bims-mepmim Biomed News
on Metabolites in pathological microenvironments and immunometabolism
Issue of 2023–01–08
twenty-two papers selected by
Erika Mariana Palmieri, NIH/NCI Laboratory of Cancer ImmunoMetabolism
  1. Lactate metabolism is essential in early-onset mitochondrial myopathy.
  2. AMPK integrates metabolite and kinase-based immunometabolic control in macrophages.
  3. Early immune pressure makes tumors metabolically stronger.
  4. Carnitine octanoyltransferase is important for the assimilation of exogenous acetyl-L-carnitine into acetyl-CoA in mammalian cells.
  5. Lactylome analysis suggests lactylation-dependent mechanisms of metabolic adaptation in hepatocellular carcinoma.
  6. Aberrant NAD synthetic flux in podocytes under diabetic conditions and effects of indoleamine 2,3-dioxygenase on promoting de novo NAD synthesis.
  7. Metabolomic analysis shows dysregulation in amino acid and NAD+ metabolism in palmitate treated hepatocytes and plasma of non-alcoholic fatty liver disease spectrum.
  8. Adipose tissue macrophages: implications for obesity-associated cancer.
  9. Thioredoxin-interacting protein is essential for memory T cell formation via the regulation of the redox metabolism.
  10. Sterol-regulated transmembrane protein TMEM86a couples LXR signaling to regulation of lysoplasmalogens in macrophages.
  11. Labeling and analyzing lipid droplets in mouse muscle stem cells.
  12. Monitoring mitochondrial translation by pulse SILAC.
  13. An assay to evaluate the capacity of cholesterol acceptors using BODIPY-cholesterol in cells.
  14. The endoplasmic reticulum kinase PERK interacts with the oxidoreductase ERO1 to metabolically adapt mitochondria.
  15. HLA-I immunopeptidome profiling of human cells infected with high-containment enveloped viruses.
  16. Expression of TRX1 optimizes the antitumor functions of human CAR T cells and confers resistance to a pro-oxidative tumor microenvironment.
  17. Metabolic regulation of species-specific developmental rates.
  18. Sodium butyrate activates HMGCS2 to promote ketone body production through SIRT5-mediated desuccinylation.
  19. An optimized protocol for the isolation of rare stromal cell populations from the mouse spleen.
  20. Adipose tissue is a source of regenerative cells that augment the repair of skeletal muscle after injury.
  21. Targeting peroxisomal fatty acid oxidation improves hepatic steatosis and insulin resistance in obese mice.
  22. Inducing trained immunity in pro-metastatic macrophages to control tumor metastasis.
  1. Sci Adv. 2023 Jan 04. 9(1): eadd3216
    Lactate metabolism is essential in early-onset mitochondrial myopathy.
    Zhenkang Chen, Bogdan Bordieanu, Rushendhiran Kesavan, Nicholas P Lesner, Siva Sai Krishna Venigalla, Spencer D Shelton, Ralph J DeBerardinis, Prashant Mishra.
      Myopathies secondary to mitochondrial electron transport chain (ETC) dysfunction can result in devastating disease. While the consequences of ETC defects have been extensively studied in culture, little in vivo data are available. Using a mouse model of severe, early-onset mitochondrial myopathy, we characterized the proteomic, transcriptomic, and metabolic characteristics of disease progression. Unexpectedly, ETC dysfunction in muscle results in reduced expression of glycolytic enzymes in our animal model and patient muscle biopsies. The decrease in glycolysis was mediated by loss of constitutive Hif1α signaling, down-regulation of the purine nucleotide cycle enzyme AMPD1, and activation of AMPK. In vivo isotope tracing experiments indicated that myopathic muscle relies on lactate import to supply central carbon metabolites. Inhibition of lactate import reduced steady-state levels of tricarboxylic acid cycle intermediates and compromised the life span of myopathic mice. These data indicate an unexpected mode of metabolic reprogramming in severe mitochondrial myopathy that regulates disease progression.
    DOI:  https://doi.org/10.1126/sciadv.add3216
  2. Mol Metab. 2022 Dec 28. pii: S2212-8778(22)00230-7. [Epub ahead of print] 101661
    AMPK integrates metabolite and kinase-based immunometabolic control in macrophages.
    Iain R Phair, Raid B Nisr, Andrew J M Howden, Marc Foretz, Douglas Lamont, Benoit Viollet, Graham Rena.
      Previous mechanistic studies on immunometabolism have focused on metabolite-based paradigms of regulation, such as itaconate. Here, we combine whole cell quantitative proteomics with gene knockout of AMPKα1, to demonstrate integration of metabolite and kinase-based immunometabolic control. Comparing macrophages with AMPKα1 catalytic subunit deletion with wild-type, inflammatory markers are largely unchanged in unstimulated cells, but with an LPS stimulus, AMPKα1 knockout leads to a striking M1 hyperpolarisation. Deletion of AMPKα1 also resulted in increased expression of rate-limiting enzymes involved in itaconate synthesis, metabolism of glucose, arginine, prostaglandins and cholesterol. Consistent with this, we observed functional changes in prostaglandin synthesis and arginine metabolism. Selective AMPKα1 activation also unlocks additional regulation of IL-6 and IL-12 in M1 macrophages. Together, our results validate AMPK as a pivotal immunometabolic regulator in macrophages.
    DOI:  https://doi.org/10.1016/j.molmet.2022.101661
  3. Cell Metab. 2023 Jan 03. pii: S1550-4131(22)00546-0. [Epub ahead of print]35(1): 3-5
    Early immune pressure makes tumors metabolically stronger.
    Sujing Yuan, Hongbo Chi.
      Metabolic communication in the tumor microenvironment underscores tumor-immune interactions and affects anti-tumor immunity, yet cell-extrinsic signals driving tumor metabolic remodeling are incompletely understood. In this issue, Tsai et al. show that during initial tumorigenesis, T cell-derived IFNγ triggers STAT3 activation and c-Myc-dependent alterations of tumor cell metabolism, which potentiates immune evasion.
    DOI:  https://doi.org/10.1016/j.cmet.2022.12.009
  4. J Biol Chem. 2022 Dec 29. pii: S0021-9258(22)01291-1. [Epub ahead of print] 102848
    Carnitine octanoyltransferase is important for the assimilation of exogenous acetyl-L-carnitine into acetyl-CoA in mammalian cells.
    Jake Hsu, Nina Fatuzzo, Nielson Weng, Wojciech Michno, Wentao Dong, Maryline Kienle, Yuqin Dai, Anca Pasca, Monther Abu-Remaileh, Natalie Rasgon, Benedetta Bigio, Carla Nasca, Chaitan Khosla.
      In eukaryotes carnitine is best known for its ability to shuttle esterified fatty acids across mitochondrial membranes for β-oxidation. It also returns to the cytoplasm, in the form of acetyl-L-carnitine (LAC), some of the resulting acetyl groups for post-translational protein modification and lipid biosynthesis. While dietary LAC supplementation has been clinically investigated, its effects on cellular metabolism are not well understood. To explain how exogenous LAC influences mammalian cell metabolism, we synthesized isotope-labeled forms of LAC and its analogs. In cultures of glucose-limited U87MG glioma cells, exogenous LAC contributed more robustly to intracellular acetyl-CoA pools than did β-hydroxybutyrate, the predominant circulating ketone body in mammals. The fact that most LAC-derived acetyl-CoA is cytosolic is evident from strong labeling of fatty acids in U87MG cells by exogenous 13C2-acetyl-L-carnitine. We found that the addition of d3-acetyl-L-carnitine increases the supply of acetyl-CoA for cytosolic post-translational modifications due to its strong kinetic isotope effect on acetyl-CoA carboxylase, the first committed step in fatty acid biosynthesis. Surprisingly, whereas cytosolic carnitine acetyltransferase (CRAT) is believed to catalyze acetyl group transfer from LAC to Coenzyme A, CRAT-/- U87MG cells were unimpaired in their ability to assimilate exogenous LAC into acetyl-CoA. We identified carnitine octanoyltransferase (CROT) as the key enzyme in this process, implicating a role for peroxisomes in efficient LAC utilization. Our work has opened the door to further biochemical investigations of a new pathway for supplying acetyl-CoA to certain glucose-starved cells.
    Keywords:  acetyl coenzyme A (acetyl‐CoA); acetylcarnitine; cell metabolism; energy metabolism; enzyme turnover; metabolic regulation; mitochondrial metabolism; peroxisome
    DOI:  https://doi.org/10.1016/j.jbc.2022.102848
  5. Nat Metab. 2023 Jan 02.
    Lactylome analysis suggests lactylation-dependent mechanisms of metabolic adaptation in hepatocellular carcinoma.
    Zijian Yang, Cong Yan, Jiaqiang Ma, Panpan Peng, Xuelian Ren, Shangli Cai, Xia Shen, Yingcheng Wu, Shu Zhang, Xiaoying Wang, Shuangjian Qiu, Jian Zhou, Jia Fan, He Huang, Qiang Gao.
      Enhanced glycolysis and accumulation of lactate is a common feature in various types of cancer. Intracellular lactate drives a recently described type of posttranslational modification, lysine lactylation (Kla), on core histones. However, the impact of lactylation on biological processes of tumour cells remains largely unknown. Here we show a global lactylome profiling on a prospectively collected hepatitis B virus-related hepatocellular carcinoma (HCC) cohort. Integrative lactylome and proteome analysis of the tumours and adjacent livers identifies 9,275 Kla sites, with 9,256 sites on non-histone proteins, indicating that Kla is a prevalent modification beyond histone proteins and transcriptional regulation. Notably, Kla preferentially affects enzymes involved in metabolic pathways, including the tricarboxylic acid cycle, and carbohydrate, amino acid, fatty acid and nucleotide metabolism. We further verify that lactylation at K28 inhibits the function of adenylate kinase 2, facilitating the proliferation and metastasis of HCC cells. Our study therefore reveals that Kla plays an important role in regulating cellular metabolism and may contribute to HCC progression.
    DOI:  https://doi.org/10.1038/s42255-022-00710-w
  6. Biochem Biophys Res Commun. 2022 Dec 21. pii: S0006-291X(22)01724-7. [Epub ahead of print]643 61-68
    Aberrant NAD synthetic flux in podocytes under diabetic conditions and effects of indoleamine 2,3-dioxygenase on promoting de novo NAD synthesis.
    Yuhua Zhang, Xingchen Zhao, Cuili Li, Yan Yang, Luan Li, Yingwen Chen, Qingying Shi, Zhilian Li, Yanhua Wu, Li Zhang, Ruizhao Li, Meijun Si, Xinling Liang, Yuanhan Chen.
      Nicotinamide adenine dinucleotide (NAD) is an essential coenzyme in the kidney. The first step in de novo NAD synthesis is regulated by indoleamine 2,3-dioxygenase (IDO), a tryptophan-catabolizing enzyme. Here, we investigated NAD synthetic flux and NAD levels in podocytes under diabetic conditions. We also studied the effects of IDO overexpression on NAD synthetic flux and high glucose (HG)-induced podocyte injury. NAD synthetases in the de novo, Preiss-Handler and salvage pathways were analyzed using in vivo single-nucleus RNA sequencing datasets (GSE131882) of control and diabetic kidney disease (DKD). The mRNA levels of these NAD synthetases were measured in vitro in HG-treated podocytes. The effects of IDO on NAD synthesis were examined by transducing cultured podocytes with an adenovirus encoding IDO, and apoptosis, podocyte markers and mobility were investigated. Cellular transcriptome analysis revealed that control podocytes had relatively low levels of NAD synthetases. In DKD podocytes, de novo NAD synthetase levels were further downregulated. IDO levels were virtually undetectable and did not increase in DKD. In vitro experiments confirmed aberrant de novo NAD synthetic flux and decreased IDO levels in HG-treated podocytes. Overexpression of IDO promoted NAD de novo synthesis, reduced NAD-bypass metabolic enzyme, increased NAD content and recovered podocyte injury markers under diabetic conditions. Taken together, our findings suggest that the de novo NAD synthetic flux is aberrant in DKD, and IDO promotes de novo NAD synthesis and NAD levels, as well as alleviates injury in HG-treated podocytes.
    Keywords:  Diabetic kidney disease; Energy metabolism; Indoleamine 2,3-dioxygenase; Nicotinamide adenine dinucleotide; Podocytes
    DOI:  https://doi.org/10.1016/j.bbrc.2022.12.059
  7. Biochem Biophys Res Commun. 2022 Dec 28. pii: S0006-291X(22)01757-0. [Epub ahead of print]643 129-138
    Metabolomic analysis shows dysregulation in amino acid and NAD+ metabolism in palmitate treated hepatocytes and plasma of non-alcoholic fatty liver disease spectrum.
    Savera Aggarwal, Vipin Yadav, Rakhi Maiwall, Archana Rastogi, Viniyendra Pamecha, Onkar Bedi, Jaswinder Singh Maras, Nirupma Trehanpati, Gayatri Ramakrishna.
      There is an alarming increase in incidence of fatty liver disease worldwide. The fatty liver disease spectrum disease ranges from simple steatosis (NAFL) to steatohepatitis (NASH) which culminates in cirrhosis and cancer. Altered metabolism is a hallmark feature associated with fatty liver disease and palmitic acid is the most abundant saturated fatty acid, therefore, the aim of this study was to compare metabolic profiles altered in hepatocytes treated with palmitic acid and also the differentially expressed plasma metabolites in spectrum of nonalcoholic fatty liver. The metabolites were analyzed by liquid chromatography-mass spectrometry (LC-MS) platform. Hepatocyte cell lines PH5CH8 and HepG2 cells when treated with 400 μM dose of palmitic acid showed typical features of steatosis. Metabolomic analysis of lipid treated hepatocyte cell lines showed differential changes in phenylalanine and tyrosine pathways, fatty acid metabolism and bile acids. The key metabolites tryptophan, kynurenine and carnitine differed significantly between subjects with NAFL, NASH and those with cirrhosis. As the tryptophan-kynurenine axis is also involved in denovo synthesis of NAD+, we found significant alterations in the NAD+ related metabolites in both palmitic acid treated and also fatty liver disease with cirrhosis. The study underscores the importance of amino acid and NAD+supplementation as promising strategies in fatty liver disorder.
    Keywords:  Amino acid; Cirrhosis; Hepatocytes; Metabolites; NAD+; NAFLD; NASH; Palmitic acid; Plasma
    DOI:  https://doi.org/10.1016/j.bbrc.2022.12.078
  8. Mil Med Res. 2023 Jan 03. 10(1): 1
    Adipose tissue macrophages: implications for obesity-associated cancer.
    Bei Li, Si Sun, Juan-Juan Li, Jing-Ping Yuan, Sheng-Rong Sun, Qi Wu.
      Obesity is one of the most serious global health problems, with an incidence that increases yearly and coincides with the development of cancer. Adipose tissue macrophages (ATMs) are particularly important in this context and contribute to linking obesity-related inflammation and tumor progression. However, the functions of ATMs on the progression of obesity-associated cancer remain unclear. In this review, we describe the origins, phenotypes, and functions of ATMs. Subsequently, we summarize the potential mechanisms on the reprogramming of ATMs in the obesity-associated microenvironment, including the direct exchange of dysfunctional metabolites, inordinate cytokines and other signaling mediators, transfer of extracellular vesicle cargo, and variations in the gut microbiota and its metabolites. A better understanding of the properties and functions of ATMs under conditions of obesity will lead to the development of new therapeutic interventions for obesity-related cancer.
    Keywords:  Adipose; Adipose tissue macrophages; Cancer; Macrophage; Obesity; Therapy
    DOI:  https://doi.org/10.1186/s40779-022-00437-5
  9. Proc Natl Acad Sci U S A. 2023 Jan 10. 120(2): e2218345120
    Thioredoxin-interacting protein is essential for memory T cell formation via the regulation of the redox metabolism.
    Kota Kokubo, Kiyoshi Hirahara, Masahiro Kiuchi, Kaori Tsuji, Yuki Shimada, Yuri Sonobe, Rie Shinmi, Takahisa Hishiya, Chiaki Iwamura, Atsushi Onodera, Toshinori Nakayama.
      CD4+ memory T cells are central to long-lasting protective immunity and are involved in shaping the pathophysiology of chronic inflammation. While metabolic reprogramming is critical for the generation of memory T cells, the mechanisms controlling the redox metabolism in memory T cell formation remain unclear. We found that reactive oxygen species (ROS) metabolism changed dramatically in T helper-2 (Th2) cells during the contraction phase in the process of memory T cell formation. Thioredoxin-interacting protein (Txnip), a regulator of oxidoreductase, regulated apoptosis by scavenging ROS via the nuclear factor erythroid 2-related factor 2 (Nrf2)-biliverdin reductase B (Blvrb) pathway. Txnip regulated the pathology of chronic airway inflammation in the lung by controlling the generation of allergen-specific pathogenic memory Th2 cells in vivo. Thus, the Txnip-Nrf2-Blvrb axis directs ROS metabolic reprogramming in Th2 cells and is a potential therapeutic target for intractable chronic inflammatory diseases.
    Keywords:  biliverdin reductase B (Blvrb); memory Th2 cells; nuclear factor-erythroid factor 2-related factor 2 (Nrf2); reactive oxygen species (ROS); thioredoxin-interacting protein (Txnip)
    DOI:  https://doi.org/10.1073/pnas.2218345120
  10. J Lipid Res. 2022 Dec 30. pii: S0022-2275(22)00158-4. [Epub ahead of print] 100325
    Sterol-regulated transmembrane protein TMEM86a couples LXR signaling to regulation of lysoplasmalogens in macrophages.
    Suzanne A E van Wouw, Marlene van den Berg, Maroua El Ouraoui, Amber Meurs, Jenina Kingma, Roelof Ottenhoff, Melanie Loix, Marten A Hoeksema, Koen Prange, Gerard Pasterkamp, Jerome J A Hendriks, Jeroen F J Bogie, Jan B van Klinken, Frederic M Vaz, Aldo Jongejan, Menno P J de Winther, Noam Zelcer.
      Lysoplasmalogens are a class of vinyl ether bioactive lipids that have a central role in plasmalogen metabolism and membrane fluidity. The liver X receptor transcription factors (LXR) are important determinants of cellular lipid homeostasis owing to their ability to regulate cholesterol and fatty acid metabolism. However, their role in governing the composition of lipid species such as lysoplasmalogens in cellular membranes is less well studied. Here, we mapped the lipidome of bone marrow-derived macrophages (BMDM) following LXR activation. We found a marked reduction in the levels of lysoplasmalogen species in the absence of changes in the levels of plasmalogens themselves. Transcriptional profiling of LXR-activated macrophages identified the gene encoding transmembrane protein TMEM86a, an integral endoplasmic reticulum protein, as a previously uncharacterized sterol-regulated gene. We demonstrate that TMEM86a is a direct transcriptional target of LXR in macrophages and microglia and that it is highly expressed in TREM2+/lipid-associated macrophages in human atherosclerotic plaques, where its expression positively correlates with other LXR-regulated genes. We further show that both murine and human TMEM86a display active lysoplasmalogenase activity that can be abrogated by inactivating mutations in the predicted catalytic site. Consequently, we demonstrate that overexpression of Tmem86a in BMDM markedly reduces lysoplasmalogen abundance and membrane fluidity, while reciprocally, silencing of Tmem86a increases basal lysoplasmalogen levels and abrogates the LXR-dependent reduction of this lipid species. Collectively, our findings implicate TMEM86a as a sterol-regulated lysoplasmalogenase in macrophages that contributes to sterol-dependent membrane remodeling.
    Keywords:  LXR; atherosclerotic plaques; bone marrow-derived macrophages; lipid metabolism; lipidomics; lysoplasmalogens; membrane fluidity; plasmalogens; sterol; transcriptional regulation
    DOI:  https://doi.org/10.1016/j.jlr.2022.100325
  11. STAR Protoc. 2022 Dec 16. pii: S2666-1667(22)00729-8. [Epub ahead of print]3(4): 101849
    Labeling and analyzing lipid droplets in mouse muscle stem cells.
    Jingjuan Chen, Feng Yue, Shihuan Kuang.
      Lipid droplets are emerging as an important and dynamic organelle whose metabolism controls stem cell behavior. Here we present a comprehensive protocol to visualize and quantify these organelles in mouse muscle satellite cells (MuSCs). This protocol includes steps for BODIPY/LipidSpot610 staining of freshly isolated MuSCs, in vitro cultured myoblasts, and single myofibers to label lipid droplets and subsequent analysis and quantification of fluorescence signals. This protocol can be modified to stain lipid droplets in other cell types of interest. For complete details on the use and execution of this protocol, please refer to Yue et al. (2022).1.
    Keywords:  Cell Biology; Cell culture; Flow Cytometry/Mass Cytometry; Microscopy; Molecular/Chemical Probes; Stem Cells
    DOI:  https://doi.org/10.1016/j.xpro.2022.101849
  12. J Biol Chem. 2023 Jan 02. pii: S0021-9258(22)01308-4. [Epub ahead of print] 102865
    Monitoring mitochondrial translation by pulse SILAC.
    Koshi Imami, Matthias Selbach, Yasushi Ishihama.
      Mitochondrial ribosomes are specialized to translate the 13 membrane proteins encoded in the mitochondrial genome, which shapes the oxidative phosphorylation (OXPHOS) complexes essential for cellular energy metabolism. Despite the importance of mitochondrial translation control, it is challenging to identify and quantify the mitochondrial-encoded proteins due to their hydrophobic nature and low abundance. Here, we introduce a mass spectrometry-based proteomic method that combines biochemical isolation of mitochondria with pulse stable isotope labeling by amino acids in cell culture (pSILAC). Our method provides the highest protein identification rate with the shortest measurement time among currently available methods, enabling us to quantify 12 out of the 13 mitochondrial-encoded proteins. We applied this method to uncover the global picture of (post-)translational regulation of both mitochondrial- and nuclear-encoded subunits of OXPHOS complexes. We found that inhibition of mitochondrial translation led to degradation of orphan nuclear-encoded subunits that are considered to form subcomplexes with the mitochondrial-encoded subunits. This method should be readily applicable to study mitochondrial translation programs in many contexts, including oxidative stress and mitochondrial disease.
    Keywords:  Mitochondria; OXPHOS; Protein complex; Proteomics; Translation; pulse SILAC
    DOI:  https://doi.org/10.1016/j.jbc.2022.102865
  13. STAR Protoc. 2023 Jan 03. pii: S2666-1667(22)00856-5. [Epub ahead of print]4(1): 101976
    An assay to evaluate the capacity of cholesterol acceptors using BODIPY-cholesterol in cells.
    Ana-Caroline Raulin, Chia-Chen Liu, Guojun Bu.
      Cholesterol is a structural component of cell membranes. Most cells are incapable of its catabolism, and intracellular cholesterol accumulation is linked to several disorders including cardiovascular and neurodegenerative diseases. Cholesterol efflux, essential to its metabolism, is dependent on acceptors such as apolipoproteins. Here, we describe an assay to evaluate the capacity of cholesterol acceptors. Cells are treated with an analog of cholesterol tagged with fluorescent BODIPY. Addition of an acceptor leads to BODIPY-cholesterol efflux, measured using a plate reader. For complete details on the use and execution of this protocol, please refer to Liu et al. (2021).1.
    Keywords:  Cell-based Assays; Molecular/Chemical Probes
    DOI:  https://doi.org/10.1016/j.xpro.2022.101976
  14. Cell Rep. 2022 Dec 23. pii: S2211-1247(22)01798-3. [Epub ahead of print] 111899
    The endoplasmic reticulum kinase PERK interacts with the oxidoreductase ERO1 to metabolically adapt mitochondria.
    Arthur Bassot, Junsheng Chen, Kei Takahashi-Yamashiro, Megan C Yap, Christine Silvia Gibhardt, Giang N T Le, Saaya Hario, Yusuke Nasu, Jack Moore, Tomas Gutiérrez, Lucas Mina, Heather Mast, Audric Moses, Rakesh Bhat, Klaus Ballanyi, Hélène Lemieux, Roberto Sitia, Ester Zito, Ivan Bogeski, Robert E Campbell, Thomas Simmen.
      Endoplasmic reticulum (ER) homeostasis requires molecular regulators that tailor mitochondrial bioenergetics to the needs of protein folding. For instance, calnexin maintains mitochondria metabolism and mitochondria-ER contacts (MERCs) through reactive oxygen species (ROS) from NADPH oxidase 4 (NOX4). However, induction of ER stress requires a quick molecular rewiring of mitochondria to adapt to new energy needs. This machinery is not characterized. We now show that the oxidoreductase ERO1⍺ covalently interacts with protein kinase RNA-like ER kinase (PERK) upon treatment with tunicamycin. The PERK-ERO1⍺ interaction requires the C-terminal active site of ERO1⍺ and cysteine 216 of PERK. Moreover, we show that the PERK-ERO1⍺ complex promotes oxidization of MERC proteins and controls mitochondrial dynamics. Using proteinaceous probes, we determined that these functions improve ER-mitochondria Ca2+ flux to maintain bioenergetics in both organelles, while limiting oxidative stress. Therefore, the PERK-ERO1⍺ complex is a key molecular machinery that allows quick metabolic adaptation to ER stress.
    Keywords:  CP: Metabolism; CP: Molecular biology; ER; ER stress; ERO1; MAMs; MERCs; PERK; bioenergetics; endoplasmic reticulum; mitochondria; mitochondria-associated membranes; mitochondria-endoplasmic reticulum contacts; oxidoreductase
    DOI:  https://doi.org/10.1016/j.celrep.2022.111899
  15. STAR Protoc. 2022 Dec 16. pii: S2666-1667(22)00790-0. [Epub ahead of print]3(4): 101910
    HLA-I immunopeptidome profiling of human cells infected with high-containment enveloped viruses.
    Shira Weingarten-Gabbay, Leah R Pearlman, Da-Yuan Chen, Susan Klaeger, Hannah B Taylor, Nicole L Welch, Derin B Keskin, Steven A Carr, Jennifer G Abelin, Mohsan Saeed, Pardis C Sabeti.
      Immunopeptidome profiling of infected cells is a powerful technique for detecting viral peptides that are naturally processed and loaded onto class I human leukocyte antigens (HLAs-I). Here, we provide a protocol for preparing samples for immunopeptidome profiling that can inactivate enveloped viruses while still preserving the integrity of the HLA-I complex. We detail steps for lysate preparation of infected cells followed by HLA-I immunoprecipitation and virus inactivation. We further describe peptide purification for mass spectrometry outside a high-containment facility. For complete details on the use and execution of this protocol, please refer to Weingarten-Gabbay et al. (2021).1.
    Keywords:  Immunology; Mass Spectrometry; Microbiology
    DOI:  https://doi.org/10.1016/j.xpro.2022.101910
  16. Front Immunol. 2022 ;13 1063313
    Expression of TRX1 optimizes the antitumor functions of human CAR T cells and confers resistance to a pro-oxidative tumor microenvironment.
    Emre Balta, Nina Janzen, Henning Kirchgessner, Vasiliki Toufaki, Christian Orlik, Jie Liang, Divya Lairikyengbam, Hinrich Abken, Beate Niesler, Karin Müller-Decker, Thomas Ruppert, Yvonne Samstag.
      Use of chimeric antigen receptor (CAR) T cells to treat B cell lymphoma and leukemia has been remarkably successful. Unfortunately, the therapeutic efficacy of CAR T cells against solid tumors is very limited, with immunosuppression by the pro-oxidative tumor microenvironment (TME) a major contributing factor. High levels of reactive oxygen species are well-tolerated by tumor cells due to their elevated expression of antioxidant proteins; however, this is not the case for T cells, which consequently become hypo-responsive. The aim of this study was to improve CAR T cell efficacy in solid tumors by empowering the antioxidant capacity of CAR T cells against the pro-oxidative TME. To this end, HER2-specific human CAR T cells stably expressing two antioxidant systems: thioredoxin-1 (TRX1), and glutaredoxin-1 (GRX1) were generated and characterized. Thereafter, antitumor functions of CAR T cells were evaluated under control or pro-oxidative conditions. To provide insights into the role of antioxidant systems, gene expression profiles as well as global protein oxidation were analyzed. Our results highlight that TRX1 is pivotal for T cell redox homeostasis. TRX1 expression allows CAR T cells to retain their cytolytic immune synapse formation, cytokine release, proliferation, and tumor cell-killing properties under pro-oxidative conditions. Evaluation of differentially expressed genes and the first comprehensive redoxosome analysis of T cells by mass spectrometry further clarified the underlying mechanisms. Taken together, enhancement of the key antioxidant TRX1 in human T cells opens possibilities to increase the efficacy of CAR T cell treatment against solid tumors.
    Keywords:  CAR T cells; ROS; cancer immunotherapy; redox regulation; thioredoxin-1; tumor microenvironment
    DOI:  https://doi.org/10.3389/fimmu.2022.1063313
  17. Nature. 2023 Jan 04.
    Metabolic regulation of species-specific developmental rates.
    Margarete Diaz-Cuadros, Teemu P Miettinen, Owen S Skinner, Dylan Sheedy, Carlos Manlio Díaz-García, Svetlana Gapon, Alexis Hubaud, Gary Yellen, Scott R Manalis, William M Oldham, Olivier Pourquié.
      Animals display substantial inter-species variation in the rate of embryonic development despite a broad conservation of the overall sequence of developmental events. Differences in biochemical reaction rates, including the rates of protein production and degradation, are thought to be responsible for species-specific rates of development1-3. However, the cause of differential biochemical reaction rates between species remains unknown. Here, using pluripotent stem cells, we have established an in vitro system that recapitulates the twofold difference in developmental rate between mouse and human embryos. This system provides a quantitative measure of developmental speed as revealed by the period of the segmentation clock, a molecular oscillator associated with the rhythmic production of vertebral precursors. Using this system, we show that mass-specific metabolic rates scale with the developmental rate and are therefore higher in mouse cells than in human cells. Reducing these metabolic rates by inhibiting the electron transport chain slowed down the segmentation clock by impairing the cellular NAD+/NADH redox balance and, further downstream, lowering the global rate of protein synthesis. Conversely, increasing the NAD+/NADH ratio in human cells by overexpression of the Lactobacillus brevis NADH oxidase LbNOX increased the translation rate and accelerated the segmentation clock. These findings represent a starting point for the manipulation of developmental rate, with multiple translational applications including accelerating the differentiation of human pluripotent stem cells for disease modelling and cell-based therapies.
    DOI:  https://doi.org/10.1038/s41586-022-05574-4
  18. Front Med. 2023 Jan 05.
    Sodium butyrate activates HMGCS2 to promote ketone body production through SIRT5-mediated desuccinylation.
    Yanhong Xu, Xiaotong Ye, Yang Zhou, Xinyu Cao, Shiqiao Peng, Yue Peng, Xiaoying Zhang, Yili Sun, Haowen Jiang, Wenying Huang, Hongkai Lian, Jiajun Yang, Jia Li, Jianping Ye.
      Ketone bodies have beneficial metabolic activities, and the induction of plasma ketone bodies is a health promotion strategy. Dietary supplementation of sodium butyrate (SB) is an effective approach in the induction of plasma ketone bodies. However, the cellular and molecular mechanisms are unknown. In this study, SB was found to enhance the catalytic activity of 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), a rate-limiting enzyme in ketogenesis, to promote ketone body production in hepatocytes. SB administrated by gavage or intraperitoneal injection significantly induced blood ß-hydroxybutyrate (BHB) in mice. BHB production was induced in the primary hepatocytes by SB. Protein succinylation was altered by SB in the liver tissues with down-regulation in 58 proteins and up-regulation in 26 proteins in the proteomics analysis. However, the alteration was mostly observed in mitochondrial proteins with 41% down- and 65% up-regulation, respectively. Succinylation status of HMGCS2 protein was altered by a reduction at two sites (K221 and K358) without a change in the protein level. The SB effect was significantly reduced by a SIRT5 inhibitor and in Sirt5-KO mice. The data suggests that SB activated HMGCS2 through SIRT5-mediated desuccinylation for ketone body production by the liver. The effect was not associated with an elevation in NAD+/NADH ratio according to our metabolomics analysis. The data provide a novel molecular mechanism for SB activity in the induction of ketone body production.
    Keywords:  HMGCS2; SIRT5; ketogenesis; sodium butyrate; succinylation
    DOI:  https://doi.org/10.1007/s11684-022-0943-0
  19. STAR Protoc. 2022 Dec 16. pii: S2666-1667(22)00803-6. [Epub ahead of print]3(4): 101923
    An optimized protocol for the isolation of rare stromal cell populations from the mouse spleen.
    Yannick O Alexandre, Scott N Mueller.
      Lymphoid tissue stromal cells are important regulators of spleen homeostasis and immune responses. Here, we present an optimized protocol that describes the digestion and enrichment steps for the isolation and analysis of rare populations of stromal cells, including fibroblastic reticular cells, perivascular cells, and glial cells found in the spleen. This protocol is suitable for subsequent analysis of spleen stromal cells by flow cytometry or single-cell RNA sequencing and to analyze different disease models. For complete details on the use and execution of this protocol, please refer to Alexandre et al. (2022).1.
    Keywords:  Cell Biology; Cell isolation; Flow Cytometry/Mass Cytometry; Immunology
    DOI:  https://doi.org/10.1016/j.xpro.2022.101923
  20. Nat Commun. 2023 Jan 05. 14(1): 80
    Adipose tissue is a source of regenerative cells that augment the repair of skeletal muscle after injury.
    Quentin Sastourné-Arrey, Maxime Mathieu, Xavier Contreras, Sylvie Monferran, Virginie Bourlier, Marta Gil-Ortega, Enda Murphy, Claire Laurens, Audrey Varin, Christophe Guissard, Corinne Barreau, Mireille André, Noémie Juin, Marie Marquès, Benoit Chaput, Cédric Moro, Donal O'Gorman, Louis Casteilla, Amandine Girousse, Coralie Sengenès.
      Fibro-adipogenic progenitors (FAPs) play a crucial role in skeletal muscle regeneration, as they generate a favorable niche that allows satellite cells to perform efficient muscle regeneration. After muscle injury, FAP content increases rapidly within the injured muscle, the origin of which has been attributed to their proliferation within the muscle itself. However, recent single-cell RNAseq approaches have revealed phenotype and functional heterogeneity in FAPs, raising the question of how this differentiation of regenerative subtypes occurs. Here we report that FAP-like cells residing in subcutaneous adipose tissue (ScAT), the adipose stromal cells (ASCs), are rapidly released from ScAT in response to muscle injury. Additionally, we find that released ASCs infiltrate the damaged muscle, via a platelet-dependent mechanism and thus contribute to the FAP heterogeneity. Moreover, we show that either blocking ASCs infiltration or removing ASCs tissue source impair muscle regeneration. Collectively, our data reveal that ScAT is an unsuspected physiological reservoir of regenerative cells that support skeletal muscle regeneration, underlining a beneficial relationship between muscle and fat.
    DOI:  https://doi.org/10.1038/s41467-022-35524-7
  21. J Biol Chem. 2022 Dec 28. pii: S0021-9258(22)01288-1. [Epub ahead of print] 102845
    Targeting peroxisomal fatty acid oxidation improves hepatic steatosis and insulin resistance in obese mice.
    Haoya Yao, Yaoqing Wang, Xiao Zhang, Ping Li, Lin Shang, Xiaocui Chen, Jia Zeng.
      Obesity and diabetes normally cause mitochondrial dysfunction and hepatic lipid accumulation, while fatty acid synthesis is suppressed and malonyl-CoA is depleted in the liver of severe obese or diabetic animals. Therefore, a negative regulatory mechanism might work for the control of mitochondrial fatty acid metabolism that is independent of malonyl-CoA in the diabetic animals. As mitochondrial β-oxidation is controlled by the acetyl-CoA/CoA ratio, and the acetyl-CoA generated in peroxisomal β-oxidation could be transported into mitochondria via carnitine shuttles, we hypothesize that peroxisomal β-oxidation might play a role in regulating mitochondrial fatty acid oxidation and inducing hepatic steatosis under the condition of obesity or diabetes. This study reveals a novel mechanism by which peroxisomal β-oxidation controls mitochondrial fatty acid oxidation in diabetic animals. We determined that excessive oxidation of fatty acids by peroxisomes generates considerable acetyl-carnitine in the liver of diabetic mice, which significantly elevates the mitochondrial acetyl-CoA/CoA ratio and causes feedback suppression of mitochondrial β-oxidation. Additionally, we found that specific suppression of peroxisomal β-oxidation enhances mitochondrial fatty acid oxidation by reducing acetyl-carnitine formation in the liver of obese mice. In conclusion, we suggest that induction of peroxisomal fatty acid oxidation serves as a mechanism for diabetes-induced hepatic lipid accumulation. Targeting peroxisomal β-oxidation might be a promising pathway in improving hepatic steatosis and insulin resistance as induced by obesity or diabetes.
    Keywords:  acetyl-carnitine; fatty acid oxidation; mitochondria; obesity; peroxisomes
    DOI:  https://doi.org/10.1016/j.jbc.2022.102845
  22. Nat Immunol. 2023 Jan 05.
    Inducing trained immunity in pro-metastatic macrophages to control tumor metastasis.
    Chuanlin Ding, Rejeena Shrestha, Xiaojuan Zhu, Anne E Geller, Shouzhen Wu, Matthew R Woeste, Wenqian Li, Haomin Wang, Fang Yuan, Raobo Xu, Julia H Chariker, Xiaoling Hu, Hong Li, David Tieri, Huang-Ge Zhang, Eric C Rouchka, Robert Mitchell, Leah J Siskind, Xiang Zhang, Xiaoji G Xu, Kelly M McMasters, Yan Yu, Jun Yan.
      Metastasis is the leading cause of cancer-related deaths and myeloid cells are critical in the metastatic microenvironment. Here, we explore the implications of reprogramming pre-metastatic niche myeloid cells by inducing trained immunity with whole beta-glucan particle (WGP). WGP-trained macrophages had increased responsiveness not only to lipopolysaccharide but also to tumor-derived factors. WGP in vivo treatment led to a trained immunity phenotype in lung interstitial macrophages, resulting in inhibition of tumor metastasis and survival prolongation in multiple mouse models of metastasis. WGP-induced trained immunity is mediated by the metabolite sphingosine-1-phosphate. Adoptive transfer of WGP-trained bone marrow-derived macrophages reduced tumor lung metastasis. Blockade of sphingosine-1-phosphate synthesis and mitochondrial fission abrogated WGP-induced trained immunity and its inhibition of lung metastases. WGP also induced trained immunity in human monocytes, resulting in antitumor activity. Our study identifies the metabolic sphingolipid-mitochondrial fission pathway for WGP-induced trained immunity and control over metastasis.
    DOI:  https://doi.org/10.1038/s41590-022-01388-8
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