bims-mepmim 2023-07-16 papers

bims-mepmim

Biomed News

on Metabolites in pathological microenvironments and immunometabolism

Issue of 2023–07–16
twenty papers selected by
Erika Mariana Palmieri, NIH/NCI Laboratory of Cancer ImmunoMetabolism

Ribonucleotide synthesis by NME6 fuels mitochondrial gene expression.
Efferocytes release extracellular vesicles to resolve inflammation and tissue injury via prosaposin-GPR37 signaling.
Liver lipophagy ameliorates nonalcoholic steatohepatitis through extracellular lipid secretion.
CRAT links cholesterol metabolism to innate immune responses in the heart.
Immunoproteasome-specific subunit PSMB9 induction is required to regulate cellular proteostasis upon mitochondrial dysfunction.
IF1 ablation prevents ATP synthase oligomerization, enhances mitochondrial ATP turnover and promotes an adenosine-mediated pro-inflammatory phenotype.
A critical role for heme synthesis and succinate in the regulation of pluripotent states transitions.
Hypoxia-reprogramed megamitochondrion contacts and engulfs lysosome to mediate mitochondrial self-digestion.
Establishing mammalian GLUT kinetics and lipid composition influences in a reconstituted-liposome system.
Glutathione limits RUNX2 oxidation and degradation to regulate bone formation.
Targeting the TCA cycle can ameliorate widespread axonal energy deficiency in neuroinflammatory lesions.
SREBP-1 upregulates lipophagy to maintain cholesterol homeostasis in brain tumor cells.
Cytochrome c lysine acetylation regulates cellular respiration and cell death in ischemic skeletal muscle.
Notch signaling regulates a metabolic switch through inhibiting PGC-1α and mitochondrial biogenesis in dedifferentiated liposarcoma.
STING is a cell-intrinsic metabolic checkpoint restricting aerobic glycolysis by targeting HK2.
Kinetic and structural characterization of carboxyspermidine dehydrogenase of polyamine biosynthesis.
Mitochondrial respiratory complex I deficiency inhibits brown adipogenesis by limiting heme regulation of histone demethylation.
Importance of glutamine in synaptic vesicles revealed by functional studies of SLC6A17 and its mutations pathogenic for intellectual disability.
Maintenance of cellular vitamin B6 levels and mitochondrial oxidative function depend on pyridoxal 5'-phosphate homeostasis protein (PLPHP).
Protocol to expand and CRISPR-Cas9 genomic edit murine MAIT cells for subsequent in vivo studies.

EMBO J. 2023 Jul 13. e113256

Ribonucleotide synthesis by NME6 fuels mitochondrial gene expression.

Nils Grotehans, Lynn McGarry, Hendrik Nolte, Vanessa Xavier, Moritz Kroker, Álvaro Jesús Narbona-Pérez, Soni Deshwal, Patrick Giavalisco, Thomas Langer, Thomas MacVicar.

  Replication of the mitochondrial genome and expression of the genes it encodes both depend on a sufficient supply of nucleotides to mitochondria. Accordingly, dysregulated nucleotide metabolism not only destabilises the mitochondrial genome, but also affects its transcription. Here, we report that a mitochondrial nucleoside diphosphate kinase, NME6, supplies mitochondria with pyrimidine ribonucleotides that are necessary for the transcription of mitochondrial genes. Loss of NME6 function leads to the depletion of mitochondrial transcripts, as well as destabilisation of the electron transport chain and impaired oxidative phosphorylation. These deficiencies are rescued by an exogenous supply of pyrimidine ribonucleosides. Moreover, NME6 is required for the maintenance of mitochondrial DNA when the access to cytosolic pyrimidine deoxyribonucleotides is limited. Our results therefore reveal an important role for ribonucleotide salvage in mitochondrial gene expression.

Keywords:  NME6; mitochondria; mitochondrial DNA; mitochondrial transcription; nucleotide metabolism

DOI:  https://doi.org/10.15252/embj.2022113256
Cell Rep. 2023 Jul 11. pii: S2211-1247(23)00819-7. [Epub ahead of print]42(7): 112808

Efferocytes release extracellular vesicles to resolve inflammation and tissue injury via prosaposin-GPR37 signaling.

Purbasha Bhattacharya, Umesh Kumar Dhawan, Mohammed Tayab Hussain, Praveen Singh, Karran Kiran Bhagat, Aarushi Singhal, Shani Austin-Williams, Shantanu Sengupta, Manikandan Subramanian.

  Macrophages release soluble mediators following efferocytic clearance of apoptotic cells to facilitate intercellular communication and promote the resolution of inflammation. However, whether inflammation resolution is modulated by extracellular vesicles (EVs) and vesicular mediators released by efferocytes is not known. We report that efferocyte-derived EVs express prosaposin, which binds to macrophage GPR37 to increase expression of the efferocytosis receptor Tim4 via an ERK-AP1-dependent signaling axis, leading to increased macrophage efferocytosis efficiency and accelerated resolution of inflammation. Neutralization and knockdown of prosaposin or blocking GRP37 abrogates the pro-resolution effects of efferocyte-derived EVs in vivo. Administration of efferocyte-derived EVs in a murine model of atherosclerosis is associated with an increase in lesional macrophage efferocytosis efficiency and a decrease in plaque necrosis and lesional inflammation. Thus, we establish a critical role for efferocyte-derived vesicular mediators in increasing macrophage efferocytosis efficiency and accelerating the resolution of inflammation and tissue injury.

Keywords:  CP: Immunology; GPCR; atherosclerosis; efferocytosis; inflammation resolution; macrophage

DOI:  https://doi.org/10.1016/j.celrep.2023.112808
Nat Commun. 2023 Jul 13. 14(1): 4084

Liver lipophagy ameliorates nonalcoholic steatohepatitis through extracellular lipid secretion.

Yoshito Minami, Atsushi Hoshino, Yusuke Higuchi, Masahide Hamaguchi, Yusaku Kaneko, Yuhei Kirita, Shunta Taminishi, Toshiyuki Nishiji, Akiyuki Taruno, Michiaki Fukui, Zoltan Arany, Satoaki Matoba.

Nonalcoholic steatohepatitis (NASH) is a progressive disorder with aberrant lipid accumulation and subsequent inflammatory and profibrotic response. Therapeutic efforts at lipid reduction via increasing cytoplasmic lipolysis unfortunately worsens hepatitis due to toxicity of liberated fatty acid. An alternative approach could be lipid reduction through autophagic disposal, i.e., lipophagy. We engineered a synthetic adaptor protein to induce lipophagy, combining a lipid droplet-targeting signal with optimized LC3-interacting domain. Activating hepatocyte lipophagy in vivo strongly mitigated both steatosis and hepatitis in a diet-induced mouse NASH model. Mechanistically, activated lipophagy promoted the excretion of lipid from hepatocytes, thereby suppressing harmful intracellular accumulation of nonesterified fatty acid. A high-content compound screen identified alpelisib and digoxin, clinically-approved compounds, as effective activators of lipophagy. Administration of alpelisib or digoxin in vivo strongly inhibited the transition to steatohepatitis. These data thus identify lipophagy as a promising therapeutic approach to prevent NASH progression.

DOI: https://doi.org/10.1038/s41467-023-39404-6
Nat Metab. 2023 Jul 13.

CRAT links cholesterol metabolism to innate immune responses in the heart.

Hua Mao, Aude Angelini, Shengyu Li, Guangyu Wang, Luge Li, Cam Patterson, Xinchun Pi, Liang Xie.

Chronic inflammation is associated with increased risk and poor prognosis of heart failure; however, the precise mechanism that provokes sustained inflammation in the failing heart remains elusive. Here we report that depletion of carnitine acetyltransferase (CRAT) promotes cholesterol catabolism through bile acid synthesis pathway in cardiomyocytes. Intracellular accumulation of bile acid or intermediate, 7α-hydroxyl-3-oxo-4-cholestenoic acid, induces mitochondrial DNA stress and triggers cGAS-STING-dependent type I interferon responses. Furthermore, type I interferon responses elicited by CRAT deficiency substantially increase AIM2 expression and AIM2-dependent inflammasome activation. Genetic deletion of cardiomyocyte CRAT in mice of both sexes results in myocardial inflammation and dilated cardiomyopathy, which can be reversed by combined depletion of caspase-1, cGAS or AIM2. Collectively, we identify a mechanism by which cardiac energy metabolism, cholesterol homeostasis and cardiomyocyte-intrinsic innate immune responses are interconnected via a CRAT-mediated bile acid synthesis pathway, which contributes to chronic myocardial inflammation and heart failure progression.

DOI: https://doi.org/10.1038/s42255-023-00844-5
Nat Commun. 2023 07 11. 14(1): 4092

Immunoproteasome-specific subunit PSMB9 induction is required to regulate cellular proteostasis upon mitochondrial dysfunction.

Minji Kim, Remigiusz A Serwa, Lukasz Samluk, Ida Suppanz, Agata Kodroń, Tomasz M Stępkowski, Praveenraj Elancheliyan, Biniyam Tsegaye, Silke Oeljeklaus, Michal Wasilewski, Bettina Warscheid, Agnieszka Chacinska.

Perturbed cellular protein homeostasis (proteostasis) and mitochondrial dysfunction play an important role in neurodegenerative diseases, however, the interplay between these two phenomena remains unclear. Mitochondrial dysfunction leads to a delay in mitochondrial protein import, causing accumulation of non-imported mitochondrial proteins in the cytosol and challenging proteostasis. Cells respond by increasing proteasome activity and molecular chaperones in yeast and C. elegans. Here, we demonstrate that in human cells mitochondrial dysfunction leads to the upregulation of a chaperone HSPB1 and, interestingly, an immunoproteasome-specific subunit PSMB9. Moreover, PSMB9 expression is dependent on the translation elongation factor EEF1A2. These mechanisms constitute a defense response to preserve cellular proteostasis under mitochondrial stress. Our findings define a mode of proteasomal activation through the change in proteasome composition driven by EEF1A2 and its spatial regulation, and are useful to formulate therapies to prevent neurodegenerative diseases.

DOI: https://doi.org/10.1038/s41467-023-39642-8
Cell Death Dis. 2023 Jul 12. 14(7): 413

IF1 ablation prevents ATP synthase oligomerization, enhances mitochondrial ATP turnover and promotes an adenosine-mediated pro-inflammatory phenotype.

Sonia Domínguez-Zorita, Inés Romero-Carramiñana, Fulvio Santacatterina, Pau B Esparza-Moltó, Carolina Simó, Araceli Del-Arco, Cristina Núñez de Arenas, Jorge Saiz, Coral Barbas, José M Cuezva.

ATPase Inhibitory Factor 1 (IF1) regulates the activity of mitochondrial ATP synthase. The expression of IF1 in differentiated human and mouse cells is highly variable. In intestinal cells, the overexpression of IF1 protects against colon inflammation. Herein, we have developed a conditional IF1-knockout mouse model in intestinal epithelium to investigate the role of IF1 in mitochondrial function and tissue homeostasis. The results show that IF1-ablated mice have increased ATP synthase/hydrolase activities, leading to profound mitochondrial dysfunction and a pro-inflammatory phenotype that impairs the permeability of the intestinal barrier compromising mouse survival upon inflammation. Deletion of IF1 prevents the formation of oligomeric assemblies of ATP synthase and alters cristae structure and the electron transport chain. Moreover, lack of IF1 promotes an intramitochondrial Ca2+ overload in vivo, minimizing the threshold to Ca2+-induced permeability transition (mPT). Removal of IF1 in cell lines also prevents the formation of oligomeric assemblies of ATP synthase, minimizing the threshold to Ca2+-induced mPT. Metabolomic analyses of mice serum and colon tissue highlight that IF1 ablation promotes the activation of de novo purine and salvage pathways. Mechanistically, lack of IF1 in cell lines increases ATP synthase/hydrolase activities and installs futile ATP hydrolysis in mitochondria, resulting in the activation of purine metabolism and in the accumulation of adenosine, both in culture medium and in mice serum. Adenosine, through ADORA2B receptors, promotes an autoimmune phenotype in mice, stressing the role of the IF1/ATP synthase axis in tissue immune responses. Overall, the results highlight that IF1 is required for ATP synthase oligomerization and that it acts as a brake to prevent ATP hydrolysis under in vivo phosphorylating conditions in intestinal cells.

DOI: https://doi.org/10.1038/s41419-023-05957-z
Elife. 2023 Jul 10. pii: e78546. [Epub ahead of print]12

A critical role for heme synthesis and succinate in the regulation of pluripotent states transitions.

Damien Detraux, Marino Caruso, Louise Feller, Maude Fransolet, Sébastien Meurant, Julie Mathieu, Thierry Arnould, Patricia Renard.

  Using embryonic stem cells (ESCs) in regenerative medicine or in disease modeling requires a complete understanding of these cells. Two main distinct developmental states of ESCs have been stabilized in vitro, a naïve pre-implantation stage and a primed post-implantation stage. Based on two recently published CRISPR-Cas9 knockout functional screens, we show here that the exit of the naïve state is impaired upon heme biosynthesis pathway blockade, linked in mESCs to the incapacity to activate MAPK- and TGFb-dependent signaling pathways after succinate accumulation. In addition, heme synthesis inhibition promotes the acquisition of 2 cell-like cells in a heme-independent manner caused by a mitochondrial succinate accumulation and leakage out of the cell. We further demonstrate that extracellular succinate acts as a paracrine/autocrine signal, able to trigger the 2C-like reprogramming through the activation of its plasma membrane receptor, SUCNR1. Overall, this study unveils a new mechanism underlying the maintenance of pluripotency under the control of heme synthesis.

Keywords:  developmental biology; human; mouse; regenerative medicine; stem cells

DOI:  https://doi.org/10.7554/eLife.78546
Nat Commun. 2023 07 11. 14(1): 4105

Hypoxia-reprogramed megamitochondrion contacts and engulfs lysosome to mediate mitochondrial self-digestion.

Tianshu Hao, Jianglong Yu, Zhida Wu, Jie Jiang, Longlong Gong, Bingjun Wang, Hanze Guo, Huabin Zhao, Bin Lu, Simone Engelender, He He, Zhiyin Song.

Mitochondria are the key organelles for sensing oxygen, which is consumed by oxidative phosphorylation to generate ATP. Lysosomes contain hydrolytic enzymes that degrade misfolded proteins and damaged organelles to maintain cellular homeostasis. Mitochondria physically and functionally interact with lysosomes to regulate cellular metabolism. However, the mode and biological functions of mitochondria-lysosome communication remain largely unknown. Here, we show that hypoxia remodels normal tubular mitochondria into megamitochondria by inducing broad inter-mitochondria contacts and subsequent fusion. Importantly, under hypoxia, mitochondria-lysosome contacts are promoted, and certain lysosomes are engulfed by megamitochondria, in a process we term megamitochondria engulfing lysosome (MMEL). Both megamitochondria and mature lysosomes are required for MMEL. Moreover, the STX17-SNAP29-VAMP7 complex contributes to mitochondria-lysosome contacts and MMEL under hypoxia. Intriguingly, MMEL mediates a mode of mitochondrial degradation, which we termed mitochondrial self-digestion (MSD). Moreover, MSD increases mitochondrial ROS production. Our results reveal a mode of crosstalk between mitochondria and lysosomes and uncover an additional pathway for mitochondrial degradation.

DOI: https://doi.org/10.1038/s41467-023-39811-9
Nat Commun. 2023 07 10. 14(1): 4070

Establishing mammalian GLUT kinetics and lipid composition influences in a reconstituted-liposome system.

Albert Suades, Aziz Qureshi, Sarah E McComas, Mathieu Coinçon, Axel Rudling, Yurie Chatzikyriakidou, Michael Landreh, Jens Carlsson, David Drew.

Glucose transporters (GLUTs) are essential for organism-wide glucose homeostasis in mammals, and their dysfunction is associated with numerous diseases, such as diabetes and cancer. Despite structural advances, transport assays using purified GLUTs have proven to be difficult to implement, hampering deeper mechanistic insights. Here, we have optimized a transport assay in liposomes for the fructose-specific isoform GLUT5. By combining lipidomic analysis with native MS and thermal-shift assays, we replicate the GLUT5 transport activities seen in crude lipids using a small number of synthetic lipids. We conclude that GLUT5 is only active under a specific range of membrane fluidity, and that human GLUT1-4 prefers a similar lipid composition to GLUT5. Although GLUT3 is designated as the high-affinity glucose transporter, in vitro D-glucose kinetics demonstrates that GLUT1 and GLUT3 actually have a similar KM, but GLUT3 has a higher turnover. Interestingly, GLUT4 has a high KM for D-glucose and yet a very slow turnover, which may have evolved to ensure uptake regulation by insulin-dependent trafficking. Overall, we outline a much-needed transport assay for measuring GLUT kinetics and our analysis implies that high-levels of free fatty acid in membranes, as found in those suffering from metabolic disorders, could directly impair glucose uptake.

DOI: https://doi.org/10.1038/s41467-023-39711-y
JCI Insight. 2023 Jul 11. pii: e166888. [Epub ahead of print]

Glutathione limits RUNX2 oxidation and degradation to regulate bone formation.

Guoli Hu, Yilin Yu, Deepika Sharma, Shondra M Pruett-Miller, Yinshi Ren, Guo-Fang Zhang, Courtney M Karner.

  Reactive oxygen species (ROS) are natural products of mitochondrial oxidative metabolism and oxidative protein folding. ROS levels must be well controlled as elevated ROS has been shown to have deleterious effects on osteoblasts. Moreover, excessive ROS is thought to underly many of the skeletal phenotypes associated with aging and sex steroid deficiency in mice and humans. The mechanisms by which osteoblasts regulate ROS and how ROS inhibits osteoblasts are not well understood. Here, we demonstrate that de novo glutathione (GSH) biosynthesis is essential to neutralize ROS and establish a pro-osteogenic REDOX environment. Using a multifaceted approach, we demonstrate that reducing GSH biosynthesis leads to acute degradation of RUNX2, impaired osteoblast differentiation and reduced bone formation. Conversely, reducing ROS using Catalase enhances RUNX2 stability and promotes osteoblast differentiation and bone formation when GSH biosynthesis is limited. Highlighting the therapeutic implications of these findings, in utero antioxidant therapy stabilizes RUNX2 and improves bone development in the Runx2+/- haploinsufficient mouse model of human Cleidocranial Dysplasia. Thus, our data establish RUNX2 as a molecular sensor of the osteoblast REDOX environment and mechanistically clarifies how ROS negatively impacts osteoblast differentiation and bone formation.

Keywords:  Amino acid metabolism; Bone Biology; Bone development; Osteoclast/osteoblast biology

DOI:  https://doi.org/10.1172/jci.insight.166888
Nat Metab. 2023 Jul 10.

Targeting the TCA cycle can ameliorate widespread axonal energy deficiency in neuroinflammatory lesions.

Yi-Heng Tai, Daniel Engels, Giuseppe Locatelli, Ioanna Emmanouilidis, Caroline Fecher, Delphine Theodorou, Stephan A Müller, Simon Licht-Mayer, Mario Kreutzfeldt, Ingrid Wagner, Natalia Prudente de Mello, Sofia-Natsouko Gkotzamani, Laura Trovò, Arek Kendirli, Almir Aljović, Michael O Breckwoldt, Ronald Naumann, Florence M Bareyre, Fabiana Perocchi, Don Mahad, Doron Merkler, Stefan F Lichtenthaler, Martin Kerschensteiner, Thomas Misgeld.

Inflammation in the central nervous system can impair the function of neuronal mitochondria and contributes to axon degeneration in the common neuroinflammatory disease multiple sclerosis (MS). Here we combine cell-type-specific mitochondrial proteomics with in vivo biosensor imaging to dissect how inflammation alters the molecular composition and functional capacity of neuronal mitochondria. We show that neuroinflammatory lesions in the mouse spinal cord cause widespread and persisting axonal ATP deficiency, which precedes mitochondrial oxidation and calcium overload. This axonal energy deficiency is associated with impaired electron transport chain function, but also an upstream imbalance of tricarboxylic acid (TCA) cycle enzymes, with several, including key rate-limiting, enzymes being depleted in neuronal mitochondria in experimental models and in MS lesions. Notably, viral overexpression of individual TCA enzymes can ameliorate the axonal energy deficits in neuroinflammatory lesions, suggesting that TCA cycle dysfunction in MS may be amendable to therapy.

DOI: https://doi.org/10.1038/s42255-023-00838-3
Cell Rep. 2023 Jul 11. pii: S2211-1247(23)00801-X. [Epub ahead of print]42(7): 112790

SREBP-1 upregulates lipophagy to maintain cholesterol homeostasis in brain tumor cells.

Feng Geng, Yaogang Zhong, Huali Su, Etienne Lefai, Shino Magaki, Timothy F Cloughesy, William H Yong, Arnab Chakravarti, Deliang Guo.

  Cholesterol is a structural component of cell membranes. How rapidly growing tumor cells maintain membrane cholesterol homeostasis is poorly understood. Here, we found that glioblastoma (GBM), the most lethal brain tumor, maintains normal levels of membrane cholesterol but with an abundant presence of cholesteryl esters (CEs) in its lipid droplets (LDs). Mechanistically, SREBP-1 (sterol regulatory element-binding protein 1), a master transcription factor that is activated upon cholesterol depletion, upregulates critical autophagic genes, including ATG9B, ATG4A, and LC3B, as well as lysosome cholesterol transporter NPC2. This upregulation promotes LD lipophagy, resulting in the hydrolysis of CEs and the liberation of cholesterol from the lysosomes, thus maintaining plasma membrane cholesterol homeostasis. When this pathway is blocked, GBM cells become quite sensitive to cholesterol deficiency with poor growth in vitro. Our study unravels an SREBP-1-autophagy-LD-CE hydrolysis pathway that plays an important role in maintaining membrane cholesterol homeostasis while providing a potential therapeutic avenue for GBM.

Keywords:  ATG4A; ATG9B; CP: Cancer; CP: Metabolism; LC3B; NPC2; SREBP-1; autophagy; cholesterol homeostasis; cholesteryl esters; glioblastoma; lipid droplets

DOI:  https://doi.org/10.1016/j.celrep.2023.112790
Nat Commun. 2023 Jul 13. 14(1): 4166

Cytochrome c lysine acetylation regulates cellular respiration and cell death in ischemic skeletal muscle.

Paul T Morse, Gonzalo Pérez-Mejías, Junmei Wan, Alice A Turner, Inmaculada Márquez, Hasini A Kalpage, Asmita Vaishnav, Matthew P Zurek, Philipp P Huettemann, Katherine Kim, Tasnim Arroum, Miguel A De la Rosa, Dipanwita Dutta Chowdhury, Icksoo Lee, Joseph S Brunzelle, Thomas H Sanderson, Moh H Malek, David Meierhofer, Brian F P Edwards, Irene Díaz-Moreno, Maik Hüttemann.

Skeletal muscle is more resilient to ischemia-reperfusion injury than other organs. Tissue specific post-translational modifications of cytochrome c (Cytc) are involved in ischemia-reperfusion injury by regulating mitochondrial respiration and apoptosis. Here, we describe an acetylation site of Cytc, lysine 39 (K39), which was mapped in ischemic porcine skeletal muscle and removed by sirtuin5 in vitro. Using purified protein and cellular double knockout models, we show that K39 acetylation and acetylmimetic K39Q replacement increases cytochrome c oxidase (COX) activity and ROS scavenging while inhibiting apoptosis via decreased binding to Apaf-1, caspase cleavage and activity, and cardiolipin peroxidase activity. These results are discussed with X-ray crystallography structures of K39 acetylated (1.50 Å) and acetylmimetic K39Q Cytc (1.36 Å) and NMR dynamics. We propose that K39 acetylation is an adaptive response that controls electron transport chain flux, allowing skeletal muscle to meet heightened energy demand while simultaneously providing the tissue with robust resilience to ischemia-reperfusion injury.

DOI: https://doi.org/10.1038/s41467-023-39820-8
Oncogene. 2023 Jul 11.

Notch signaling regulates a metabolic switch through inhibiting PGC-1α and mitochondrial biogenesis in dedifferentiated liposarcoma.

Pei-Chieh Tien, Xiyue Chen, Bennett D Elzey, Raphael E Pollock, Shihuan Kuang.

Human dedifferentiated liposarcoma (DDLPS) is a rare but lethal cancer with no driver mutations being identified, hampering the development of targeted therapies. We and others recently reported that constitutive activation of Notch signaling through overexpression of the Notch1 intracellular domain (NICDOE) in murine adipocytes leads to tumors resembling human DDLPS. However, the mechanisms underlying the oncogenic functions of Notch activation in DDLPS remains unclear. Here, we show that Notch signaling is activated in a subset of human DDLPS and correlates with poor prognosis and expression of MDM2, a defining marker of DDLPS. Metabolic analyses reveal that murine NICDOE DDLPS cells exhibit markedly reduced mitochondrial respiration and increased glycolysis, mimicking the Warburg effect. This metabolic switch is associated with diminished expression of peroxisome proliferator-activated receptor gamma coactivator 1α (Ppargc1a, encoding PGC-1α protein), a master regulator of mitochondrial biogenesis. Genetic ablation of the NICDOE cassette rescues the expression of PGC-1α and mitochondrial respiration. Similarly, overexpression of PGC-1α is sufficient to rescue mitochondria biogenesis, inhibit the growth and promote adipogenic differentiation of DDLPS cells. Together, these data demonstrate that Notch activation inhibits PGC-1α to suppress mitochondrial biogenesis and drive a metabolic switch in DDLPS.

DOI: https://doi.org/10.1038/s41388-023-02768-6
Nat Cell Biol. 2023 Jul 13.

STING is a cell-intrinsic metabolic checkpoint restricting aerobic glycolysis by targeting HK2.

Liting Zhang, Congqing Jiang, Yunhong Zhong, Kongliang Sun, Huiru Jing, Jiayu Song, Jun Xie, Yaru Zhou, Mao Tian, Chuchu Zhang, Xiaona Sun, Shaowei Wang, Xi Cheng, Yuelan Zhang, Wei Wei, Xiang Li, Bishi Fu, Pinghui Feng, Bing Wu, Hong-Bing Shu, Junjie Zhang.

Evasion of antitumour immunity is a hallmark of cancer. STING, a putative innate immune signalling adaptor, has a pivotal role in mounting antitumour immunity by coordinating innate sensing and adaptive immune surveillance in myeloid cells. STING is markedly silenced in various human malignancies and acts as a cell-intrinsic tumour suppressor. How STING exerts intrinsic antitumour activity remains unclear. Here, we report that STING restricts aerobic glycolysis independent of its innate immune function. Mechanistically, STING targets hexokinase II (HK2) to block its hexokinase activity. As such, STING inhibits HK2 to restrict tumour aerobic glycolysis and promote antitumour immunity in vivo. In human colorectal carcinoma samples, lactate, which can be used as a surrogate for aerobic glycolysis, is negatively correlated with STING expression level and antitumour immunity. Taken together, this study reveals that STING functions as a cell-intrinsic metabolic checkpoint that restricts aerobic glycolysis to promote antitumour immunity. These findings have important implications for the development of STING-based therapeutic modalities to improve antitumour immunotherapy.

DOI: https://doi.org/10.1038/s41556-023-01185-x
J Biol Chem. 2023 Jul 10. pii: S0021-9258(23)02061-6. [Epub ahead of print] 105033

Kinetic and structural characterization of carboxyspermidine dehydrogenase of polyamine biosynthesis.

Danielle F Lee, Nicole Atencio, Shade Bouchey, Madeline R Shoemaker, Joshua S Dodd, Meredith Satre, Kenneth A Miller, Jeffrey S McFarlane.

  Polyamines are positively charged alkylamines ubiquitous among eukaryotes, prokaryotes and archaea. Humans obtain polyamines through dietary intake, metabolic production, or uptake of polyamines made by gut microbes. The polyamine biosynthetic pathway used by most gut microbes differs from that used by human cells. This alternative pathway employs carboxyspermidine dehydrogenase (CASDH), an enzyme with limited characterization. Here, we solved a 1.94 Å X-ray crystal structure of Bacteroides fragilis CASDH was solved by molecular replacement. BfCASDH is composed of three domains with a fold similar to saccharopine dehydrogenase, but with a distinct active site arrangement. Using steady-state methods, we determined kcat and kcat/Km for BfCASDH and Clostridium leptum CASDH using putrescine, diaminopropane, aspartate semi-aldehyde, NADH and NADPH as substrates. These data revealed evidence of cooperativity in BfCASDH. Putrescine is the likely polyamine substrate and NADPH is the coenzyme used to complete the reaction, forming carboxyspermidine as a product. These data provide the first kinetic characterization of CASDH - a key enzyme in the production of microbial polyamines.

Keywords:  aspartate semialdehyde; carboxyspermidine; dehydrogenase; gut microbiome; norspermidine; polyamine; putrescine; spermidine

DOI:  https://doi.org/10.1016/j.jbc.2023.105033
Mitochondrion. 2023 Jul 12. pii: S1567-7249(23)00067-3. [Epub ahead of print]

Mitochondrial respiratory complex I deficiency inhibits brown adipogenesis by limiting heme regulation of histone demethylation.

Jingjing Liu, Wen Lu, Dongyue Yan, Junyuan Guo, Li Zhou, Bimin Shi, Xiong Su.

  Mitochondrial functions play a crucial role in determining the metabolic and thermogenic status of brown adipocytes. Increasing evidence reveals that the mitochondrial oxidative phosphorylation (OXPHOS) system plays an important role in brown adipogenesis, but the mechanistic insights are limited. Herein, we explored the potential metabolic mechanisms leading to OXPHOS regulation of brown adipogenesis in pharmacological and genetic models of mitochondrial respiratory complex I deficiency. OXPHOS deficiency inhibits brown adipogenesis through disruption of the brown adipogenic transcription circuit without affecting ATP levels. Neither blockage of calcium signaling nor antioxidant treatment can rescue the suppressed brown adipogenesis. Metabolomics analysis revealed a decrease in levels of tricarboxylic acid cycle intermediates and heme. Heme supplementation specifically enhances respiratory complex I activity without affecting complex II and partially reverses the inhibited brown adipogenesis by OXPHOS deficiency. Moreover, the regulation of brown adipogenesis by the OXPHOS-heme axis may be due to the suppressed histone methylation status by increasing histone demethylation. In summary, our findings identified a heme-sensing retrograde signaling pathway that connects mitochondrial OXPHOS to the regulation of brown adipocyte differentiation and metabolic functions.

Keywords:  brown adipocytes; differentiation; heme; histone methylation; oxidative phosphorylation

DOI:  https://doi.org/10.1016/j.mito.2023.07.004
Elife. 2023 Jul 13. pii: RP86972. [Epub ahead of print]12

Importance of glutamine in synaptic vesicles revealed by functional studies of SLC6A17 and its mutations pathogenic for intellectual disability.

Xiaobo Jia, Jiemin Zhu, Xiling Bian, Sulin Liu, Sihan Yu, Wenjun Liang, Lifen Jiang, Renbo Mao, Wenxia Zhang, Yi Rao.

  Human mutations in the gene encoding the solute carrier (SLC) 6A17 caused intellectual disability (ID). The physiological role of SLC6A17 and pathogenesis of SLC6A17-based-ID were both unclear. Here, we report learning deficits in Slc6a17 knockout and point mutant mice. Biochemistry, proteomic, and electron microscopy (EM) support SLC6A17 protein localization in synaptic vesicles (SVs). Chemical analysis of SVs by liquid chromatography coupled to mass spectrometry (LC-MS) revealed glutamine (Gln) in SVs containing SLC6A17. Virally mediated overexpression of SLC6A17 increased Gln in SVs. Either genetic or virally mediated targeting of Slc6a17 reduced Gln in SVs. One ID mutation caused SLC6A17 mislocalization while the other caused defective Gln transport. Multidisciplinary approaches with seven types of genetically modified mice have shown Gln as an endogenous substrate of SLC6A17, uncovered Gln as a new molecule in SVs, established the necessary and sufficient roles of SLC6A17 in Gln transport into SVs, and suggested SV Gln decrease as the key pathogenetic mechanism in human ID.

Keywords:  SLC6A17; glutamine; learning; memory; mouse; neuroscience; synaptic vesicle

DOI:  https://doi.org/10.7554/eLife.86972
J Biol Chem. 2023 Jul 12. pii: S0021-9258(23)02075-6. [Epub ahead of print] 105047

Maintenance of cellular vitamin B6 levels and mitochondrial oxidative function depend on pyridoxal 5'-phosphate homeostasis protein (PLPHP).

Jolita Ciapaite, Carlo W T van Roermund, Marjolein Bosma, Johan Gerrits, Sander M Houten, Lodewijk IJlst, Hans R Waterham, Clara D M van Karnebeek, Ronald J A Wanders, Fried J T Zwartkruis, Judith J Jans, Nanda M Verhoeven-Duif.

  Recently, biallelic variants in PLPBP coding for pyridoxal 5'-phosphate homeostasis protein (PLPHP) were identified as a novel cause of early-onset vitamin B6-dependent epilepsy. The molecular function and precise role of PLPHP in vitamin B6 metabolism are not well understood. To address these questions we used PLPHP deficient patient skin fibroblasts and HEK293 cells, and YBL036C (PLPHP ortholog) deficient yeast. We showed that independent of extracellular B6 vitamer type (pyridoxine, pyridoxamine or pyridoxal), intracellular PLP was lower in PLPHP deficient fibroblasts and HEK293 cells compared to controls. Culturing cells with pyridoxine or pyridoxamine led to the concentration-dependent accumulation of pyridoxine 5'-phosphate and pyridoxamine 5'-phosphate (PMP), respectively, suggesting insufficient pyridox(am)ine 5'-phosphate oxidase (PNPO) activity. Experiments utilizing 13C4-pyridoxine confirmed lower PNPO activity and revealed increased fractional turnovers of PLP and pyridoxal, indicating increased PLP hydrolysis to pyridoxal in PLPHP deficient cells. This effect could be partly counteracted by inactivation of pyridoxal phosphatase. PLPHP deficiency had a distinct effect on mitochondrial PLP and PMP, suggesting impaired activity of mitochondrial transaminases. Moreover, in YBL036C deficient yeast PLP was depleted and PMP accumulated only with carbon sources requiring mitochondrial metabolism. Lactate and pyruvate accumulation along with the decrease of tricarboxylic acid cycle intermediates downstream of α-ketoglutarate suggested impaired mitochondrial oxidative metabolism in PLPHP deficient HEK293 cells. We hypothesize that impaired activity of mitochondrial transaminases may contribute to this depletion. Taken together, our study provides new insights into the pathomechanisms of PLPBP deficiency and reinforces the link between PLPHP function, vitamin B6 metabolism and mitochondrial oxidative metabolism.

Keywords:  Pyridoxal 5’-phosphate homeostasis protein PLPHP (PROSC); mitochondrial dysfunction; pyridox(am)ine 5′-phosphate oxidase (PNPO); transaminases; vitamin B(6) metabolism; α-ketoglutarate

DOI:  https://doi.org/10.1016/j.jbc.2023.105047
STAR Protoc. 2023 Jul 09. pii: S2666-1667(23)00386-6. [Epub ahead of print]4(3): 102419

Protocol to expand and CRISPR-Cas9 genomic edit murine MAIT cells for subsequent in vivo studies.

Anastasia du Halgouet, Aurélie Darbois, Aurélia Alphonse, Thomas Yvorra, Ludovic Colombeau, Raphaël Rodriguez, Olivier Lantz, Marion Salou.

  Generating knockout mice for target molecules in specific T cell populations, without subset-specific promoters, is time-consuming and costly. Here, we describe steps for enriching mucosal-associated invariant T cells from the thymus, expanding them in vitro and performing a CRISPR-Cas9 knockout. We then detail procedure for injecting the knockout cells into wounded Cd3ε-/- mice and characterizing them in the skin. For complete details on the use and execution of this protocol, please refer to du Halgouet et al. (2023).1.

Keywords:  CRISPR; Cell culture; Immunology

DOI:  https://doi.org/10.1016/j.xpro.2023.102419