bims-mepmim 2024-03-31 papers

bims-mepmim

Biomed News

on Metabolites in pathological microenvironments and immunometabolism

Issue of 2024–03–31
twenty-one papers selected by
Erika Mariana Palmieri, NIH/NCI Laboratory of Cancer ImmunoMetabolism

Pyrimidines maintain mitochondrial pyruvate oxidation to support de novo lipogenesis.
Rab4A-directed endosome traffic shapes pro-inflammatory mitochondrial metabolism in T cells via mitophagy, CD98 expression, and kynurenine-sensitive mTOR activation.
Acetyl-CoA synthetase 2 contributes to a better prognosis for liver cancer by switching acetate-glucose metabolism.
Acod1 expression in cancer cells promotes immune evasion through the generation of inhibitory peptides.
Fetal liver macrophages contribute to the hematopoietic stem cell niche by controlling granulopoiesis.
Thioredoxin is a metabolic rheostat controlling regulatory B cells.
Cholesterol trafficking to the ER leads to activation of CaMKII/JNK/NLRP3 and promotes atherosclerosis.
ATF4-SLC7A11-GSH axis mediates the acquisition of immunosuppressive properties by activated CD4+ T cells in low arginine condition.
Naked mole-rats have distinctive cardiometabolic and genetic adaptations to their underground low-oxygen lifestyles.
The constitutively active form of a key cholesterol synthesis enzyme is lipid droplet-localized and upregulated in endometrial cancer tissues.
iNOS is necessary for GBP-mediated T. gondii clearance in murine macrophages via vacuole nitration and intravacuolar network collapse.
BCAA metabolism in pancreatic cancer affects lipid balance by regulating fatty acid import into mitochondria.
Transketolase promotes MAFLD by limiting inosine-induced mitochondrial activity.
Mass spectrometry imaging protocol for spatial mapping of lipids, N-glycans and peptides in murine lung tissue.
Stem cell-derived vessels-on-chip for cardiovascular disease modeling.
The pleiotropic functions of reactive oxygen species in cancer.
A stress sensor, IRE1α, is required for bacterial-exotoxin-induced interleukin-1β production in tissue-resident macrophages.
Nitric oxide and tumor necrosis factor-⍺ levels are negatively correlated in endotoxin tolerance recovery in vitro.
TRIM13 reduces cholesterol efflux and increases oxidized LDL uptake leading to foam cell formation and atherosclerosis.
Adhesion energy controls lipid binding-mediated endocytosis.
A palmitoylation-depalmitoylation relay spatiotemporally controls GSDMD activation in pyroptosis.

Science. 2024 Mar 29. 383(6690): 1484-1492

Pyrimidines maintain mitochondrial pyruvate oxidation to support de novo lipogenesis.

Umakant Sahu, Elodie Villa, Colleen R Reczek, Zibo Zhao, Brendan P O'Hara, Michael D Torno, Rohan Mishra, William D Shannon, John M Asara, Peng Gao, Ali Shilatifard, Navdeep S Chandel, Issam Ben-Sahra.

Cellular purines, particularly adenosine 5'-triphosphate (ATP), fuel many metabolic reactions, but less is known about the direct effects of pyrimidines on cellular metabolism. We found that pyrimidines, but not purines, maintain pyruvate oxidation and the tricarboxylic citric acid (TCA) cycle by regulating pyruvate dehydrogenase (PDH) activity. PDH activity requires sufficient substrates and cofactors, including thiamine pyrophosphate (TPP). Depletion of cellular pyrimidines decreased TPP synthesis, a reaction carried out by TPP kinase 1 (TPK1), which reportedly uses ATP to phosphorylate thiamine (vitamin B1). We found that uridine 5'-triphosphate (UTP) acts as the preferred substrate for TPK1, enabling cellular TPP synthesis, PDH activity, TCA-cycle activity, lipogenesis, and adipocyte differentiation. Thus, UTP is required for vitamin B1 utilization to maintain pyruvate oxidation and lipogenesis.

DOI: https://doi.org/10.1126/science.adh2771
Nat Commun. 2024 Mar 22. 15(1): 2598

Rab4A-directed endosome traffic shapes pro-inflammatory mitochondrial metabolism in T cells via mitophagy, CD98 expression, and kynurenine-sensitive mTOR activation.

Nick Huang, Thomas Winans, Brandon Wyman, Zachary Oaks, Tamas Faludi, Gourav Choudhary, Zhi-Wei Lai, Joshua Lewis, Miguel Beckford, Manuel Duarte, Daniel Krakko, Akshay Patel, Joy Park, Tiffany Caza, Mahsa Sadeghzadeh, Laurence Morel, Mark Haas, Frank Middleton, Katalin Banki, Andras Perl.

Activation of the mechanistic target of rapamycin (mTOR) is a key metabolic checkpoint of pro-inflammatory T-cell development that contributes to the pathogenesis of autoimmune diseases, such as systemic lupus erythematosus (SLE), however, the underlying mechanisms remain poorly understood. Here, we identify a functional role for Rab4A-directed endosome traffic in CD98 receptor recycling, mTOR activation, and accumulation of mitochondria that connect metabolic pathways with immune cell lineage development and lupus pathogenesis. Based on integrated analyses of gene expression, receptor traffic, and stable isotope tracing of metabolic pathways, constitutively active Rab4AQ72L exerts cell type-specific control over metabolic networks, dominantly impacting CD98-dependent kynurenine production, mTOR activation, mitochondrial electron transport and flux through the tricarboxylic acid cycle and thus expands CD4+ and CD3+CD4-CD8- double-negative T cells over CD8+ T cells, enhancing B cell activation, plasma cell development, antinuclear and antiphospholipid autoantibody production, and glomerulonephritis in lupus-prone mice. Rab4A deletion in T cells and pharmacological mTOR blockade restrain CD98 expression, mitochondrial metabolism and lineage skewing and attenuate glomerulonephritis. This study identifies Rab4A-directed endosome traffic as a multilevel regulator of T cell lineage specification during lupus pathogenesis.

DOI: https://doi.org/10.1038/s41467-024-46441-2
Exp Mol Med. 2024 Mar 25.

Acetyl-CoA synthetase 2 contributes to a better prognosis for liver cancer by switching acetate-glucose metabolism.

Kyung Hee Jung, Sujin Lee, Han Sun Kim, Jin-Mo Kim, Yun Ji Lee, Min Seok Park, Myeong-Seong Seo, Misu Lee, Mijin Yun, Sunghyouk Park, Soon-Sun Hong.

Acetyl-CoA synthetase 2 (ACSS2)-dependent acetate usage has generally been associated with tumorigenesis and increased malignancy in cancers under nutrient-depleted conditions. However, the nutrient usage and metabolic characteristics of the liver differ from those of other organs; therefore, the mechanism of ACSS2-mediated acetate metabolism may also differ in liver cancer. To elucidate the underlying mechanisms of ACSS2 in liver cancer and acetate metabolism, the relationships between patient acetate uptake and metabolic characteristics and between ACSS2 and tumor malignancies were comprehensively studied in vitro, in vivo and in humans. Clinically, we initially found that ACSS2 expression was decreased in liver cancer patients. Moreover, PET-CT imaging confirmed that lower-grade cancer cells take up more 11C-acetate but less 18F-fluorodeoxyglucose (18F-FDG); however, this trend was reversed in higher-grade cancer. Among liver cancer cells, those with high ACSS2 expression avidly absorbed acetate even in a glucose-sufficient environment, whereas those with low ACSS2 expression did not, thereby showing correlations with their respective ACSS2 expression. Metabolomic isotope tracing in vitro and in vivo revealed greater acetate incorporation, greater lipid anabolic metabolism, and less malignancy in high-ACSS2 tumors. Notably, ACSS2 downregulation in liver cancer cells was associated with increased tumor occurrence in vivo. In human patient cohorts, patients in the low-ACSS2 subgroup exhibited reduced anabolism, increased glycolysis/hypoxia, and poorer prognosis. We demonstrated that acetate uptake by ACSS2 in liver cancer is independent of glucose depletion and contributes to lipid anabolic metabolism and reduced malignancy, thereby leading to a better prognosis for liver cancer patients.

DOI: https://doi.org/10.1038/s12276-024-01185-3
Cell Rep. 2024 Mar 22. pii: S2211-1247(24)00312-7. [Epub ahead of print]43(4): 113984

Acod1 expression in cancer cells promotes immune evasion through the generation of inhibitory peptides.

James H Schofield, Joseph Longo, Ryan D Sheldon, Emma Albano, Abigail E Ellis, Mark A Hawk, Sean Murphy, Loan Duong, Sharif Rahmy, Xin Lu, Russell G Jones, Zachary T Schafer.

  Targeting programmed cell death protein 1 (PD-1) is an important component of many immune checkpoint blockade (ICB) therapeutic approaches. However, ICB is not an efficacious strategy in a variety of cancer types, in part due to immunosuppressive metabolites in the tumor microenvironment. Here, we find that αPD-1-resistant cancer cells produce abundant itaconate (ITA) due to enhanced levels of aconitate decarboxylase (Acod1). Acod1 has an important role in the resistance to αPD-1, as decreasing Acod1 levels in αPD-1-resistant cancer cells can sensitize tumors to αPD-1 therapy. Mechanistically, cancer cells with high Acod1 inhibit the proliferation of naive CD8+ T cells through the secretion of inhibitory factors. Surprisingly, inhibition of CD8+ T cell proliferation is not dependent on the secretion of ITA but is instead a consequence of the release of small inhibitory peptides. Our study suggests that strategies to counter the activity of Acod1 in cancer cells may sensitize tumors to ICB therapy.

Keywords:  Acod1; CP: Cancer; CP: Immunology; TCA cycle; immune evasion; itaconate

DOI:  https://doi.org/10.1016/j.celrep.2024.113984
Elife. 2024 Mar 25. pii: e86493. [Epub ahead of print]13

Fetal liver macrophages contribute to the hematopoietic stem cell niche by controlling granulopoiesis.

Amir Hossein Kayvanjoo, Iva Splichalova, David Alejandro Bejarano, Hao Huang, Katharina Mauel, Nikola Makdissi, David Heider, Hui Ming Tew, Nora Reka Balzer, Eric Greto, Collins Osei-Sarpong, Kevin Baßler, Joachim L Schultze, Stefan Uderhardt, Eva Kiermaier, Marc Beyer, Andreas Schlitzer, Elvira Mass.

  During embryogenesis, the fetal liver becomes the main hematopoietic organ, where stem and progenitor cells as well as immature and mature immune cells form an intricate cellular network. Hematopoietic stem cells (HSCs) reside in a specialized niche, which is essential for their proliferation and differentiation. However, the cellular and molecular determinants contributing to this fetal HSC niche remain largely unknown. Macrophages are the first differentiated hematopoietic cells found in the developing liver, where they are important for fetal erythropoiesis by promoting erythrocyte maturation and phagocytosing expelled nuclei. Yet, whether macrophages play a role in fetal hematopoiesis beyond serving as a niche for maturing erythroblasts remains elusive. Here, we investigate the heterogeneity of macrophage populations in the murine fetal liver to define their specific roles during hematopoiesis. Using a single-cell omics approach combined with spatial proteomics and genetic fate-mapping models, we found that fetal liver macrophages cluster into distinct yolk sac-derived subpopulations and that long-term HSCs are interacting preferentially with one of the macrophage subpopulations. Fetal livers lacking macrophages show a delay in erythropoiesis and have an increased number of granulocytes, which can be attributed to transcriptional reprogramming and altered differentiation potential of long-term HSCs. Together, our data provide a detailed map of fetal liver macrophage subpopulations and implicate macrophages as part of the fetal HSC niche.

Keywords:  developmental biology; immunology; inflammation; mouse

DOI:  https://doi.org/10.7554/eLife.86493
Nat Immunol. 2024 Mar 29.

Thioredoxin is a metabolic rheostat controlling regulatory B cells.

Hannah F Bradford, Thomas C R McDonnell, Alexander Stewart, Andrew Skelton, Joseph Ng, Zara Baig, Franca Fraternali, Deborah Dunn-Walters, David A Isenberg, Adnan R Khan, Claudio Mauro, Claudia Mauri.

Metabolic programming is important for B cell fate, but the bioenergetic requirement for regulatory B (Breg) cell differentiation and function is unknown. Here we show that Breg cell differentiation, unlike non-Breg cells, relies on mitochondrial electron transport and homeostatic levels of reactive oxygen species (ROS). Single-cell RNA sequencing analysis revealed that TXN, encoding the metabolic redox protein thioredoxin (Trx), is highly expressed by Breg cells, unlike Trx inhibitor TXNIP which was downregulated. Pharmacological inhibition or gene silencing of TXN resulted in mitochondrial membrane depolarization and increased ROS levels, selectively suppressing Breg cell differentiation and function while favoring pro-inflammatory B cell differentiation. Patients with systemic lupus erythematosus (SLE), characterized by Breg cell deficiencies, present with B cell mitochondrial membrane depolarization, elevated ROS and fewer Trx+ B cells. Exogenous Trx stimulation restored Breg cells and mitochondrial membrane polarization in SLE B cells to healthy B cell levels, indicating Trx insufficiency underlies Breg cell impairment in patients with SLE.

DOI: https://doi.org/10.1038/s41590-024-01798-w
J Lipid Res. 2024 Mar 22. pii: S0022-2275(24)00039-7. [Epub ahead of print] 100534

Cholesterol trafficking to the ER leads to activation of CaMKII/JNK/NLRP3 and promotes atherosclerosis.

Mustafa Yalcinkaya, Wenli Liu, Tong Xiao, Sandra Abramowicz, Ranran Wang, Nan Wang, Marit Westerterp, Alan R Tall.

  The deposition of cholesterol-rich lipoproteins in the arterial wall triggers macrophage inflammatory responses, which promote atherosclerosis. The NLRP3 inflammasome aggravates atherosclerosis; however, cellular mechanisms connecting macrophage cholesterol accumulation to inflammasome activation are poorly understood. We investigated the mechanisms of NLRP3 inflammasome activation in cholesterol loaded macrophages and in atherosclerosis-prone Ldlr-/- mice with defects in macrophage cholesterol efflux. We found that accumulation of cholesterol in macrophages treated with modified LDL or cholesterol crystals, or in macrophages defective in the cholesterol efflux promoting transporters ABCA1 and ABCG1, leads to activation of NLRP3 inflammasomes as a result of increased cholesterol trafficking from the plasma membrane to the ER, via Aster-B. In turn, the accumulation of cholesterol in the ER activates the inositol triphosphate-3 receptor, CaMKII/JNK and induces NLRP3 deubiquitylation by BRCC3. An NLRP3 deubiquitylation inhibitor or deficiency of Abro1, an essential scaffolding protein in the BRCC3-containing cytosolic complex, suppressed inflammasome activation, neutrophil extracellular trap formation (NETosis) and atherosclerosis in vivo. These results identify a link between trafficking of cholesterol to the ER, NLRP3 deubiquitylation, inflammasome activation and atherosclerosis.

Keywords:  CaMKII; Cholesterol; ER; JNK; NLRP3 inflammasome

DOI:  https://doi.org/10.1016/j.jlr.2024.100534
Cell Rep. 2024 Mar 23. pii: S2211-1247(24)00323-1. [Epub ahead of print]43(4): 113995

ATF4-SLC7A11-GSH axis mediates the acquisition of immunosuppressive properties by activated CD4+ T cells in low arginine condition.

Ziqi Zou, Qian Cheng, Jiajie Zhou, Chenyao Guo, Andreas V Hadjinicolaou, Mariolina Salio, Xinghua Liang, Cuiyu Yang, Yue Du, Weiran Yao, Dongrui Wang, Vincenzo Cerundolo, Qingqing Wang, Meng Xia.

  The tumor microenvironment (TME) is restricted in metabolic nutrients including the semi-essential amino acid arginine. While complete arginine deprivation causes T cell dysfunction, it remains unclear how arginine levels fluctuate in the TME to shape T cell fates. Here, we find that the 20-μM low arginine condition, representing the levels found in the plasma of patients with cancers, confers Treg-like immunosuppressive capacities upon activated T cells. In vivo mouse tumor models and human single-cell RNA-sequencing datasets reveal positive correlations between low arginine condition and intratumoral Treg accumulation. Mechanistically, low arginine-activated T cells engage in metabolic and transcriptional reprogramming, using the ATF4-SLC7A11-GSH axis, to preserve their suppressive function. These findings improve our understanding of the role of arginine in human T cell biology with potential applications for immunotherapy strategies.

Keywords:  CP: Immunology; SLC7A11; T cells; arginine; immunosuppression; tumor microenvironment

DOI:  https://doi.org/10.1016/j.celrep.2024.113995
Nat Commun. 2024 Mar 27. 15(1): 2204

Naked mole-rats have distinctive cardiometabolic and genetic adaptations to their underground low-oxygen lifestyles.

Chris G Faulkes, Thomas R Eykyn, Jan Lj Miljkovic, James D Gilbert, Rebecca L Charles, Hiran A Prag, Nikayla Patel, Daniel W Hart, Michael P Murphy, Nigel C Bennett, Dunja Aksentijevic.

The naked mole-rat Heterocephalus glaber is a eusocial mammal exhibiting extreme longevity (37-year lifespan), extraordinary resistance to hypoxia and absence of cardiovascular disease. To identify the mechanisms behind these exceptional traits, metabolomics and RNAseq of cardiac tissue from naked mole-rats was compared to other African mole-rat genera (Cape, Cape dune, Common, Natal, Mahali, Highveld and Damaraland mole-rats) and evolutionarily divergent mammals (Hottentot golden mole and C57/BL6 mouse). We identify metabolic and genetic adaptations unique to naked mole-rats including elevated glycogen, thus enabling glycolytic ATP generation during cardiac ischemia. Elevated normoxic expression of HIF-1α is observed while downstream hypoxia responsive-genes are down-regulated, suggesting adaptation to low oxygen environments. Naked mole-rat hearts show reduced succinate levels during ischemia compared to C57/BL6 mouse and negligible tissue damage following ischemia-reperfusion injury. These evolutionary traits reflect adaptation to a unique hypoxic and eusocial lifestyle that collectively may contribute to their longevity and health span.

DOI: https://doi.org/10.1038/s41467-024-46470-x
J Biol Chem. 2024 Mar 25. pii: S0021-9258(24)01729-0. [Epub ahead of print] 107232

The constitutively active form of a key cholesterol synthesis enzyme is lipid droplet-localized and upregulated in endometrial cancer tissues.

Hudson W Coates, Tina B Nguyen, Ximing Du, Ellen M Olzomer, Rhonda Farrell, Frances L Byrne, Hongyuan Yang, Andrew J Brown.

  Cholesterol is essential for both normal cell viability and cancer cell proliferation. Aberrant activity of squalene monooxygenase (SM, also known as squalene epoxidase or SQLE), the rate-limiting enzyme of the committed cholesterol synthesis pathway, is accordingly implicated in a growing list of cancers. We previously reported that hypoxia triggers the truncation of SM to a constitutively active form, thus preserving sterol synthesis during oxygen shortfalls. Here, we show SM truncation is upregulated and correlates with the magnitude of hypoxia in endometrial cancer tissues, supporting the in vivo relevance of our earlier work. To further investigate the pathophysiological consequences of SM truncation, we examine using complementary immunofluorescence and cell fractionation approaches its lipid droplet-localized pool and find that it exclusively comprises the truncated enzyme. This partitioning is facilitated by the loss of an endoplasmic reticulum-embedded region at the SM N-terminus, whereas the catalytic domain containing membrane-associated C-terminal helices is spared. Moreover, we determined multiple amphipathic helices contribute to the lipid droplet localization of trunSM. Taken together, our results expand on the striking differences between the two forms of SM and suggest upregulated truncation may contribute to SM-related oncogenesis.

Keywords:  cancer; cholesterol; lipid droplets; protein degradation; squalene monooxygenase

DOI:  https://doi.org/10.1016/j.jbc.2024.107232
Nat Commun. 2024 Mar 27. 15(1): 2698

iNOS is necessary for GBP-mediated T. gondii clearance in murine macrophages via vacuole nitration and intravacuolar network collapse.

Xiao-Yu Zhao, Samantha L Lempke, Jan C Urbán Arroyo, Isabel G Brown, Bocheng Yin, Magdalena M Magaj, Nadia K Holness, Jamison Smiley, Stefanie Redemann, Sarah E Ewald.

Toxoplasma gondii is an obligate intracellular parasite of rodents and humans. Interferon-inducible guanylate binding proteins (GBPs) are mediators of T. gondii clearance, however, this mechanism is incomplete. Here, using automated spatially targeted optical micro proteomics we demonstrate that inducible nitric oxide synthetase (iNOS) is highly enriched at GBP2+ parasitophorous vacuoles (PV) in murine macrophages. iNOS expression in macrophages is necessary to limit T. gondii load in vivo and in vitro. Although iNOS activity is dispensable for GBP2 recruitment and PV membrane ruffling; parasites can replicate, egress and shed GBP2 when iNOS is inhibited. T. gondii clearance by iNOS requires nitric oxide, leading to nitration of the PV and collapse of the intravacuolar network of membranes in a chromosome 3 GBP-dependent manner. We conclude that reactive nitrogen species generated by iNOS cooperate with GBPs to target distinct structures in the PV that are necessary for optimal parasite clearance in macrophages.

DOI: https://doi.org/10.1038/s41467-024-46790-y
Cancer Metab. 2024 Mar 26. 12(1): 10

BCAA metabolism in pancreatic cancer affects lipid balance by regulating fatty acid import into mitochondria.

Klára Gotvaldová, Jitka Špačková, Jiří Novotný, Kamila Baslarová, Petr Ježek, Lenka Rossmeislová, Jan Gojda, Katarína Smolková.

   BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) has been associated with the host dysmetabolism of branched-chain amino acids (BCAAs), however, the implications for the role of BCAA metabolism in PDAC development or progression are not clear. The mitochondrial catabolism of valine, leucine, and isoleucine is a multistep process leading to the production of short-chain R-CoA species. They can be subsequently exported from mitochondria as short-chain carnitines (SC-CARs), utilized in anabolic pathways, or released from the cells.
METHODS: We examined the specificities of BCAA catabolism and cellular adaptation strategies to BCAA starvation in PDAC cells in vitro. We used metabolomics and lipidomics to quantify major metabolic changes in response to BCAA withdrawal. Using confocal microscopy and flow cytometry we quantified the fluorescence of BODIPY probe and the level of lipid droplets (LDs). We used BODIPY-conjugated palmitate to evaluate transport of fatty acids (FAs) into mitochondria. Also, we have developed a protocol for quantification of SC-CARs, BCAA-derived metabolites.
RESULTS: Using metabolic profiling, we found that BCAA starvation leads to massive triglyceride (TG) synthesis and LD accumulation. This was associated with the suppression of activated FA transport into the mitochondrial matrix. The suppression of FA import into mitochondria was rescued with the inhibitor of the acetyl-CoA carboxylase (ACC) and the activator of AMP kinase (AMPK), which both regulate carnitine palmitoyltransferase 1A (CPT1) activation status.
CONCLUSIONS: Our data suggest that BCAA catabolism is required for the import of long chain carnitines (LC-CARs) into mitochondria, whereas the disruption of this link results in the redirection of activated FAs into TG synthesis and its deposition into LDs. We propose that this mechanism protects cells against mitochondrial overload with LC-CARs and it might be part of the universal reaction to amino acid perturbations during cancer growth, regulating FA handling and storage.

Keywords:  BCAA metabolism; Fatty acid/Transport; Fluorescence microscopy; Lipid droplets; Lipidomics; Mitochondria; Pancreatic cancer; Triglycerides

DOI:  https://doi.org/10.1186/s40170-024-00335-5
Cell Metab. 2024 Mar 23. pii: S1550-4131(24)00082-2. [Epub ahead of print]

Transketolase promotes MAFLD by limiting inosine-induced mitochondrial activity.

Lingfeng Tong, Zhangbing Chen, Yangyang Li, Xinxia Wang, Changjie Yang, Yakui Li, Yemin Zhu, Ying Lu, Qi Liu, Nannan Xu, Sijia Shao, Lifang Wu, Ping Zhang, Guangyu Wu, Xiaoyu Wu, Xiaosong Chen, Junwei Fang, Renbing Jia, Tianle Xu, Bin Li, Liang Zheng, Junling Liu, Xuemei Tong.

  Metabolic dysfunction-associated fatty liver disease (MAFLD) has a global prevalence of about 25% and no approved therapy. Using metabolomic and proteomic analyses, we identified high expression of hepatic transketolase (TKT), a metabolic enzyme of the pentose phosphate pathway, in human and mouse MAFLD. Hyperinsulinemia promoted TKT expression through the insulin receptor-CCAAT/enhancer-binding protein alpha axis. Utilizing liver-specific TKT overexpression and knockout mouse models, we demonstrated that TKT was sufficient and required for MAFLD progression. Further metabolic flux analysis revealed that Tkt deletion increased hepatic inosine levels to activate the protein kinase A-cAMP response element binding protein cascade, promote phosphatidylcholine synthesis, and improve mitochondrial function. Moreover, insulin induced hepatic TKT to limit inosine-dependent mitochondrial activity. Importantly, N-acetylgalactosamine (GalNAc)-siRNA conjugates targeting hepatic TKT showed promising therapeutic effects on mouse MAFLD. Our study uncovers how hyperinsulinemia regulates TKT-orchestrated inosine metabolism and mitochondrial function and provides a novel therapeutic strategy for MAFLD prevention and treatment.

Keywords:  GalNAc-siRNA conjugates; MAFLD; inosine; metabolic dysfunction-associated fatty liver disease; mitochondrial function; transketolase

DOI:  https://doi.org/10.1016/j.cmet.2024.03.003
Rapid Commun Mass Spectrom. 2024 May 15. 38(9): e9721

Mass spectrometry imaging protocol for spatial mapping of lipids, N-glycans and peptides in murine lung tissue.

Yuen T Ngai, Matthew T Briggs, Parul Mittal, Clifford Young, Emma Parkinson-Lawrence, Manuela Klingler-Hoffmann, Sandra Orgeig, Peter Hoffmann.

RATIONALE: The application of matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to murine lungs is challenging due to the spongy nature of the tissue. Lungs consist of interconnected air sacs (alveoli) lined by a single layer of flattened epithelial cells, which requires inflation to maintain its natural structure. Therefore, a protocol that is compatible with both lung instillation and high spatial resolution is essential to enable multi-omic studies on murine lung disease models using MALDI-MSI.
METHODS AND RESULTS: To maintain the structural integrity of the tissue, murine lungs were inflated with 8% (w/v) gelatin for lipid MSI of fresh frozen tissues or 4% (v/v) paraformaldehyde neutral buffer for N-glycan and peptide MSI of FFPE tissues. Tissues were sectioned and prepared for enzymatic digestion and/or matrix deposition. Glycerol-free PNGase F was applied for N-glycan MSI, while Trypsin Gold was applied for peptide MSI using the iMatrixSpray and ImagePrep Station, respectively. For lipid, N-glycan and peptide MSI, α-cyano-4-hydroxycinnamic acid matrix was deposited using the iMatrixSpray. MS data were acquired with 20 μm spatial resolution using a timsTOF fleX MS instrument followed by MS fragmentation of lipids, N-glycans and peptides. For lipid MSI, trapped ion mobility spectrometry was used to separate isomeric/isobaric lipid species. SCiLS™ Lab was used to visualize all MSI data. For analyte identification, MetaboScape®, GlycoMod and Mascot were used to annotate MS fragmentation spectra of lipids, N-glycans and tryptic peptides, respectively.
CONCLUSIONS: Our protocol provides instructions on sample preparation for high spatial resolution MALDI-MSI, MS/MS data acquisition and lipid, N-glycan and peptide annotation and identification from murine lungs. This protocol will allow non-biased analyses of diseased lungs from preclinical murine models and provide further insight into disease models.

DOI: https://doi.org/10.1002/rcm.9721
Cell Rep. 2024 Mar 26. pii: S2211-1247(24)00336-X. [Epub ahead of print]43(4): 114008

Stem cell-derived vessels-on-chip for cardiovascular disease modeling.

Maren Marder, Caroline Remmert, Julius A Perschel, Munkhtur Otgonbayar, Christine von Toerne, Stefanie Hauck, Judith Bushe, Annette Feuchtinger, Bilal Sheikh, Michel Moussus, Matthias Meier.

  The metabolic syndrome is accompanied by vascular complications. Human in vitro disease models are hence required to better understand vascular dysfunctions and guide clinical therapies. Here, we engineered an open microfluidic vessel-on-chip platform that integrates human pluripotent stem cell-derived endothelial cells (SC-ECs). The open microfluidic design enables seamless integration with state-of-the-art analytical technologies, including single-cell RNA sequencing, proteomics by mass spectrometry, and high-resolution imaging. Beyond previous systems, we report SC-EC maturation by means of barrier formation, arterial toning, and high nitric oxide synthesis levels under gravity-driven flow. Functionally, we corroborate the hallmarks of early-onset atherosclerosis with low sample volumes and cell numbers under flow conditions by determining proteome and secretome changes in SC-ECs stimulated with oxidized low-density lipoprotein and free fatty acids. More broadly, our organ-on-chip platform enables the modeling of patient-specific human endothelial tissue and has the potential to become a general tool for animal-free vascular research.

Keywords:  CP: Stem cell research; atherosclerosis; disease modeling; hiPSC-derived endothelial cells; open microfluidics; single-cell sequencing; vessel-on-chip

DOI:  https://doi.org/10.1016/j.celrep.2024.114008
Nat Cancer. 2024 Mar;5(3): 384-399

The pleiotropic functions of reactive oxygen species in cancer.

Katherine Wu, Ahmed Ezat El Zowalaty, Volkan I Sayin, Thales Papagiannakopoulos.

Cellular redox homeostasis is an essential, dynamic process that ensures the balance between reducing and oxidizing reactions within cells and thus has implications across all areas of biology. Changes in levels of reactive oxygen species can disrupt redox homeostasis, leading to oxidative or reductive stress that contributes to the pathogenesis of many malignancies, including cancer. From transformation and tumor initiation to metastatic dissemination, increasing reactive oxygen species in cancer cells can paradoxically promote or suppress the tumorigenic process, depending on the extent of redox stress, its spatiotemporal characteristics and the tumor microenvironment. Here we review how redox regulation influences tumorigenesis, highlighting therapeutic opportunities enabled by redox-related alterations in cancer cells.

DOI: https://doi.org/10.1038/s43018-024-00738-9
Cell Rep. 2024 Mar 22. pii: S2211-1247(24)00309-7. [Epub ahead of print]43(4): 113981

A stress sensor, IRE1α, is required for bacterial-exotoxin-induced interleukin-1β production in tissue-resident macrophages.

Izumi Sasaki, Yuri Fukuda-Ohta, Chihiro Nakai, Naoko Wakaki-Nishiyama, Chizuyo Okamoto, Daisuke Okuzaki, Shuhei Morita, Shiori Kaji, Yuki Furuta, Hiroaki Hemmi, Takashi Kato, Asumi Yamamoto, Emi Tosuji, Shin-Ichiroh Saitoh, Takashi Tanaka, Katsuaki Hoshino, Shinji Fukuda, Kensuke Miyake, Etsushi Kuroda, Ken J Ishii, Takao Iwawaki, Koichi Furukawa, Tsuneyasu Kaisho.

  Cholera toxin (CT), a bacterial exotoxin composed of one A subunit (CTA) and five B subunits (CTB), functions as an immune adjuvant. CTB can induce production of interleukin-1β (IL-1β), a proinflammatory cytokine, in synergy with a lipopolysaccharide (LPS), from resident peritoneal macrophages (RPMs) through the pyrin and NLRP3 inflammasomes. However, how CTB or CT activates these inflammasomes in the macrophages has been unclear. Here, we clarify the roles of inositol-requiring enzyme 1 alpha (IRE1α), an endoplasmic reticulum (ER) stress sensor, in CT-induced IL-1β production in RPMs. In RPMs, CTB is incorporated into the ER and induces ER stress responses, depending on GM1, a cell membrane ganglioside. IRE1α-deficient RPMs show a significant impairment of CT- or CTB-induced IL-1β production, indicating that IRE1α is required for CT- or CTB-induced IL-1β production in RPMs. This study demonstrates the critical roles of IRE1α in activation of both NLRP3 and pyrin inflammasomes in tissue-resident macrophages.

Keywords:  CP: Cell biology; CP: Immunology; IL-1β; IRE1α; Pyrin inflammasome; resident peritoneal macrophages

DOI:  https://doi.org/10.1016/j.celrep.2024.113981
Nitric Oxide. 2024 Mar 26. pii: S1089-8603(24)00042-9. [Epub ahead of print]

Nitric oxide and tumor necrosis factor-⍺ levels are negatively correlated in endotoxin tolerance recovery in vitro.

Ya-Ying Chang, Yuh-Huey Chao, Wei-Horng Jean, Tzu-Yu Lin, Cheng-Wei Lu.

  Endotoxin tolerance (ET) is the hyporesponsiveness to lipopolysaccharide (LPS) after prior exposure. It is characterized by the downregulation of pro-inflammatory cytokine levels. Although ET protects against inflammation, its abolishment or recovery is critical for immunity. Nitric oxide (NO) plays various roles in the development of ET; however, its specific role in ET recovery remains unknown. To induce ET, RAW264.7 cells (a murine macrophage cell line) were pre-exposed to LPS (LPS1, 100 ng/mL for 24 h) and subsequently re-stimulated with LPS (LPS2, 100 ng/mL for 24 h). Expression of cytokines, NO, nitrite and inducible NO synthase (iNOS) were measured after 0, 12, 24, and 36 h of resting after LPS1 treatment with or without the iNOS-specific inhibitor, 1400W. LPS2-induced tumor necrosis factor-⍺ (TNF-⍺) and interleukin-6 (IL-6) were downregulated after LPS1 treatment, confirming the development of ET. Notably, TNF-⍺ and IL-6 levels spontaneously rebounded after 12-24 h of resting following LPS1 treatment. In contrast, levles of NO, nitrite and iNOS increased during ET development and decreased during ET recovery. Moreover, 1400W inhibited ET development and blocked the early production of NO (<12 h) during ET recovery. Our findings suggest a negative correlation between iNOS-induced NO and cytokine levels in the abolishment of ET.

Keywords:  Endotoxin tolerance; Inducible nitric oxide synthase; Lipopolysaccharide; Nitric oxide; Tumor necrosis factor-⍺

DOI:  https://doi.org/10.1016/j.niox.2024.03.005
J Biol Chem. 2024 Mar 25. pii: S0021-9258(24)01719-8. [Epub ahead of print] 107224

TRIM13 reduces cholesterol efflux and increases oxidized LDL uptake leading to foam cell formation and atherosclerosis.

Suresh Govatati, Raj Kumar, Monoranjan Boro, James G Traylor, A Wayne Orr, Aldons J Lusis, Gadiparthi N Rao.

  Accumulation of lipids within the arterial wall is the key initiating event in the development of atherosclerosis. Impaired cholesterol efflux and/or uptake can influence arterial lipid accumulation. Here, we report that tripartite motif-containing protein 13 (TRIM13), a RING-type E3 ubiquitin ligase, plays a role in arterial lipid accumulation leading to atherosclerosis. Using molecular approaches and knockout mouse model, we found that TRIM13 expression was induced both in the aorta and peritoneal macrophages (pMφ) of ApoE-/- mice in response to western diet (WD) in vivo. Furthermore, proatherogenic cytokine IL-1β also induced TRIM13 expression both in pMφ and vascular smooth muscle cells (VSMCs). Furthermore, we found that TRIM13 via ubiquitination and degradation of LXRα/β downregulates the expression of their target genes ABCA1/G1 and thereby inhibits cholesterol efflux. In addition, TRIM13 by ubiquitinating and degrading SOCS1/3 mediates STAT1 activation, CD36 expression and foam cell formation. In line with these observations, genetic deletion of TRIM13 by rescuing cholesterol efflux and inhibiting foam cell formation protects against diet-induced atherosclerosis. We also found that while TRIM13 and CD36 levels were increased, LXRα/β, ABCA1/G1 and SOCS3 levels were decreased both in Mφ and SMCs of stenotic human coronary arteries as compared to non-stenotic arteries. More intriguingly, the expression levels of TRIM13 and its downstream signaling molecules were correlated with the severity of stenotic lesions. Together, these observations reveal for the first time that TRIM13 plays a crucial role in diet-induced atherosclerosis, and that it could be a potential drug target against this vascular lesion.

Keywords:  ABCA1/G1; LXRα/β; TRIM13; atherosclerosis; cholesterol efflux; foam cell

DOI:  https://doi.org/10.1016/j.jbc.2024.107224
Nat Commun. 2024 Mar 29. 15(1): 2767

Adhesion energy controls lipid binding-mediated endocytosis.

Raluca Groza, Kita Valerie Schmidt, Paul Markus Müller, Paolo Ronchi, Claire Schlack-Leigers, Ursula Neu, Dmytro Puchkov, Rumiana Dimova, Claudia Matthaeus, Justin Taraska, Thomas R Weikl, Helge Ewers.

Several bacterial toxins and viruses can deform membranes through multivalent binding to lipids for clathrin-independent endocytosis. However, it remains unclear, how membrane deformation and endocytic internalization are mechanistically linked. Here we show that many lipid-binding virions induce membrane deformation and clathrin-independent endocytosis, suggesting a common mechanism based on multivalent lipid binding by globular particles. We create a synthetic cellular system consisting of a lipid-anchored receptor in the form of GPI-anchored anti-GFP nanobodies and a multivalent globular binder exposing 180 regularly-spaced GFP molecules on its surface. We show that these globular, 40 nm diameter, particles bind to cells expressing the receptor, deform the plasma membrane upon adhesion and become endocytosed in a clathrin-independent manner. We explore the role of the membrane adhesion energy in endocytosis by using receptors with affinities varying over 7 orders of magnitude. Using this system, we find that once a threshold in adhesion energy is overcome to allow for membrane deformation, endocytosis occurs reliably. Multivalent, binding-induced membrane deformation by globular binders is thus sufficient for internalization to occur and we suggest it is the common, purely biophysical mechanism for lipid-binding mediated endocytosis of toxins and pathogens.

DOI: https://doi.org/10.1038/s41467-024-47109-7
Nat Cell Biol. 2024 Mar 27.

A palmitoylation-depalmitoylation relay spatiotemporally controls GSDMD activation in pyroptosis.

Na Zhang, Jian Zhang, Yuanxin Yang, Hengyue Shan, Shouqiao Hou, Hongwen Fang, Min Ma, Zhongwen Chen, Li Tan, Daichao Xu.

Gasdermin D (GSDMD) is the executor of pyroptosis, which is important for host defence against pathogen infection. Following activation, caspase-mediated cleavage of GSDMD releases an amino-terminal fragment (GSDMD-NT), which oligomerizes and forms pores in the plasma membrane, leading to cell death and release of proinflammatory cytokines. The spatial and temporal regulation of this process in cells remains unclear. Here we identify GSDMD as a substrate for reversible S-palmitoylation on C192 during pyroptosis. The palmitoyl acyltransferase DHHC7 palmitoylates GSDMD to direct its cleavage by caspases. Subsequently, palmitoylation of GSDMD-NT promotes its translocation to the plasma membrane, where APT2 depalmitoylates GSDMD-NT to unmask the C192 residue and promote GSDMD-NT oligomerization. Perturbation of either palmitoylation or depalmitoylation suppresses pyroptosis, leading to increased survival of mice with lipopolysaccharide-induced lethal septic shock and increased sensitivity to bacterial infection. Our findings reveal a model through which a palmitoylation-depalmitoylation relay spatiotemporally controls GSDMD activation during pyroptosis.

DOI: https://doi.org/10.1038/s41556-024-01397-9