherapeutic treatment of cancer13, host responsiveness to CDN-producing pathogenic microorganisms11 and-potentially-for some inflammatory diseases.

DOI: https://doi.org/10.1038/s41586-019-1553-0
J Biol Chem. 2019 Sep 12. pii: jbc.RA119.010099. [Epub ahead of print]

Molecular basis for the function of the αβ heterodimer of human NAD-dependent isocitrate dehydrogenase.

Pengkai Sun, Tengfei Ma, Tianlong Zhang, Hanwen Zhu, Jianyang Zhang, Yabing Liu, Jianping Ding.

  Mammalian mitochondrial NAD- dependent isocitrate dehydrogenase (NAD-IDH) catalyzes the decarboxylation of isocitrate into α-ketoglutarate in the tricarboxylic acid cycle. It exists as the α2βγ heterotetramer composed of the αβ and αγ heterodimers. Different from the αγ heterodimer that can be allosterically activated by CIT and ADP, the αβ heterodimer cannot be allosterically regulated by the activators; however, the molecular mechanism is unclear. We report here the crystal structures of the αβ heterodimer of human NAD-IDH with the α subunit in apo form, and in a Ca2+-bound, an NAD-bound, and an NADH-bound forms. Structural analyses and comparisons reveal that the αβ heterodimer has a similar yet more compact overall structure compared to the αγ heterodimer, and contains a pseudo allosteric site which is structurally different from the allosteric site. In particular, the β3-α3 and β12-α8 loops of the β subunit at the pseudo allosteric site adopt significantly different conformations from those of the γ subunit at the allosteric site, and hence impede the binding of the activators, explaining why the αβ heterodimer cannot be allosterically regulated by the activators. The structural data also show that NADH can compete with NAD to bind to the active site and inhibits the activity of the αβ heterodimer. These findings together with the biochemical data reveal the molecular basis for the function of the αβ heterodimer of human NAD-IDH.

Keywords:  NAD-IDH; allosteric regulation; conformational change; crystal structure; enzyme mechanism; enzyme structure; inhibition mechanism; isocitrate dehydrogenase; tricarboxylic acid cycle (TCA cycle) (Krebs cycle)

DOI:  https://doi.org/10.1074/jbc.RA119.010099
Cell. 2019 Aug 28. pii: S0092-8674(19)30900-6. [Epub ahead of print]

Proteomics of Melanoma Response to Immunotherapy Reveals Mitochondrial Dependence.

Michal Harel, Rona Ortenberg, Siva Karthik Varanasi, Kailash Chandra Mangalhara, Mariya Mardamshina, Ettai Markovits, Erez N Baruch, Victoria Tripple, May Arama-Chayoth, Eyal Greenberg, Anjana Shenoy, Ruveyda Ayasun, Naama Knafo, Shihao Xu, Liat Anafi, Gali Yanovich-Arad, Georgina D Barnabas, Shira Ashkenazi, Michal J Besser, Jacob Schachter, Marcus Bosenberg, Gerald S Shadel, Iris Barshack, Susan M Kaech, Gal Markel, Tamar Geiger.

  Immunotherapy has revolutionized cancer treatment, yet most patients do not respond. Here, we investigated mechanisms of response by profiling the proteome of clinical samples from advanced stage melanoma patients undergoing either tumor infiltrating lymphocyte (TIL)-based or anti- programmed death 1 (PD1) immunotherapy. Using high-resolution mass spectrometry, we quantified over 10,300 proteins in total and ∼4,500 proteins across most samples in each dataset. Statistical analyses revealed higher oxidative phosphorylation and lipid metabolism in responders than in non-responders in both treatments. To elucidate the effects of the metabolic state on the immune response, we examined melanoma cells upon metabolic perturbations or CRISPR-Cas9 knockouts. These experiments indicated lipid metabolism as a regulatory mechanism that increases melanoma immunogenicity by elevating antigen presentation, thereby increasing sensitivity to T cell mediated killing both in vitro and in vivo. Altogether, our proteomic analyses revealed association between the melanoma metabolic state and the response to immunotherapy, which can be the basis for future improvement of therapeutic response.

Keywords:  anti-PD-1; cancer metabolism; immune checkpoint inhibitors; immunotherapy; lipid metabolism; mass spectrometry; melanoma; mitochondrial metabolism; proteomics; tumor-infiltrating lymphocytes

DOI:  https://doi.org/10.1016/j.cell.2019.08.012
Blood. 2019 Sep 11. pii: blood.2019000162. [Epub ahead of print]

JAK2 mutant hematopoietic cells display metabolic alterations that can be targeted to treat myeloproliferative neoplasms.

Tata Nageswara Rao, Nils Hansen, Julian Hilfiker, Shivam Rai, Julia-Magdalena Majewska, Danijela Leković, Deniz Gezer, Nicola Andina, Serena Galli, Teresa Cassel, Florian Geier, Julien Delezie, Ronny Nienhold, Hui Hao-Shen, Christian Beisel, Serena Di Palma, Sarah Dimeloe, Jonel Trebicka, Dominik Wolf, Max Gassmann, Teresa W-M Fan, Andrew N Lane, Christoph Handschin, Stefan Dirnhofer, Nicolaus Kröger, Christoph Hess, Thomas Radimerski, Steffen Koschmieder, Vladan P Čokić, Radek C Skoda.

Increased energy requirement and metabolic reprograming are hallmarks of cancer cells. We show that metabolic alterations in hematopoietic cells are fundamental to the pathogenesis of mutant JAK2 driven myeloproliferative neoplasms (MPNs). We found that expression of mutant JAK2 augmented and subverted metabolic activity of MPN cells resulting in systemic metabolic changes in vivo, including hypoglycemia, adipose tissue atrophy and early mortality. Hypoglycemia in MPN mouse models correlated with hyperactive erythropoiesis and was due to a combination of elevated glycolysis and increased oxidative phosphorylation. Modulating nutrient supply through high fat diet improved survival, while high glucose diet augmented the MPN phenotype. Transcriptomic and metabolomic analyses identified numerous metabolic nodes in JAK2 mutant hematopoietic stem and progenitor cells that were altered in comparison with wildtype controls. We studied the consequences of elevated levels of Pfkfb3, a key regulatory enzyme of glycolysis, and found that pharmacological inhibition of Pfkfb3 with the small molecule 3PO reversed hypoglycemia and reduced hematopoietic manifestations of MPN. These effects were additive with the JAK1/2 inhibitor, Ruxolitinib, in vivo and in vitro. Inhibition of glycolysis by 3PO altered the redox homeostasis, leading to accumulation of reactive oxygen species and augmented apoptosis rate. Our findings reveal the contribution of metabolic alterations to the pathogenesis of MPN and suggest that metabolic dependencies of mutant cells represent vulnerabilities that can be targeted for treating MPN.

DOI: https://doi.org/10.1182/blood.2019000162
Biol Chem. 2019 Sep 11. pii: /j/bchm.ahead-of-print/hsz-2019-0310/hsz-2019-0310.xml. [Epub ahead of print]

Coupling of import and assembly pathways in mitochondrial protein biogenesis.

Alexander Grevel, Nikolaus Pfanner, Thomas Becker.

  Biogenesis and function of mitochondria depend on the import of about 1000 precursor proteins that are produced on cytosolic ribosomes. The translocase of the outer membrane (TOM) forms the entry gate for most proteins. After passage through the TOM channel, dedicated preprotein translocases sort the precursor proteins into the mitochondrial subcompartments. Many proteins have to be assembled into oligomeric membrane-integrated complexes in order to perform their functions. In this review, we discuss a dual role of mitochondrial preprotein translocases in protein translocation and oligomeric assembly, focusing on the biogenesis of the TOM complex and the respiratory chain. The sorting and assembly machinery (SAM) of the outer mitochondrial membrane forms a dynamic platform for coupling transport and assembly of TOM subunits. The biogenesis of the cytochrome c oxidase of the inner membrane involves a molecular circuit to adjust translation of mitochondrial-encoded core subunits to the availability of nuclear-encoded partner proteins. Thus, mitochondrial protein translocases not only import precursor proteins but can also support their assembly into functional complexes.

Keywords:  TOM complex; mitochondria; protein assembly; protein import; respiratory chain

DOI:  https://doi.org/10.1515/hsz-2019-0310
Autophagy. 2019 Sep 12.

ATG2 regulation of phagophore expansion at mitochondria-associated ER membranes.

Zhenyuan Tang, Yoshinori Takahashi, Hong-Gang Wang.

  The mechanism by which ATG2 (ATG2A and ATG2B in mammals) regulates autophagosome biogenesis remains largely unknown. In our recent study, we showed that ATG2A translocates to the mitochondria-associated ER membranes (MAM) to promote phagophore growth during nutrient starvation. Mechanistically, the mitochondrial translocase TOMM40 binds to a C-terminal domain of ATG2A, termed the MAM localization domain (MLD), and mediates its MAM translocation in a manner dependent on the TOMM receptor TOMM70. Moreover, ATG2A associates with ATG9A through its N-terminal domain and this interaction is required for phagophore expansion and efficient autophagic flux. These observations suggest that ATG2 operates a mechanism for phagophore expansion at the MAM through the TOMM40-TOMM70 complex and ATG9 during autophagy.

Keywords:  ATG2; ATG9; TOM40; TOM70; mitochondria-associated ER membranes (MAM); phagophore expansion

DOI:  https://doi.org/10.1080/15548627.2019.1666594
Cell Rep. 2019 Sep 10. pii: S2211-1247(19)31063-0. [Epub ahead of print]28(11): 2837-2850.e5

Tracing Tumor Evolution in Sarcoma Reveals Clonal Origin of Advanced Metastasis.

Yuning J Tang, Jianguo Huang, Hidetoshi Tsushima, Ga I Ban, Hongyuan Zhang, Kristianne M Oristian, Vijitha Puviindran, Nerissa Williams, Xiruo Ding, Jianhong Ou, Sin-Ho Jung, Chang-Lung Lee, Yiqun Jiao, Benny J Chen, David G Kirsch, Benjamin A Alman.

  Cellular heterogeneity is frequently observed in cancer, but the biological significance of heterogeneous tumor clones is not well defined. Using multicolor reporters and CRISPR-Cas9 barcoding, we trace clonal dynamics in a mouse model of sarcoma. We show that primary tumor growth is associated with a reduction in clonal heterogeneity. Local recurrence of tumors following surgery or radiation therapy is driven by multiple clones. In contrast, advanced metastasis to the lungs is driven by clonal selection of a single metastatic clone (MC). Using RNA sequencing (RNA-seq) and in vivo assays, we identify candidate suppressors of metastasis, namely, Rasd1, Reck, and Aldh1a2. These genes are downregulated in MCs of the primary tumors prior to the formation of metastases. Overexpression of these suppressors of metastasis impair the ability of sarcoma cells to colonize the lungs. Overall, this study reveals clonal dynamics during each step of tumor progression, from initiation to growth, recurrence, and distant metastasis.

Keywords:  cancer metastasis; clonal evolution; lineage tracing; sarcoma; suppressors of metastasis; tumor heterogeneity

DOI:  https://doi.org/10.1016/j.celrep.2019.08.029
Methods Cell Biol. 2019 ;pii: S0091-679X(19)30078-0. [Epub ahead of print]154 15-29

Kidney harvesting and metabolite extraction for metabolomics studies in rodents.

Igor Maksimovic, Song Zhang, Ivan Vuckovic, Maria V Irazabal.

  Elucidating the metabolic changes that accompany disease states via metabolomics analysis of tissues has become an important avenue of exploration in biomarker and therapeutic target discovery. Conventional harvesting techniques rely on post-euthanasia tissue harvest which introduces ischemic conditions and subsequent metabolome changes that may ultimately introduce artifacts into final analyses. In this chapter, we present protocols for low-ischemia time rapid kidney tissue harvest followed by metabolite extraction for metabolomics studies in rodents.

Keywords:  Ischemia/reperfusion; Metabolomics; Tissue harvest

DOI:  https://doi.org/10.1016/bs.mcb.2019.05.009
Front Microbiol. 2019 ;10 1929

Isocitrate Lyase and Succinate Semialdehyde Dehydrogenase Mediate the Synthesis of α-Ketoglutarate in Pseudomonas fluorescens.

Azhar A Alhasawi, Sean C Thomas, Sujeethar Tharmalingam, Felix Legendre, Vasu D Appanna.

  Glycerol is an important by-product of the biodiesel industry and its transformation into value-added products like keto acids is being actively pursued in order to improve the efficacy of this renewable energy sector. Here, we report that the enhanced production of α-ketoglutarate (KG) effected by Pseudomonas fluorescens in a mineral medium supplemented with manganese (Mn) is propelled by the increased activities of succinate semialdehyde dehydrogenase (SSADH), γ-aminobutyric acid aminotransaminase (GABAT), and isocitrate lyase (ICL). The latter generates glyoxylate and succinate two key metabolites involved in this process. Fumarate reductase (FRD) also aids in augmenting the pool of succinate, a precursor of succinate semialdehyde (SSA). The latter is then carboxylated to KG with the assistance of α-ketoglutarate decarboxylase (KDC). These enzymes work in tandem to ensure copious secretion of the keto acid. When incubated with glycerol in the presence of bicarbonate ( HCO3- ), cell-free extracts readily produce KG with a metabolite fingerprint attributed to glutamate, γ-aminobutyric acid (GABA), succinate and succinate semialdehyde. Further targeted metabolomic and functional proteomic studies with high-performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR) and gel electrophoresis techniques provided molecular insights into this KG-generating machinery. Real-time quantitative polymerase chain reaction (RT-qPCR) analyses revealed the transcripts responsible for ICL and SSADH were elevated in the Mn-supplemented cultures. This hitherto unreported metabolic network where ICL and SSADH orchestrate the enhanced production of KG from glycerol, provides an elegant means of converting an industrial waste into a keto acid with wide-ranging application in the medical, cosmetic, and chemical sectors.

Keywords:  biofuel; enzymes; isocitrate; metabolic engineering; succinate semialdehyde; α-Ketoglutarate; γ-Amino butyric acid

DOI:  https://doi.org/10.3389/fmicb.2019.01929