bims-mepmim 2023-02-19 papers

bims-mepmim

Biomed News

on Metabolites in pathological microenvironments and immunometabolism

Issue of 2023–02–19
28 papers selected by
Erika Mariana Palmieri, NIH/NCI Laboratory of Cancer ImmunoMetabolism

Cytoskeletal association of ATP citrate lyase controls the mechanodynamics of macropinocytosis.
Innate immune cell-intrinsic ketogenesis is dispensable for organismal metabolism and age-related inflammation.
The outer mitochondrial membrane protein TMEM11 demarcates spatially restricted BNIP3/BNIP3L-mediated mitophagy.
A systematic evaluation of quenching and extraction procedures for quantitative metabolome profiling of Hela carcinoma cell under 2D and 3D cell culture conditions.
PUFAs dictate the balance of power in ferroptosis.
SLC3A2 Promotes Tumor Associated Macrophage Polarization via Metabolic Reprogramming in Lung Cancer.
CD5 expression by dendritic cells directs T cell immunity and sustains immunotherapy responses.
Branched chain amino acids alter cellular redox to induce lipid oxidation and reduce de novo lipogenesis in the liver.
Dynamic differentiation of F4/80+ tumor-associated macrophage and its role in tumor vascularization in a syngeneic mouse model of colorectal liver metastasis.
Mitochondrial function in macrophages controls cardiac repair after myocardial infarction.
Human alveolar macrophage metabolism is compromised during Mycobacterium tuberculosis infection.
β-cell selective inhibition of DNA damage response signaling by nitric oxide is associated with an attenuation in glucose uptake.
TMEM164 is an acyltransferase that forms ferroptotic C20:4 ether phospholipids.
Hydrogen sulfide as an anti-calcification stratagem in human aortic valve: Altered biogenesis and mitochondrial metabolism of H2S lead to H2S deficiency in calcific aortic valve disease.
PFKFB2-mediated glycolysis promotes lactate-driven continual efferocytosis by macrophages.
Repair of airway epithelia requires metabolic rewiring towards fatty acid oxidation.
Caspase-11 contributes to site-1 protease cleavage and SREBP1 activation in the inflammatory response of macrophages.
Integrated landscape of cardiac metabolism in end-stage human nonischemic dilated cardiomyopathy.
Spatial Omics DataBase (SODB): increasing accessibility to spatial omics data.
Retinoids stored locally in the lung are required to attenuate the severity of acute lung injury in male mice.
Loss of Lkb1 in CD11c+ myeloid cells protects mice from diet-induced obesity while enhancing glucose intolerance and IL-17/IFN-γ imbalance.
Immunosuppressive reprogramming of neutrophils by lung mesenchymal cells promotes breast cancer metastasis.
Protocol for Plasma Extracellular Vesicle and Particle Isolation and Mass Spectrometry-Based Proteomic Identification.
Harnessing the reverse cholesterol transport pathway to favor differentiation of monocyte-derived APCs and antitumor responses.
Dynamics of co-substrate pools can constrain and regulate metabolic fluxes.
Xanthine dehydrogenase rewires metabolism and the survival of nutrient deprived lung adenocarcinoma cells by facilitating UPR and autophagic degradation.
Lipid induced alteration in retinoic acid signaling leads to mitochondrial dysfunction in HepG2 and Huh7 cells.
Metabolic heterogeneity and cross-feeding within isogenic yeast populations captured by DILAC.

Proc Natl Acad Sci U S A. 2023 Feb 21. 120(8): e2213272120

Cytoskeletal association of ATP citrate lyase controls the mechanodynamics of macropinocytosis.

Joseph Puccini, Jia Wei, Liang Tong, Dafna Bar-Sagi.

  Macropinocytosis is an actin-dependent mode of nonselective endocytosis that mediates the uptake of extracellular fluid-phase cargoes. It is now well recognized that tumor cells exploit macropinocytosis to internalize macromolecules that can be catabolized and used to support cell growth and proliferation under nutrient-limiting conditions. Therefore, the identification of molecular mechanisms that control macropinocytosis is fundamental to the understanding of the metabolic adaptive landscape of tumor cells. Here, we report that the acetyl-CoA-producing enzyme, ATP citrate lyase (ACLY), is a key regulator of macropinocytosis and describes a heretofore-unappreciated association of ACLY with the actin cytoskeleton. The cytoskeletal tethering of ACLY is required for the spatially defined acetylation of heterodimeric actin capping protein, which we identify as an essential mediator of the actin remodeling events that drive membrane ruffling and macropinocytosis. Furthermore, we identify a requirement for mitochondrial-derived citrate, an ACLY substrate, for macropinocytosis, and show that mitochondria traffic to cell periphery regions juxtaposed to plasma membrane ruffles. Collectively, these findings establish a mode of metabolite compartmentalization that supports the spatiotemporal modulation of membrane-cytoskeletal interactions required for macropinocytosis by coupling regional acetyl-CoA availability with dynamic protein acetylation.

Keywords:  actin cytoskeleton; macropinocytosis; membrane ruffling

DOI:  https://doi.org/10.1073/pnas.2213272120
J Biol Chem. 2023 Feb 10. pii: S0021-9258(23)00137-0. [Epub ahead of print] 103005

Innate immune cell-intrinsic ketogenesis is dispensable for organismal metabolism and age-related inflammation.

Emily L Goldberg, Anudari Letian, Tamara Dlugos, Claire Leveau, Vishwa Deep Dixit.

  Aging is accompanied by chronic low-grade inflammation, but the mechanisms that allow this to persist are not well understood. Ketone bodies are alternative fuels produced when glucose is limited and improve indicators of healthspan in aging mouse models. Moreover, the most abundant ketone body, β-hydroxybutyrate (BHB), inhibits the NLRP3 inflammasome in myeloid cells, a key potentiator of age-related inflammation. Given that myeloid cells express ketogenic machinery, we hypothesized this pathway may serve as a metabolic checkpoint of inflammation. To test this hypothesis, we conditionally ablated ketogenesis by disrupting expression of the terminal enzyme required for ketogenesis, 3-Hydroxy-3-Methylglutaryl-CoA Lyase (HMGCL). By deleting HMGCL in the liver, we validated the functional targeting and establish that the liver is the only organ that can produce the life-sustaining quantities of ketone bodies required for survival during fasting or ketogenic diet feeding. Conditional ablation of HMGCL in neutrophils and macrophages had modest effects on body weight and glucose tolerance in aging, but worsened glucose homeostasis in myeloid cell-specific Hmgcl-deficient mice fed a high-fat diet. Our results suggest that during aging, liver-derived circulating ketone bodies might be more important for deactivating the NLRP3 inflammasome and controlling organismal metabolism.

Keywords:  aging; inflammation; innate immunity; ketone; metabolism

DOI:  https://doi.org/10.1016/j.jbc.2023.103005
J Cell Biol. 2023 Apr 03. pii: e202204021. [Epub ahead of print]222(4):

The outer mitochondrial membrane protein TMEM11 demarcates spatially restricted BNIP3/BNIP3L-mediated mitophagy.

Mehmet Oguz Gok, Olivia M Connor, Xun Wang, Cameron J Menezes, Claire B Llamas, Prashant Mishra, Jonathan R Friedman.

Mitochondria play critical roles in cellular metabolism and to maintain their integrity, they are regulated by several quality control pathways, including mitophagy. During BNIP3/BNIP3L-dependent receptor-mediated mitophagy, mitochondria are selectively targeted for degradation by the direct recruitment of the autophagy protein LC3. BNIP3 and/or BNIP3L are upregulated situationally, for example during hypoxia and developmentally during erythrocyte maturation. However, it is not well understood how they are spatially regulated within the mitochondrial network to locally trigger mitophagy. Here, we find that the poorly characterized mitochondrial protein TMEM11 forms a complex with BNIP3 and BNIP3L and co-enriches at sites of mitophagosome formation. We find that mitophagy is hyper-active in the absence of TMEM11 during both normoxia and hypoxia-mimetic conditions due to an increase in BNIP3/BNIP3L mitophagy sites, supporting a model that TMEM11 spatially restricts mitophagosome formation.

DOI: https://doi.org/10.1083/jcb.202204021
Biotechnol J. 2023 Feb 16. e2200444

A systematic evaluation of quenching and extraction procedures for quantitative metabolome profiling of Hela carcinoma cell under 2D and 3D cell culture conditions.

Tong Wang, Xueting Wang, Yingping Zhuang, Guan Wang.

  Metabolic reprogramming has been coined as a hallmark of cancer, accompanied by which the alterations in metabolite levels have profound effects on gene expression, cellular differentiation and the tumor environment. Yet a systematic evaluation of quenching and extraction procedures for quantitative metabolome profiling of tumor cells is currently lacking. To achieve this, this study is aimed at establishing an unbiased and leakage-free metabolome preparation protocol for Hela carcinoma cell. We evaluated 12 combinations of quenching and extraction methods from three quenchers (liquid nitrogen, -40°C 50% methanol, 0.5°C normal saline) and four extractants (-80°C 80% methanol, 0.5°C methanol: chloroform: water (1:1:1, v/v/v), 0.5°C 50% acetonitrile, 75°C 70% ethanol) for global metabolite profiling of adherent Hela carcinoma cells. Based on the isotope dilution mass spectrometry (IDMS) method, gas/liquid chromatography in tandem with mass spectrometry was used to quantitatively determine 43 metabolites including sugar phosphates, organic acids, amino acids, adenosine nucleotides and coenzymes involved in central carbon metabolism. The results showed that the total amount of the intracellular metabolites in cell extracts obtained using different sample preparation procedures with the IDMS method ranged from 21.51 to 295.33 nmol/million cells. Among 12 combinations, cells that washed twice with phosphate buffered saline (PBS), quenched with liquid nitrogen, and then extracted with 50% acetonitrile was found to be the most optimal method to acquire intracellular metabolites with high efficiency of metabolic arrest and minimal loss during sample preparation. In addition, the same conclusion was drawn as these 12 combinations were applied to obtain quantitative metabolome data from three-dimensional (3D) tumor spheroids. Furthermore, a case study was carried out to evaluate the effect of doxorubicin (DOX) on both adherent cells and 3D tumor spheroids using quantitative metabolite profiling. Pathway enrichment analysis using targeted metabolomics data showed that DOX exposure would significantly affect amino acid metabolism-related pathways, which might be related to the mitigation of redox stress. Strikingly, our data suggested that compared to 2D cells the increased intracellular glutamine level in 3D cells benefited replenishing the tricarboxylic acid (TCA) cycle when the glycolysis was limited after dosing with DOX. Taken together, this study provides a well-established quenching and extraction protocol for quantitative metabolome profiling of Hela carcinoma cell under 2D and 3D cell culture conditions. Based on this, quantitative time-resolved metabolite data can serve to the generation of hypotheses on metabolic reprogramming to reveal its important role in tumor development and treatment. This article is protected by copyright. All rights reserved.

Keywords:  3D tumor spheroids; Hela; extraction; isotope dilution mass spectrometry; metabolomics; quenching; sample preparation

DOI:  https://doi.org/10.1002/biot.202200444
Cell Calcium. 2023 Feb 06. pii: S0143-4160(23)00015-5. [Epub ahead of print]110 102703

PUFAs dictate the balance of power in ferroptosis.

Shan Xin, Joel A Schick.

  Ferroptosis is an iron-dependent form of cell death triggered by dysregulation of biochemical processes that culminate in lethal lipid peroxidation. Lipid metabolism is fundamental for determining ferroptotic fate, however, the mechanisms that alter lipid components to shape ferroptosis susceptibility remains elusive. A recent article by Lin and colleagues in Nature Communications systematically analyzed phospholipid transporters (phospholipid scramblases, flippases, and floppases), and identified that the lipid flippase solute carrier family 47 member 1 (SLC47A1) functions as a regulator of lipid remodeling and promotes ferroptosis resistance. SLC47A1 is transactivated by peroxisome proliferator activated receptor alpha (PPARA). Upon ferroptosis induction, SLC47A1 upregulation inhibits DHA/DPA polyunsaturated fatty acid containing glycerophospholipids (PUFA-PLs) accumulation to block ferroptosis. Depletion of either PPARA or SLC47A1 sensitized cells to ferroptosis by favoring ACSL4-SOAT1-mediated production of polyunsaturated fatty acid containing (PUFA) cholesterol esters. Ferroptosis has been widely linked to degenerative processes and tumor suppression. These findings indicate that lipid transporters may provide yet another means by which PUFA-containing membrane lipids convey ferroptosis sensitivity.

Keywords:  CE; Ferroptosis; Flippase; Lipid remodeling; MUFA; PUFA; SFA; SlcLC47A1

DOI:  https://doi.org/10.1016/j.ceca.2023.102703
Cancer Sci. 2023 Feb 15.

SLC3A2 Promotes Tumor Associated Macrophage Polarization via Metabolic Reprogramming in Lung Cancer.

Zhuan Li, Songming Chen, Xiang He, Siyuan Gong, Lunquan Sun, Liang Weng.

  Tumor associated macrophages (TAMs) are one of the most abundant immunosuppressive cells in the tumor microenvironment and possess crucial functions in facilitating tumor progression. Emerging evidences indicate that altered metabolic properties in cancer cell support the tumorigenic functions of TAMs. However, mechanisms and mediators underly crosstalk between cancer cell and TAMs remain largely unknown. In present study, we revealed that high Solute Carrier Family 3 Member 2 (SLC3A2) expression in lung cancer patients were associated with TAMs and poor prognosis. Knockdown of SLC3A2 in lung adenocarcinoma cells impaired M2 polarization of macrophages in co-culture system. By using metabolome analysis, we identified that knockdown SLC3A2 altered metabolism of lung cancer cells and changed multiple metabolites including arachidonic acid in the tumor microenvironment. More importantly, we demonstrated that arachidonic acid was responsible for SLC3A2 mediated macrophage polarization in the tumor microenvironment to differentiate into M2 type both in vitro and in vivo. Our data illustrate previously undescribed mechanisms responsible for TAMs polarization and suggest that SLC3A2 acts as a metabolic switch on lung adenocarcinoma cells to induce macrophage phenotypic reprogramming via arachidonic acid.

Keywords:  Arachidonic acid; Lung adenocarcinoma; Macrophage polarization; SLC3A2; Tumor associated macrophage

DOI:  https://doi.org/10.1111/cas.15760
Science. 2023 Feb 17. 379(6633): eabg2752

CD5 expression by dendritic cells directs T cell immunity and sustains immunotherapy responses.

Mingyu He, Kate Roussak, Feiyang Ma, Nicholas Borcherding, Vince Garin, Mike White, Charles Schutt, Trine I Jensen, Yun Zhao, Courtney A Iberg, Kairav Shah, Himanshi Bhatia, Daniel Korenfeld, Sabrina Dinkel, Judah Gray, Alina Ulezko Antonova, Stephen Ferris, David Donermeyer, Cecilia Lindestam Arlehamn, Matthew M Gubin, Jingqin Luo, Laurent Gorvel, Matteo Pellegrini, Alessandro Sette, Thomas Tung, Rasmus Bak, Robert L Modlin, Ryan C Fields, Robert D Schreiber, Paul M Allen, Eynav Klechevsky.

The induction of proinflammatory T cells by dendritic cell (DC) subtypes is critical for antitumor responses and effective immune checkpoint blockade (ICB) therapy. Here, we show that human CD1c+CD5+ DCs are reduced in melanoma-affected lymph nodes, with CD5 expression on DCs correlating with patient survival. Activating CD5 on DCs enhanced T cell priming and improved survival after ICB therapy. CD5+ DC numbers increased during ICB therapy, and low interleukin-6 (IL-6) concentrations promoted their de novo differentiation. Mechanistically, CD5 expression by DCs was required to generate optimally protective CD5hi T helper and CD8+ T cells; further, deletion of CD5 from T cells dampened tumor elimination in response to ICB therapy in vivo. Thus, CD5+ DCs are an essential component of optimal ICB therapy.

DOI: https://doi.org/10.1126/science.abg2752
Am J Physiol Endocrinol Metab. 2023 Feb 15.

Branched chain amino acids alter cellular redox to induce lipid oxidation and reduce de novo lipogenesis in the liver.

Chaitra Surugihalli, Vaishna Muralidaran, Caitlin E Ryan, Kruti Patel, David Zhao, Nishanth E Sunny.

  Metabolic and molecular interactions between branched chain amino acid (BCAA) and lipid metabolism are evident in insulin resistant tissues. However, it remains unclear whether insulin resistance is a prerequisite for these relationships and whether BCAAs or their metabolic intermediates can modulate hepatic lipid oxidation and synthesis. We hypothesized that BCAAs can alter hepatic oxidative function and de novo lipogenesis, independent of them being anaplerotic substrates for the mitochondria. Mice (C57BL/6NJ) were reared on a low-fat (LF), LF diet plus 1.5X BCAAs (LB), high-fat (HF) or HF diet plus 1.5X BCAAs (HB) for 12-wks. Hepatic metabolism was profiled utilizing stable isotopes coupled to mass-spectrometry and nuclear magnetic resonance, together with fed-to-fasted changes in gene and protein expression. A greater induction of lipid oxidation and ketogenesis upon fasting was evident in the BCAA supplemented, insulin sensitive livers from LB mice, while their rates of hepatic de novo lipogenesis remained lower than their LF counterparts. Onset of insulin resistance in HF and HB mice livers blunted these responses. Whole-body turnover of BCAAs and their ketoacids, their serum concentrations, and the ketogenic flux from BCAA catabolism, all remained similar between fasted LF and LB mice. This suggested that the impact of BCAAs on lipid metabolism can occur independent of them or their degradation products fueling anaplerosis through the liver mitochondria. Further, the greater induction of lipid oxidation in the LB livers accompanied higher mitochondrial NADH/NAD+ ratio and higher fed-to-fasting phosphorylation of AMPKα and ACC. Taken together, our results provide evidence that BCAA supplementation, under conditions of insulin sensitivity, improved the feeding-to-fasting induction of hepatic lipid oxidation through changes in cellular redox, thus providing a favorable biochemical environment for flux through β-oxidation and lower de novo lipogenesis.

Keywords:  branched chain amino acids; fatty acid oxidation; lipogenesis; liver metabolism; mitochondria

DOI:  https://doi.org/10.1152/ajpendo.00307.2022
Cell Death Dis. 2023 Feb 13. 14(2): 117

Dynamic differentiation of F4/80+ tumor-associated macrophage and its role in tumor vascularization in a syngeneic mouse model of colorectal liver metastasis.

Ting Qiao, Wanli Yang, Xiangchuan He, Ping Song, Xiao Chen, Ruijie Liu, Jian Xiao, Xiaoli Yang, Mingqi Li, Yudan Gao, Guoan Chen, Yi Lu, Jian Zhang, Jing Leng, Huan Ren.

Tumor-associated macrophages (TAMs) are highly heterogeneous and play vital roles in tumor progression. Here we adopted a C57BL/6 mouse model imitating the late-stage colorectal liver metastasis (CRLM) by Mc38 colorectal cancer cell injection via the portal vein. With serial sections of CRLM biopsies, we defined 7-9 days post-injection as the critical period for tumor neovascularization, which was initiated from the innate liver vessels via vessel cooption and extended by vascular mimicry and thereof growth of CD34+cells. In samples with increasing-sized liver metastases, the infiltrated Ly6C+ CD11b+ F4/80- monocytes steadily gained the expression of F4/80, a Kupffer cell marker, before transformed into Ly6C- CD11bint F4/80+ cells, which, the same phenotype was also adapted by Ly6C- CD11b- F4/80+ Kupffer cells. F4/80+ TAMs showed proximity to neovascularization and tumor vessels, functionally angiogenic in vivo; and greatly promoted the activation of a few key angiogenic markers such as VEGFA, Ki67, etc. in endothelial cells in vitro. Depletion of macrophages or diversion of macrophage polarization during neovascularization impeded tumor growth and vascularization and resulted in greatly reduced F4/80+ TAMs, yet increased CD11b+ cells due to inhibition of TAM differentiation. In summary, our results showed dynamic and spatial-temporal F4/80+ TAM transformation within the tumor microenvironment and strengthened its role as perivascular and angiogenic TAMs in CRLM.

DOI: https://doi.org/10.1038/s41419-023-05626-1
J Clin Invest. 2023 Feb 15. pii: e167079. [Epub ahead of print]133(4):

Mitochondrial function in macrophages controls cardiac repair after myocardial infarction.

David Weissman, Christoph Maack.

Cardiac healing following acute myocardial infarction (MI) involves the mobilization and activation of immune cells, including macrophages. In the early phase after MI, macrophages adopt a proinflammatory phenotype, while polarizing toward a reparative one in the late stage. Although metabolic reprogramming has been observed during this transition, the mechanistic links to macrophage differentiation are still poorly understood. In this issue of the JCI, Cai, Zhao and colleagues demonstrate that mitochondrial function in macrophages governed the resolution of inflammation and tissue repair by modulating the phagocytic removal of apoptotic cells (so-called efferocytosis) as well as myofibroblast activation. These findings provide important mechanistic insights into the potential relevance of metabolic modulation of macrophage functions following MI, which might lead to alternative therapeutic strategies for MI.

DOI: https://doi.org/10.1172/JCI167079
Front Immunol. 2022 ;13 1044592

Human alveolar macrophage metabolism is compromised during Mycobacterium tuberculosis infection.

Laura E Mendonca, Erwan Pernet, Nargis Khan, Joaquin Sanz, Eva Kaufmann, Jeffrey Downey, Alexandre Grant, Marianna Orlova, Erwin Schurr, Connie Krawczyk, Russell G Jones, Luis B Barreiro, Maziar Divangahi.

  Pulmonary macrophages have two distinct ontogenies: long-lived embryonically-seeded alveolar macrophages (AM) and bone marrow-derived macrophages (BMDM). Here, we show that after infection with a virulent strain of Mycobacterium tuberculosis (H37Rv), primary murine AM exhibit a unique transcriptomic signature characterized by metabolic reprogramming distinct from conventional BMDM. In contrast to BMDM, AM failed to shift from oxidative phosphorylation (OXPHOS) to glycolysis and consequently were unable to control infection with an avirulent strain (H37Ra). Importantly, healthy human AM infected with H37Ra equally demonstrated diminished energetics, recapitulating our observation in the murine model system. However, the results from seahorse showed that the shift towards glycolysis in both AM and BMDM was inhibited by H37Rv. We further demonstrated that pharmacological (e.g. metformin or the iron chelator desferrioxamine) reprogramming of AM towards glycolysis reduced necrosis and enhanced AM capacity to control H37Rv growth. Together, our results indicate that the unique bioenergetics of AM renders these cells a perfect target for Mtb survival and that metabolic reprogramming may be a viable host targeted therapy against TB.

Keywords:  cell death programs; immunity; immunometabolism; metabolic reprograming; pulmonary macrophages; tuberculosis

DOI:  https://doi.org/10.3389/fimmu.2022.1044592
J Biol Chem. 2023 Feb 09. pii: S0021-9258(23)00126-6. [Epub ahead of print] 102994

β-cell selective inhibition of DNA damage response signaling by nitric oxide is associated with an attenuation in glucose uptake.

Chay Teng Yeo, Erin M Kropp, Polly A Hansen, Michael Pereckas, Bryndon J Oleson, Aaron Naatz, Jennifer S Stancill, Kyle A Ross, Rebekah L Gundry, John A Corbett.

Nitric oxide plays a dual role in regulating DNA damage response (DDR) signaling in pancreatic β-cells. As a genotoxic agent, nitric oxide activates two types of DDR signaling; however, when produced at micromolar levels by the inducible isoform of nitric oxide synthase (iNOS), nitric oxide inhibits DDR signaling and DDR-induced apoptosis in a β-cell-selective manner. DDR signaling inhibition by nitric oxide correlates with mitochondrial oxidative metabolism inhibition and decreases in ATP and NAD+. Unlike most cell types, β-cells do not compensate for impaired mitochondrial oxidation by increasing glycolytic flux, and this metabolic inflexibility leads to a decrease in ATP and NAD+. Here, we used multiple analytical approaches to determine changes in intermediary metabolites in β-cells and non-β-cells treated with nitric oxide or the complex I inhibitor rotenone. In addition to ATP and NAD+, glycolytic and TCA cycle intermediates as well as NADPH are significantly decreased in β-cells treated with nitric oxide or rotenone. Consistent with glucose-6-phosphate residing at the metabolic branchpoint for glycolysis and the pentose phosphate pathway (NADPH), we show that mitochondrial oxidation inhibitors limit glucose uptake in a β-cell-selective manner. Our findings indicate that the β-cell selective inhibition of DDR signaling by nitric oxide is associated with a decrease in ATP to levels that fall significantly below the Km for ATP of glucokinase (glucose uptake) and suggest that this action places the β-cell in a state of suspended animation where it is metabolically inert until nitric oxide is removed, and metabolic function can be restored.

DOI: https://doi.org/10.1016/j.jbc.2023.102994
Nat Chem Biol. 2023 Feb 13.

TMEM164 is an acyltransferase that forms ferroptotic C20:4 ether phospholipids.

Alex Reed, Timothy Ware, Haoxin Li, J Fernando Bazan, Benjamin F Cravatt.

Ferroptosis is an iron-dependent form of cell death driven by oxidation of polyunsaturated fatty acid (PUFA) phospholipids. Large-scale genetic screens have uncovered a specialized role for PUFA ether phospholipids (ePLs) in promoting ferroptosis. Understanding of the enzymes involved in PUFA-ePL production, however, remains incomplete. Here we show, using a combination of pathway mining of genetic dependency maps, AlphaFold-guided structure predictions and targeted lipidomics, that the uncharacterized transmembrane protein TMEM164-the genetic ablation of which has been shown to protect cells from ferroptosis-is a cysteine active center enzyme that selectively transfers C20:4 acyl chains from phosphatidylcholine to lyso-ePLs to produce PUFA ePLs. Genetic deletion of TMEM164 across a set of ferroptosis-sensitive cancer cell lines caused selective reductions in C20:4 ePLs with minimal effects on C20:4 diacyl PLs, and this lipid profile produced a variable range of protection from ferroptosis, supportive of an important but contextualized role for C20:4 ePLs in this form of cell death.

DOI: https://doi.org/10.1038/s41589-022-01253-7
Redox Biol. 2023 Apr;pii: S2213-2317(23)00030-7. [Epub ahead of print]60 102629

Hydrogen sulfide as an anti-calcification stratagem in human aortic valve: Altered biogenesis and mitochondrial metabolism of H2S lead to H2S deficiency in calcific aortic valve disease.

Zsolt Combi, László Potor, Péter Nagy, Katalin Éva Sikura, Tamás Ditrói, Eszter Petra Jurányi, Klaudia Galambos, Tamás Szerafin, Péter Gergely, Matthew Whiteman, Roberta Torregrossa, Yuchao Ding, Lívia Beke, Zoltán Hendrik, Gábor Méhes, György Balla, József Balla.

  Hydrogen sulfide (H2S) was previously revealed to inhibit osteoblastic differentiation of valvular interstitial cells (VICs), a pathological feature in calcific aortic valve disease (CAVD). This study aimed to explore the metabolic control of H2S levels in human aortic valves. Lower levels of bioavailable H2S and higher levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were detected in aortic valves of CAVD patients compared to healthy individuals, accompanied by higher expression of cystathionine γ-lyase (CSE) and same expression of cystathionine β-synthase (CBS). Increased biogenesis of H2S by CSE was found in the aortic valves of CAVD patients which is supported by increased production of lanthionine. In accordance, healthy human aortic VICs mimic human pathology under calcifying conditions, as elevated CSE expression is associated with low levels of H2S. The expression of mitochondrial enzymes involved in H2S catabolism including sulfide quinone oxidoreductase (SQR), the key enzyme in mitochondrial H2S oxidation, persulfide dioxygenase (ETHE1), sulfite oxidase (SO) and thiosulfate sulfurtransferase (TST) were up-regulated in calcific aortic valve tissues, and a similar expression pattern was observed in response to high phosphate levels in VICs. AP39, a mitochondria-targeting H2S donor, rescued VICs from an osteoblastic phenotype switch and reduced the expression of IL-1β and TNF-α in VICs. Both pro-inflammatory cytokines aggravated calcification and osteoblastic differentiation of VICs derived from the calcific aortic valves. In contrast, IL-1β and TNF-α provided an early and transient inhibition of VICs calcification and osteoblastic differentiation in healthy cells and that effect was lost as H2S levels decreased. The benefit was mediated via CSE induction and H2S generation. We conclude that decreased levels of bioavailable H2S in human calcific aortic valves result from an increased H2S metabolism that facilitates the development of CAVD. CSE/H2S represent a pathway that reverses the action of calcifying stimuli.

Keywords:  Arteriosclerosis; Chronic kidney disease; Hydrogen sulfide; Mitochondrial H(2)S catabolism; Phosphate; Valvular inflammation; Vascular calcification

DOI:  https://doi.org/10.1016/j.redox.2023.102629
Nat Metab. 2023 Feb 16.

PFKFB2-mediated glycolysis promotes lactate-driven continual efferocytosis by macrophages.

Maaike Schilperoort, David Ngai, Marina Katerelos, David A Power, Ira Tabas.

Resolving-type macrophages prevent chronic inflammation by clearing apoptotic cells through efferocytosis. These macrophages are thought to rely mainly on oxidative phosphorylation, but emerging evidence suggests a possible link between efferocytosis and glycolysis. To gain further insight into this issue, we investigated molecular-cellular mechanisms involved in efferocytosis-induced macrophage glycolysis and its consequences. We found that efferocytosis promotes a transient increase in macrophage glycolysis that is dependent on rapid activation of the enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 2 (PFKFB2), which distinguishes this process from glycolysis in pro-inflammatory macrophages. Mice transplanted with activation-defective PFKFB2 bone marrow and then subjected to dexamethasone-induced thymocyte apoptosis exhibit impaired thymic efferocytosis, increased thymic necrosis, and lower expression of the efferocytosis receptors MerTK and LRP1 on thymic macrophages compared with wild-type control mice. In vitro mechanistic studies revealed that glycolysis stimulated by the uptake of a first apoptotic cell promotes continual efferocytosis through lactate-mediated upregulation of MerTK and LRP1. Thus, efferocytosis-induced macrophage glycolysis represents a unique metabolic process that sustains continual efferocytosis in a lactate-dependent manner. The differentiation of this process from inflammatory macrophage glycolysis raises the possibility that it could be therapeutically enhanced to promote efferocytosis and resolution in chronic inflammatory diseases.

DOI: https://doi.org/10.1038/s42255-023-00736-8
Nat Commun. 2023 Feb 13. 14(1): 721

Repair of airway epithelia requires metabolic rewiring towards fatty acid oxidation.

Stefania Crotta, Matteo Villa, Jack Major, Katja Finsterbusch, Miriam Llorian, Peter Carmeliet, Joerg Buescher, Andreas Wack.

Epithelial tissues provide front-line barriers shielding the organism from invading pathogens and harmful substances. In the airway epithelium, the combined action of multiciliated and secretory cells sustains the mucociliary escalator required for clearance of microbes and particles from the airways. Defects in components of mucociliary clearance or barrier integrity are associated with recurring infections and chronic inflammation. The timely and balanced differentiation of basal cells into mature epithelial cell subsets is therefore tightly controlled. While different growth factors regulating progenitor cell proliferation have been described, little is known about the role of metabolism in these regenerative processes. Here we show that basal cell differentiation correlates with a shift in cellular metabolism from glycolysis to fatty acid oxidation (FAO). We demonstrate both in vitro and in vivo that pharmacological and genetic impairment of FAO blocks the development of fully differentiated airway epithelial cells, compromising the repair of airway epithelia. Mechanistically, FAO links to the hexosamine biosynthesis pathway to support protein glycosylation in airway epithelial cells. Our findings unveil the metabolic network underpinning the differentiation of airway epithelia and identify novel targets for intervention to promote lung repair.

DOI: https://doi.org/10.1038/s41467-023-36352-z
Front Immunol. 2023 ;14 1009973

Caspase-11 contributes to site-1 protease cleavage and SREBP1 activation in the inflammatory response of macrophages.

Yinglan Cheng, Ichiro Manabe, Sumio Hayakawa, Yusuke Endo, Yumiko Oishi.

  Sterol regulatory element-binding proteins (SREBPs) are key transcription factors that control fatty acid and cholesterol metabolism. As the major SREBP isoform in macrophages, SREBP1a is also required for inflammatory and phagocytotic functions. However, it is insufficiently understood how SREBP1a is activated by the innate immune response in macrophages. Here, we show that mouse caspase-11 is a novel inflammatory activator of SREBP1a in macrophages. Upon LPS treatment, caspase-11 was found to promote the processing of site-1 protease (S1P), an enzyme that mediates the cleavage and activation of SREBP1. We also determined that caspase-11 directly associates with S1P and cleaves it at a specific site. Furthermore, deletion of the Casp4 gene, which encodes caspase-11, impaired the activation of S1P and SREBP1 as well as altered the expression of genes regulated by SREBP1 in macrophages. These results demonstrate that the caspase-11/S1P pathway activates SREBP1 in response to LPS, thus regulating subsequent macrophage activation.

Keywords:  SREBP (sterol regulatory element-binding protein) pathway; caspase; inflammatory response; macrophage; site-1 protease (S1P)

DOI:  https://doi.org/10.3389/fimmu.2023.1009973
Nat Cardiovasc Res. 2022 Sep;1(9): 817-829

Integrated landscape of cardiac metabolism in end-stage human nonischemic dilated cardiomyopathy.

Emily Flam, Cholsoon Jang, Danielle Murashige, Yifan Yang, Michael P Morley, Sunhee Jung, Daniel S Kantner, Hannah Pepper, Kenneth C Bedi, Jeff Brandimarto, Benjamin L Prosser, Thomas Cappola, Nathaniel W Snyder, Joshua D Rabinowitz, Kenneth B Margulies, Zolt Arany.

Heart failure (HF) is a leading cause of mortality. Failing hearts undergo profound metabolic changes, but a comprehensive evaluation in humans is lacking. We integrate plasma and cardiac tissue metabolomics of 678 metabolites, genome-wide RNA-sequencing, and proteomic studies to examine metabolic status in 87 explanted human hearts from 39 patients with end-stage HF compared with 48 nonfailing donors. We confirm bioenergetic defects in human HF and reveal selective depletion of adenylate purines required for maintaining ATP levels. We observe substantial reductions in fatty acids and acylcarnitines in failing tissue, despite plasma elevations, suggesting defective import of fatty acids into cardiomyocytes. Glucose levels, in contrast, are elevated. Pyruvate dehydrogenase, which gates carbohydrate oxidation, is de-repressed, allowing increased lactate and pyruvate burning. Tricarboxylic acid cycle intermediates are significantly reduced. Finally, bioactive lipids are profoundly reprogrammed, with marked reductions in ceramides and elevations in lysoglycerophospholipids. These data unveil profound metabolic abnormalities in human failing hearts.

DOI: https://doi.org/10.1038/s44161-022-00117-6
Nat Methods. 2023 Feb 16.

Spatial Omics DataBase (SODB): increasing accessibility to spatial omics data.

DOI: https://doi.org/10.1038/s41592-023-01772-8
Nat Commun. 2023 Feb 15. 14(1): 851

Retinoids stored locally in the lung are required to attenuate the severity of acute lung injury in male mice.

Igor O Shmarakov, Galina A Gusarova, Mohammad N Islam, María Marhuenda-Muñoz, Jahar Bhattacharya, William S Blaner.

Retinoids are potent transcriptional regulators that act in regulating cell proliferation, differentiation, and other cellular processes. We carried out studies in male mice to establish the importance of local cellular retinoid stores within the lung alveolus for maintaining its health in the face of an acute inflammatory challenge induced by intranasal instillation of lipopolysaccharide. We also undertook single cell RNA sequencing and bioinformatic analyses to identify roles for different alveolar cell populations involved in mediating these retinoid-dependent responses. Here we show that local retinoid stores and uncompromised metabolism and signaling within the lung are required to lessen the severity of an acute inflammatory challenge. Unexpectedly, our data also establish that alveolar cells other than lipofibroblasts, specifically microvascular endothelial and alveolar epithelial cells, are able to take up lipoprotein-transported retinoid and to accumulate cellular retinoid stores that are directly used to respond to an acute inflammatory challenge.

DOI: https://doi.org/10.1038/s41467-023-36475-3
Cell Mol Life Sci. 2023 Feb 13. 80(3): 63

Loss of Lkb1 in CD11c+ myeloid cells protects mice from diet-induced obesity while enhancing glucose intolerance and IL-17/IFN-γ imbalance.

Yunyan Sun, Bing Wang, Qianwen Hu, Haixiao Zhang, Xun Lai, Tier Wang, Chunxiao Zhao, Jiali Wang, Xi Zhang, Qing Niu, Baolin He, Erlie Jiang, Mingxia Shi, Xiaoming Feng, Yuechen Luo.

  Adipose tissue CD11c+ myeloid cell is an independent risk factor associated with obesity and metabolic disorders. However, the underlying molecular basis remains elusive. Here, we demonstrated that liver kinase B1 (Lkb1), a key bioenergetic sensor, is involved in CD11c+ cell-mediated immune responses in diet-induced obesity. Loss of Lkb1 in CD11c+ cells results in obesity resistance but lower glucose tolerance, which accompanies tissue-specific immune abnormalities. The accumulation and CD80's expression of Lkb1 deficient adipose-tissue specific dendritic cells but not macrophages is restrained. Additionally, the balance of IL-17A and IFN-γ remarkably tips towards the latter in fat T cells and CD11c- macrophages. Mechanistically, IFN-γ promotes apoptosis of preadipocytes and inhibits their adipogenesis while IL-17A promotes the adipogenesis in vitro, which might account in part for the fat gain resistant phenotype. In summary, these findings reveal that Lkb1 is essential for fat CD11c+ dendritic cells responding to HFD exposure and provides new insights into the IL-17A/IFN-γ balance in HFD-induced obesity.

Keywords:  Dendritic cells; High-fat-diet-induced obesity; IFN-γ; IL-17A; Liver kinase B1; Macrophages; T cells; Visceral adipose tissue

DOI:  https://doi.org/10.1007/s00018-023-04707-w
Sci Immunol. 2023 Feb 24. 8(80): eadd5204

Immunosuppressive reprogramming of neutrophils by lung mesenchymal cells promotes breast cancer metastasis.

Zheng Gong, Qing Li, Jiayuan Shi, Peishan Li, Li Hua, Leonard D Shultz, Guangwen Ren.

Neutrophils, the most abundant innate immune cells, function as crucial regulators of the adaptive immune system in diverse pathological conditions, including metastatic cancer. However, it remains largely unknown whether their immunomodulatory functions are intrinsic or acquired within the pathological tissue environment. Here, using mouse models of metastatic breast cancer in the lungs, we show that, although neutrophils isolated from bone marrow (BM) or blood are minimally immunosuppressive, lung-infiltrating neutrophils are robustly suppressive of both T cells and natural killer (NK) cells. We found that this tissue-specific immunosuppressive capacity of neutrophils exists in the steady state and is reinforced by tumor-associated inflammation. Acquisition of potent immunosuppression activity by lung-infiltrating neutrophils was endowed by the lung-resident stroma, specifically CD140a+ mesenchymal cells (MCs) and largely via prostaglandin-endoperoxide synthase 2 (PTGS2), the rate-limiting enzyme for prostaglandin E2 (PGE2) biosynthesis. MC-specific deletion of Ptgs2 or pharmacological inhibition of PGE2 receptors reversed lung neutrophil-mediated immunosuppression and mitigated lung metastasis of breast cancer in vivo. These lung stroma-targeting strategies substantially improved the therapeutic efficacy of adoptive T cell-based immunotherapy in treating metastatic disease in mice. Collectively, our results reveal that the immunoregulatory effects of neutrophils are induced by tissue-resident stroma and that targeting tissue-specific stromal factors represents an effective approach to boost tissue-resident immunity against metastatic disease.

DOI: https://doi.org/10.1126/sciimmunol.add5204
Methods Mol Biol. 2023 ;2628 291-300

Protocol for Plasma Extracellular Vesicle and Particle Isolation and Mass Spectrometry-Based Proteomic Identification.

Amirmohammad Nasiri Kenari, Linda Bojmar, Søren Heissel, Henrik Molina, David Lyden, Ayuko Hoshino.

  Plasma extracellular vesicles and particles (EVPs) are enriched in biomolecules that reflect individuals' physiological and pathological states. Several studies have demonstrated the potential of human plasma EVPs as a novel liquid biopsy. Here we describe a protocol for human plasma EVPs isolation and proteomic characterization. We isolated human plasma EVPs by the classical ultracentrifugation method and performed mass spectrometry-based proteomic profiling. Using this protocol, researchers can reveal the plasma EVPs proteome and explore the clinical application of plasma EVPs proteins for developing disease biomarkers.

Keywords:  Extracellular vesicles and particles; Mass spectrometry; Plasma

DOI:  https://doi.org/10.1007/978-1-0716-2978-9_19
Cell Death Dis. 2023 Feb 15. 14(2): 129

Harnessing the reverse cholesterol transport pathway to favor differentiation of monocyte-derived APCs and antitumor responses.

Laura Raccosta, Maura Marinozzi, Susan Costantini, Daniela Maggioni, Lorena Maria Ferreira, Gianfranca Corna, Paola Zordan, Angela Sorice, Diego Farinello, Silvia Bianchessi, Michela Riba, Dejan Lazarevic, Paolo Provero, Matthias Mack, Attilio Bondanza, Ivan Nalvarte, J-A Gustafsson, Valeria Ranzani, Francesco De Sanctis, Stefano Ugel, Silvère Baron, Jean-Marc A Lobaccaro, Lorenzo Pontini, Manuela Pacciarini, Catia Traversari, Massimiliano Pagani, Vincenzo Bronte, Giovanni Sitia, Per Antonson, Andrea Brendolan, Alfredo Budillon, Vincenzo Russo.

Lipid and cholesterol metabolism play a crucial role in tumor cell behavior and in shaping the tumor microenvironment. In particular, enzymatic and non-enzymatic cholesterol metabolism, and derived metabolites control dendritic cell (DC) functions, ultimately impacting tumor antigen presentation within and outside the tumor mass, dampening tumor immunity and immunotherapeutic attempts. The mechanisms accounting for such events remain largely to be defined. Here we perturbed (oxy)sterol metabolism genetically and pharmacologically and analyzed the tumor lipidome landscape in relation to the tumor-infiltrating immune cells. We report that perturbing the lipidome of tumor microenvironment by the expression of sulfotransferase 2B1b crucial in cholesterol and oxysterol sulfate synthesis, favored intratumoral representation of monocyte-derived antigen-presenting cells, including monocyte-DCs. We also found that treating mice with a newly developed antagonist of the oxysterol receptors Liver X Receptors (LXRs), promoted intratumoral monocyte-DC differentiation, delayed tumor growth and synergized with anti-PD-1 immunotherapy and adoptive T cell therapy. Of note, looking at LXR/cholesterol gene signature in melanoma patients treated with anti-PD-1-based immunotherapy predicted diverse clinical outcomes. Indeed, patients whose tumors were poorly infiltrated by monocytes/macrophages expressing LXR target genes showed improved survival over the course of therapy. Thus, our data support a role for (oxy)sterol metabolism in shaping monocyte-to-DC differentiation, and in tumor antigen presentation critical for responsiveness to immunotherapy. The identification of a new LXR antagonist opens new treatment avenues for cancer patients.

DOI: https://doi.org/10.1038/s41419-023-05620-7
Elife. 2023 Feb 17. pii: e84379. [Epub ahead of print]12

Dynamics of co-substrate pools can constrain and regulate metabolic fluxes.

Robert West, Hadrien Delattre, Elad Noor, Elisenda Feliu, Orkun Soyer.

  Cycling of co-substrates, whereby a metabolite is converted among alternate forms via different reactions, is ubiquitous in metabolism. Several cycled co-substrates are well known as energy and electron carriers (e.g. ATP and NAD(P)H), but there are also other metabolites that act as cycled co-substrates in different parts of central metabolism. Here, we develop a mathematical framework to analyse the effect of co-substrate cycling on metabolic flux. In the cases of a single reaction and linear pathways, we find that co-substrate cycling imposes an additional flux limit on a reaction, distinct to the limit imposed by the kinetics of the primary enzyme catalysing that reaction. Using analytical methods, we show that this additional limit is a function of the total pool size and turnover rate of the cycled co-substrate. Expanding from this insight and using simulations, we show that regulation of these two parameters can allow regulation of flux dynamics in branched and coupled pathways. To support these theoretical insights, we analysed existing flux measurements and enzyme levels from the central carbon metabolism and identified several reactions that could be limited by the dynamics of co-substrate cycling. We discuss how the limitations imposed by co-substrate cycling provide experimentally testable hypotheses on specific metabolic phenotypes. We conclude that measuring and controlling co-substrate dynamics is crucial for understanding and engineering metabolic fluxes in cells.

Keywords:  E. coli; computational biology; systems biology

DOI:  https://doi.org/10.7554/eLife.84379
Int J Biol Sci. 2023 ;19(3): 772-788

Xanthine dehydrogenase rewires metabolism and the survival of nutrient deprived lung adenocarcinoma cells by facilitating UPR and autophagic degradation.

Man-Man Chen, Wei Guo, Si-Meng Chen, Xiao-Zhen Guo, Lan Xu, Xiao-Yu Ma, Yu-Xiang Wang, Cen Xie, Ling-Hua Meng.

  Xanthine dehydrogenase (XDH) is the rate-limiting enzyme in purine catabolism by converting hypoxanthine to xanthine and xanthine to uric acid. The altered expression and activity of XDH are associated with the development and prognosis of multiple types of cancer, while its role in lung adenocarcinoma (LUAD) remains unknown. Herein, we demonstrated that XDH was highly expressed in LUAD and was significantly correlated with poor prognosis. Though inhibition of XDH displayed moderate effect on the viability of LUAD cells cultured in the complete medium, it significantly attenuated the survival of starved cells. Similar results were obtained in XDH-knockout cells. Nucleosides supplementation rescued the survival of starved LUAD cells upon XDH inhibition, while inhibition of purine nucleoside phosphorylase abrogated the process, indicating that nucleoside degradation is required for the XDH-mediated survival of LUAD cells. Accordingly, metabolic flux revealed that ribose derived from nucleoside fueled key carbon metabolic pathways to sustain the survival of starved LUAD cells. Mechanistically, down-regulation of XDH suppressed unfolded protein response (UPR) and autophagic flux in starved LUAD cells. Inhibition of XDH decreased the level of amino acids produced by autophagic degradation, which was accompanied with down-regulation of mTORC1 signaling. Supplementation of amino acids including glutamine or glutamate rescued the survival of starved LUAD cells upon knockout or inhibition of XDH. Finally, XDH inhibitors potentiated the anti-cancer activity of 2-deoxy-D-glucose that induced UPR and/or autophagy in vitro and in vivo. In summary, XDH plays a crucial role in the survival of starved LUAD cells and targeting XDH may improve the efficacy of drugs that induce UPR and autophagy in the therapy of LUAD.

Keywords:  LUAD; UPR; Xanthine dehydrogenase; autophagy; cell survival; nucleoside degradation

DOI:  https://doi.org/10.7150/ijbs.78948
Biochem Cell Biol. 2023 Feb 14.

Lipid induced alteration in retinoic acid signaling leads to mitochondrial dysfunction in HepG2 and Huh7 cells.

Eshani Karmakar, Nabanita Das, Bidisha Mukherjee, Prosenjit Das, Satinath Mukhopadhyay, Sib Sankar Roy.

A surfeit of mitochondrial ROS and inflammation serve as obligatory mediators of lipid-associated hepatocellular maladies. While retinoid homeostasis is essential in restoring systemic energy balance, its role in hepatic mitochondrial function remains elusive. The role of Lecithin: Retinol Acyl Transferase (LRAT) in maintenance of retinoid homeostasis is appreciated earlier, however its role in modulating retinoic acid (RA) bioavailability upon lipid-imposition is unexplored. We identified LRAT overexpression in HFD-fed rats and palmitate-treated hepatoma cells. Elevation in LRAT expression depletes RA production and deregulates RA signaling. This altered RA metabolism enhances fat accumulation, accompanied by inflammation that leads to impaired mitochondrial function through enhanced ROS generation. Hence, LRAT inhibition could be a novel approach preventing lipid-induced mitochondrial dysfunction in hepatoma cells.

DOI: https://doi.org/10.1139/bcb-2022-0266
Nat Microbiol. 2023 Feb 16.

Metabolic heterogeneity and cross-feeding within isogenic yeast populations captured by DILAC.

Stephan Kamrad, Clara Correia-Melo, Lukasz Szyrwiel, Simran Kaur Aulakh, Jürg Bähler, Vadim Demichev, Michael Mülleder, Markus Ralser.

Genetically identical cells are known to differ in many physiological parameters such as growth rate and drug tolerance. Metabolic specialization is believed to be a cause of such phenotypic heterogeneity, but detection of metabolically divergent subpopulations remains technically challenging. We developed a proteomics-based technology, termed differential isotope labelling by amino acids (DILAC), that can detect producer and consumer subpopulations of a particular amino acid within an isogenic cell population by monitoring peptides with multiple occurrences of the amino acid. We reveal that young, morphologically undifferentiated yeast colonies contain subpopulations of lysine producers and consumers that emerge due to nutrient gradients. Deconvoluting their proteomes using DILAC, we find evidence for in situ cross-feeding where rapidly growing cells ferment and provide the more slowly growing, respiring cells with ethanol. Finally, by combining DILAC with fluorescence-activated cell sorting, we show that the metabolic subpopulations diverge phenotypically, as exemplified by a different tolerance to the antifungal drug amphotericin B. Overall, DILAC captures previously unnoticed metabolic heterogeneity and provides experimental evidence for the role of metabolic specialization and cross-feeding interactions as a source of phenotypic heterogeneity in isogenic cell populations.

DOI: https://doi.org/10.1038/s41564-022-01304-8