bims-mepmim 2023-08-27 papers

bims-mepmim

Biomed News

on Metabolites in pathological microenvironments and immunometabolism

Issue of 2023–08–27
eightteen papers selected by
Erika Mariana Palmieri, NIH/NCI Laboratory of Cancer ImmunoMetabolism

Comprehensive isotopomer analysis of glutamate and aspartate in small tissue samples.
Pyruvate dehydrogenase operates as an intramolecular nitroxyl generator during macrophage metabolic reprogramming.
Metabolic reprogramming contributes to radioprotection by protein kinase Cδ.
Long-chain dicarboxylic acids play a critical role in inducing peroxisomal β-oxidation and hepatic triacylglycerol accumulation.
Glutarate regulates T cell metabolism and anti-tumour immunity.
The Transcription Factor ChREBP Links Mitochondrial Lipidomes to Mitochondrial Morphology and Progression of Diabetic Kidney Disease.
Structural mechanism of mitochondrial membrane remodelling by human OPA1.
Hepatic SREBP signaling requires SPRING to govern systemic lipid metabolism in mice and humans.
Single-cell lipidomics enabled by dual-polarity ionization and ion mobility-mass spectrometry imaging.
RaMALDI: Enabling simultaneous Raman and MALDI imaging of the same tissue section.
Deciphering the decline of metabolic elasticity in aging and obesity.
Glycerol 3-phosphate phosphatase/PGPH-2 counters metabolic stress and promotes healthy aging via a glycogen sensing-AMPK-HLH-30-autophagy axis in C. elegans.
Spatially resolved visualization of reprogrammed metabolism in hepatocellular carcinoma by mass spectrometry imaging.
Allopurinol and oxypurinol differ in their strength and mechanisms of inhibition of xanthine oxidoreductase.
Succinylation of a KEAP1 sensor lysine promotes NRF2 activation.
Alveolar type II epithelial cell FASN maintains lipid homeostasis in experimental COPD.
Peroxisome disruption alters lipid metabolism and potentiates anti-tumor response with MAPK-targeted therapy in melanoma.
The iron chaperone poly C binding protein 1 regulates iron efflux through intestinal ferroportin in mice.

Cell Metab. 2023 Aug 15. pii: S1550-4131(23)00272-3. [Epub ahead of print]

Comprehensive isotopomer analysis of glutamate and aspartate in small tissue samples.

Feng Cai, Divya Bezwada, Ling Cai, Rohit Mahar, Zheng Wu, Mario C Chang, Panayotis Pachnis, Chendong Yang, Sherwin Kelekar, Wen Gu, Bailey Brooks, Bookyung Ko, Hieu S Vu, Thomas P Mathews, Lauren G Zacharias, Misty Martin-Sandoval, Duyen Do, K Celeste Oaxaca, Eunsook S Jin, Vitaly Margulis, Craig R Malloy, Matthew E Merritt, Ralph J DeBerardinis.

  Stable isotopes are powerful tools to assess metabolism. 13C labeling is detected using nuclear magnetic resonance (NMR) spectroscopy or mass spectrometry (MS). MS has excellent sensitivity but generally cannot discriminate among different 13C positions (isotopomers), whereas NMR is less sensitive but reports some isotopomers. Here, we develop an MS method that reports all 16 aspartate and 32 glutamate isotopomers while requiring less than 1% of the sample used for NMR. This method discriminates between pathways that result in the same number of 13C labels in aspartate and glutamate, providing enhanced specificity over conventional MS. We demonstrate regional metabolic heterogeneity within human tumors, document the impact of fumarate hydratase (FH) deficiency in human renal cancers, and investigate the contributions of tricarboxylic acid (TCA) cycle turnover and CO2 recycling to isotope labeling in vivo. This method can accompany NMR or standard MS to provide outstanding sensitivity in isotope-labeling experiments, particularly in vivo.

Keywords:  aspartate; cancer; glutamate; isotopomer; mass spectrometry; nuclear magnetic resonance; pyruvate carboxylase; stable isotope; tricarboxylic acid cycle

DOI:  https://doi.org/10.1016/j.cmet.2023.07.013
Nat Commun. 2023 Aug 22. 14(1): 5114

Pyruvate dehydrogenase operates as an intramolecular nitroxyl generator during macrophage metabolic reprogramming.

Erika M Palmieri, Ronald Holewinski, Christopher L McGinity, Ciro L Pierri, Nunziata Maio, Jonathan M Weiss, Vincenzo Tragni, Katrina M Miranda, Tracey A Rouault, Thorkell Andresson, David A Wink, Daniel W McVicar.

M1 macrophages enter a glycolytic state when endogenous nitric oxide (NO) reprograms mitochondrial metabolism by limiting aconitase 2 and pyruvate dehydrogenase (PDH) activity. Here, we provide evidence that NO targets the PDH complex by using lipoate to generate nitroxyl (HNO). PDH E2-associated lipoate is modified in NO-rich macrophages while the PDH E3 enzyme, also known as dihydrolipoamide dehydrogenase (DLD), is irreversibly inhibited. Mechanistically, we show that lipoate facilitates NO-mediated production of HNO, which interacts with thiols forming irreversible modifications including sulfinamide. In addition, we reveal a macrophage signature of proteins with reduction-resistant modifications, including in DLD, and identify potential HNO targets. Consistently, DLD enzyme is modified in an HNO-dependent manner at Cys477 and Cys484, and molecular modeling and mutagenesis show these modifications impair the formation of DLD homodimers. In conclusion, our work demonstrates that HNO is produced physiologically. Moreover, the production of HNO is dependent on the lipoate-rich PDH complex facilitating irreversible modifications that are critical to NO-dependent metabolic rewiring.

DOI: https://doi.org/10.1038/s41467-023-40738-4
J Biol Chem. 2023 Aug 21. pii: S0021-9258(23)02214-7. [Epub ahead of print] 105186

Metabolic reprogramming contributes to radioprotection by protein kinase Cδ.

Angela M Ohm, Trisiani Affandi, Julie A Reisz, M Cecilia Caino, Angelo D'Alessandro, Mary E Reyland.

  Loss of protein kinase Cδ (PKCδ) activity renders cells resistant to DNA damaging agents, including irradiation, however the mechanism(s) underlying resistance is poorly understood. Here we have asked if metabolic reprogramming by PKCδ contributes to radioprotection. Analysis of global metabolomics showed that depletion of PKCδ affects metabolic pathways that control energy production, and antioxidant, nucleotide, and amino acid biosynthesis. Increased NADPH and nucleotide production in PKCδ depleted cells is associated with upregulation of the pentose phosphate pathway (PPP) as evidenced by increased activation of G6PD and an increase in the nucleotide precursor, 5-phosphoribosyl-1-pyrophosphate. Stable isotope tracing with U-[13C6] glucose showed reduced utilization of glucose for glycolysis in PKCδ depleted cells, and no increase in U-[13C6] glucose incorporation into purines or pyrimidines. In contrast, isotope tracing with [13C5, 15N2] glutamine showed increased utilization of glutamine for synthesis of nucleotides, glutathione and TCA intermediates, and increased incorporation of labelled glutamine into pyruvate and lactate. Using a glycolytic rate assay, we confirmed that anaerobic glycolysis is increased in PKCδ depleted cells; this was accompanied by a reduction in oxidative phosphorylation, as assayed using a mitochondrial stress assay. Importantly, pretreatment of cells with specific inhibitors of the PPP or glutaminase prior to irradiation reversed radioprotection in PKCδ depleted cells, indicating that these cells have acquired co-dependency on the PPP and glutamine for survival. Our studies demonstrate that metabolic reprogramming to increase utilization of glutamine and nucleotide synthesis contributes to radioprotection in the context of PKCδ inhibition.

Keywords:  apoptosis; metabolism; protein kinase Cδ; radioprotection; salivary gland

DOI:  https://doi.org/10.1016/j.jbc.2023.105186
J Biol Chem. 2023 Aug 18. pii: S0021-9258(23)02202-0. [Epub ahead of print] 105174

Long-chain dicarboxylic acids play a critical role in inducing peroxisomal β-oxidation and hepatic triacylglycerol accumulation.

Wei Zhang, Lina Zhang, Haoya Yao, Yaoqing Wang, Xiao Zhang, Lin Shang, Xiaocui Chen, Jia Zeng.

  Recent studies provide evidence that peroxisomal β-oxidation negatively regulates mitochondrial fatty acid oxidation, and induction of peroxisomal β-oxidation causes hepatic lipid accumulation. However, whether there exists a triggering mechanism inducing peroxisomal β-oxidation is not clear. Long-chain dicarboxylic acids are the product of mono fatty acids subjected to ω-oxidation, and both fatty acids ω-oxidation and peroxisomal β-oxidation are induced under ketogenic conditions, indicating there might be a crosstalk between. Here, we revealed that administration of long-chain dicarboxylic acids strongly induces peroxisomal fatty acids β-oxidation and causes hepatic steatosis in mice through the metabolites acetyl-CoA and hydrogen peroxide. Under ketogenic conditions, upregulation of fatty acids ω-oxidation resulted in increased generation of long-chain dicarboxylic acids and induction of peroxisomal β-oxidation, which causes hepatic accumulation of lipid droplets in animals. Inhibition of fatty acids ω-oxidation reduced long-chain dicarboxylic acids formation and significantly lowered peroxisomal β-oxidation and improved hepatic steatosis. Our results suggest that endogenous long-chain dicarboxylic acids act as triggering molecules inducing peroxisomal β-oxidation and hepatic triacylglycerol deposition. Targeting fatty acids ω-oxidation might be an effective pathway in treating fatty liver and related metabolic diseases through regulating peroxisomal β-oxidation.

Keywords:  dicarboxylic acids; hepatic steatosis; mitochondria; peroxisomes; ω-oxidation

DOI:  https://doi.org/10.1016/j.jbc.2023.105174
Nat Metab. 2023 Aug 21.

Glutarate regulates T cell metabolism and anti-tumour immunity.

Eleanor Minogue, Pedro P Cunha, Brennan J Wadsworth, Guinevere L Grice, Shiv K Sah-Teli, Rob Hughes, David Bargiela, Alessandro Quaranta, Javier Zurita, Robin Antrobus, Pedro Velica, Laura Barbieri, Craig E Wheelock, Peppi Koivunen, James A Nathan, Iosifina P Foskolou, Randall S Johnson.

T cell function and fate can be influenced by several metabolites: in some cases, acting through enzymatic inhibition of α-ketoglutarate-dependent dioxygenases, in others, through post-translational modification of lysines in important targets. We show here that glutarate, a product of amino acid catabolism, has the capacity to do both, and has potent effects on T cell function and differentiation. We found that glutarate exerts those effects both through α-ketoglutarate-dependent dioxygenase inhibition, and through direct regulation of T cell metabolism via glutarylation of the pyruvate dehydrogenase E2 subunit. Administration of diethyl glutarate, a cell-permeable form of glutarate, alters CD8+ T cell differentiation and increases cytotoxicity against target cells. In vivo administration of the compound is correlated with increased levels of both peripheral and intratumoural cytotoxic CD8+ T cells. These results demonstrate that glutarate is an important regulator of T cell metabolism and differentiation with a potential role in the improvement of T cell immunotherapy.

DOI: https://doi.org/10.1038/s42255-023-00855-2
J Biol Chem. 2023 Aug 21. pii: S0021-9258(23)02213-5. [Epub ahead of print] 105185

The Transcription Factor ChREBP Links Mitochondrial Lipidomes to Mitochondrial Morphology and Progression of Diabetic Kidney Disease.

Li Li, Jianyin Long, Koki Mise, Naravat Poungavrin, Philip L Lorenzi, Iqbal Mahmud, Lin Tan, Pradip K Saha, Yashpal S Kanwar, Benny H Chang, Farhad R Danesh.

  A substantial body of evidence has established the contributions of both mitochondrial dynamics and lipid metabolism to the pathogenesis of diabetic kidney disease (DKD). However, the precise interplay between these two key metabolic regulators of DKD is not fully understood. Here, we uncover a link between mitochondrial dynamics and lipid metabolism by investigating the role of carbohydrate-response element-binding protein (ChREBP), a glucose-responsive transcription factor and a master regulator of lipogenesis, in kidney podocytes. We find that inducible podocyte-specific knockdown of ChREBP in diabetic db/db mice improves key biochemical and histological features of DKD in addition to significantly reducing mitochondrial fragmentation. Because of the critical role of ChREBP in lipid metabolism, we interrogated whether and how mitochondrial lipidomes play a role in ChREBP-mediated mitochondrial fission. Our findings suggest a key role for a family of ether phospholipids in ChREBP-induced mitochondrial remodeling. We find that overexpression of glyceronephosphate O-acyltransferase (GNPAT), a critical enzyme in the biosynthesis of plasmalogens, reverses the protective phenotype of ChREBP deficiency on mitochondrial fragmentation. Finally, our data also points to Gnpat as a direct transcriptional target of ChREBP. Taken together, our results uncover a distinct mitochondrial lipid signature as the link between ChREBP-induced mitochondrial dynamics and progression of DKD.

Keywords:  diabetic nephropathy; kidney metabolism; lipid metabolism; mitochondria; phospholipid

DOI:  https://doi.org/10.1016/j.jbc.2023.105185
Nature. 2023 Aug 23.

Structural mechanism of mitochondrial membrane remodelling by human OPA1.

Alexander von der Malsburg, Gracie M Sapp, Kelly E Zuccaro, Alexander von Appen, Frank R Moss, Raghav Kalia, Jeremy A Bennett, Luciano A Abriata, Matteo Dal Peraro, Martin van der Laan, Adam Frost, Halil Aydin.

Distinct morphologies of the mitochondrial network support divergent metabolic and regulatory processes that determine cell function and fate1-3. The mechanochemical GTPase optic atrophy 1 (OPA1) influences the architecture of cristae and catalyses the fusion of the mitochondrial inner membrane4,5. Despite its fundamental importance, the molecular mechanisms by which OPA1 modulates mitochondrial morphology are unclear. Here, using a combination of cellular and structural analyses, we illuminate the molecular mechanisms that are key to OPA1-dependent membrane remodelling and fusion. Human OPA1 embeds itself into cardiolipin-containing membranes through a lipid-binding paddle domain. A conserved loop within the paddle domain inserts deeply into the bilayer, further stabilizing the interactions with cardiolipin-enriched membranes. OPA1 dimerization through the paddle domain promotes the helical assembly of a flexible OPA1 lattice on the membrane, which drives mitochondrial fusion in cells. Moreover, the membrane-bending OPA1 oligomer undergoes conformational changes that pull the membrane-inserting loop out of the outer leaflet and contribute to the mechanics of membrane remodelling. Our findings provide a structural framework for understanding how human OPA1 shapes mitochondrial morphology and show us how human disease mutations compromise OPA1 functions.

DOI: https://doi.org/10.1038/s41586-023-06441-6
Nat Commun. 2023 Aug 25. 14(1): 5181

Hepatic SREBP signaling requires SPRING to govern systemic lipid metabolism in mice and humans.

Sebastian Hendrix, Jenina Kingma, Roelof Ottenhoff, Masoud Valiloo, Monika Svecla, Lobke F Zijlstra, Vinay Sachdev, Kristina Kovac, Johannes H M Levels, Aldo Jongejan, Jan F de Boer, Folkert Kuipers, Antoine Rimbert, Giuseppe D Norata, Anke Loregger, Noam Zelcer.

The sterol regulatory element binding proteins (SREBPs) are transcription factors that govern cholesterol and fatty acid metabolism. We recently identified SPRING as a post-transcriptional regulator of SREBP activation. Constitutive or inducible global ablation of Spring in mice is not tolerated, and we therefore develop liver-specific Spring knockout mice (LKO). Transcriptomics and proteomics analysis reveal attenuated SREBP signaling in livers and hepatocytes of LKO mice. Total plasma cholesterol is reduced in male and female LKO mice in both the low-density lipoprotein and high-density lipoprotein fractions, while triglycerides are unaffected. Loss of Spring decreases hepatic cholesterol and triglyceride content due to diminished biosynthesis, which coincides with reduced very-low-density lipoprotein secretion. Accordingly, LKO mice are protected from fructose diet-induced hepatosteatosis. In humans, we find common genetic SPRING variants that associate with circulating high-density lipoprotein cholesterol and ApoA1 levels. This study positions SPRING as a core component of hepatic SREBP signaling and systemic lipid metabolism in mice and humans.

DOI: https://doi.org/10.1038/s41467-023-40943-1
Nat Commun. 2023 Aug 25. 14(1): 5185

Single-cell lipidomics enabled by dual-polarity ionization and ion mobility-mass spectrometry imaging.

Hua Zhang, Yuan Liu, Lauren Fields, Xudong Shi, Penghsuan Huang, Haiyan Lu, Andrew J Schneider, Xindi Tang, Luigi Puglielli, Nathan V Welham, Lingjun Li.

Single-cell (SC) analysis provides unique insight into individual cell dynamics and cell-to-cell heterogeneity. Here, we utilize trapped ion mobility separation coupled with dual-polarity ionization mass spectrometry imaging (MSI) to enable high-throughput in situ profiling of the SC lipidome. Multimodal SC imaging, in which dual-polarity-mode MSI is used to perform serial data acquisition runs on individual cells, significantly enhanced SC lipidome coverage. High-spatial resolution SC-MSI identifies both inter- and intracellular lipid heterogeneity; this heterogeneity is further explicated by Uniform Manifold Approximation and Projection and machine learning-driven classifications. We characterize SC lipidome alteration in response to stearoyl-CoA desaturase 1 inhibition and, additionally, identify cell-layer specific lipid distribution patterns in mouse cerebellar cortex. This integrated multimodal SC-MSI technology enables high-resolution spatial mapping of intercellular and cell-to-cell lipidome heterogeneity, SC lipidome remodeling induced by pharmacological intervention, and region-specific lipid diversity within tissue.

DOI: https://doi.org/10.1038/s41467-023-40512-6
Biosens Bioelectron. 2023 Aug 12. pii: S0956-5663(23)00539-0. [Epub ahead of print]239 115597

RaMALDI: Enabling simultaneous Raman and MALDI imaging of the same tissue section.

Ethan Yang, Jeong Hee Kim, Caitlin M Tressler, Xinyi Elaine Shen, Dalton R Brown, Cole C Johnson, Tae-Hun Hahm, Ishan Barman, Kristine Glunde.

  Multimodal tissue imaging techniques that integrate two complementary modalities are powerful discovery tools for unraveling biological processes and identifying biomarkers of disease. Combining Raman spectroscopic imaging (RSI) and matrix-assisted laser-desorption/ionization (MALDI) mass spectrometry imaging (MSI) to obtain fused images with the advantages of both modalities has the potential of providing spatially resolved, sensitive, specific biomolecular information, but has so far involved two separate sample preparations, or even consecutive tissue sections for RSI and MALDI MSI, resulting in images with inherent disparities. We have developed RaMALDI, a streamlined, integrated, multimodal imaging workflow of RSI and MALDI MSI, performed on a single tissue section with one sample preparation protocol. We show that RaMALDI imaging of various tissues effectively integrates molecular information acquired from both RSI and MALDI MSI of the same sample, which will drive discoveries in cell biology, biomedicine, and pathology, and advance tissue diagnostics.

Keywords:  MALDI; Mass spectrometry imaging; Matrix; Multimodal imaging; Raman spectroscopy

DOI:  https://doi.org/10.1016/j.bios.2023.115597
Cell Metab. 2023 Aug 18. pii: S1550-4131(23)00296-6. [Epub ahead of print]

Deciphering the decline of metabolic elasticity in aging and obesity.

Qiuzhong Zhou, Lexiang Yu, Joshua R Cook, Li Qiang, Lei Sun.

  Organisms must adapt to fluctuating nutrient availability to maintain energy homeostasis. Here, we term the capacity for such adaptation and restoration "metabolic elasticity" and model it through ad libitum-fasting-refeeding cycles. Metabolic elasticity is achieved by coordinate versatility in gene expression, which we call "gene elasticity." We have developed the gene elasticity score as a systematic method to quantify the elasticity of the transcriptome across metabolically active tissues in mice and non-human primates. Genes involved in lipid and carbohydrate metabolism show high gene elasticity, and their elasticity declines with age, particularly with PPARγ dysregulation in adipose tissue. Synchronizing PPARγ activity with nutrient conditions through feeding-timed agonism optimizes their metabolic benefits and safety. We further broaden the conceptual scope of metabolic and gene elasticity to dietary challenges, revealing declines in diet-induced obesity similar to those in aging. Altogether, our findings provide a dynamic perspective on the dysmetabolic consequences of aging and obesity.

Keywords:  adipocyte; adipose tissue; aging; gene elasticity; liver; metabolic decline; metabolic elasticity; muscle; nutrient challenge; obesity

DOI:  https://doi.org/10.1016/j.cmet.2023.08.001
Nat Commun. 2023 Aug 25. 14(1): 5214

Glycerol 3-phosphate phosphatase/PGPH-2 counters metabolic stress and promotes healthy aging via a glycogen sensing-AMPK-HLH-30-autophagy axis in C. elegans.

Elite Possik, Laura-Lee Klein, Perla Sanjab, Ruyuan Zhu, Laurence Côté, Ying Bai, Dongwei Zhang, Howard Sun, Anfal Al-Mass, Abel Oppong, Rasheed Ahmad, Alex Parker, S R Murthy Madiraju, Fahd Al-Mulla, Marc Prentki.

Metabolic stress caused by excess nutrients accelerates aging. We recently demonstrated that the newly discovered enzyme glycerol-3-phosphate phosphatase (G3PP; gene Pgp), which operates an evolutionarily conserved glycerol shunt that hydrolyzes glucose-derived glycerol-3-phosphate to glycerol, counters metabolic stress and promotes healthy aging in C. elegans. However, the mechanism whereby G3PP activation extends healthspan and lifespan, particularly under glucotoxicity, remained unknown. Here, we show that the overexpression of the C. elegans G3PP homolog, PGPH-2, decreases fat levels and mimics, in part, the beneficial effects of calorie restriction, particularly in glucotoxicity conditions, without reducing food intake. PGPH-2 overexpression depletes glycogen stores activating AMP-activate protein kinase, which leads to the HLH-30 nuclear translocation and activation of autophagy, promoting healthy aging. Transcriptomics reveal an HLH-30-dependent longevity and catabolic gene expression signature with PGPH-2 overexpression. Thus, G3PP overexpression activates three key longevity factors, AMPK, the TFEB homolog HLH-30, and autophagy, and may be an attractive target for age-related metabolic disorders linked to excess nutrients.

DOI: https://doi.org/10.1038/s41467-023-40857-y
Cancer Cell Int. 2023 Aug 24. 23(1): 177

Spatially resolved visualization of reprogrammed metabolism in hepatocellular carcinoma by mass spectrometry imaging.

Bangzhen Ma, Yang Zhang, Jiwei Ma, Xinguo Chen, Chenglong Sun, Chengkun Qin.

   BACKGROUND: Metabolic reprogramming refers to tumor-associated metabolic alterations during tumorigenesis and has been regarded as one of the most important features of cancer. Profiling the altered metabolites and lipids in hepatocellular carcinoma with spatial signature will not only enhance our understanding of tumor metabolic reprogramming, but also offer potential metabolic liabilities that might be exploited for hepatocellular carcinoma therapy.
METHODS: We perform matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) analysis on both hepatocellular carcinoma xenograft mouse model and hepatocellular carcinoma patients. Discriminatory metabolites that altered during the development of hepatocellular carcinoma are screened and imaged in xenograft mouse model and are further validated in 21 hepatocellular carcinoma patients.
RESULTS: We discover stepwise metabolic alterations and progressively increasing metabolic heterogeneity during the growth of hepatocellular carcinoma. Arginine and its metabolites spermine and spermidine, choline and phosphatidylcholine metabolism, and fatty acids were found to be significantly reprogrammed in hepatocellular carcinoma tissues.
CONCLUSIONS: The spatially resolved profiling of the metabolites and lipids in highly heterogeneous hepatocellular carcinoma tissue will contribute to obtaining precise metabolic information for the understanding of tumor metabolic reprogramming.

Keywords:  Hepatocellular carcinoma, MALDI-MS; Metabolites, lipids; Spatially resolved imaging

DOI:  https://doi.org/10.1186/s12935-023-03027-0
J Biol Chem. 2023 Aug 23. pii: S0021-9258(23)02217-2. [Epub ahead of print] 105189

Allopurinol and oxypurinol differ in their strength and mechanisms of inhibition of xanthine oxidoreductase.

Mai Sekine, Ken Okamoto, Emil F Pai, Koji Nagata, Kimiyoshi Ichida, Russ Hille, Takeshi Nishino.

  Xanthine oxidoreductase (XOR) is a metalloenzyme that catalyzes the final steps in purine metabolism by converting hypoxanthine to xanthine and then uric acid. Allopurinol, an analog of hypoxanthine, is widely used as an anti-gout drug, as XOR-mediated metabolism of allopurinol to oxypurinol leads to oxypurinol rotation in the enzyme active site and reduction of the molybdenum Mo(VI) active center to Mo(IV), inhibiting subsequent urate production. However, when oxypurinol is administered directly to a mouse model of hyperuricemia, it yields a weaker urate-lowering effect than allopurinol. To better understand its mechanism of inhibition and inform patient dosing strategies, here we performed kinetic and structural analyses of the inhibitory activity of oxypurinol. Our results demonstrated that oxypurinol was less effective than allopurinol both in vivo and in vitro. We show that upon reoxidation to Mo(VI), oxypurinol binding is greatly weakened, and reduction by xanthine, hypoxanthine, or allopurinol is required for reformation of the inhibitor-enzyme complex. In addition, we show oxypurinol only weakly inhibits the conversion of hypoxanthine to xanthine and is therefore unlikely to effect feedback inhibition of de novo purine synthesis. Furthermore, we observed weak allosteric inhibition of purine nucleoside phosphorylase by oxypurinol, which has potentially adverse effects for patients. Considering these results, we propose the single-dose method currently used to treat hyperuricemia can result in unnecessarily high levels of allopurinol. While the short half-life of allopurinol in blood suggests that oxypurinol is responsible for enzyme inhibition, we anticipate multiple, smaller doses of allopurinol would reduce the total allopurinol patient load.

Keywords:  Allopurinol; Gout; Oxypurinol; Xanthine oxidase; Xanthine oxidoreductase

DOI:  https://doi.org/10.1016/j.jbc.2023.105189
Cell Chem Biol. 2023 Aug 11. pii: S2451-9456(23)00243-X. [Epub ahead of print]

Succinylation of a KEAP1 sensor lysine promotes NRF2 activation.

Lara Ibrahim, Caroline Stanton, Kayla Nutsch, Thu Nguyen, Chloris Li-Ma, Yeonjin Ko, Gabriel C Lander, R Luke Wiseman, Michael J Bollong.

Cross talk between metabolism and stress-responsive signaling is essential for maintaining cellular homeostasis. This cross talk is often achieved through covalent modification of proteins by endogenous, reactive metabolites that regulate key stress-responsive transcription factors like NRF2. Metabolites including methylglyoxal, glyceraldehyde 3-phosphate, fumarate, and itaconate covalently modify sensor cysteines of the NRF2 repressor KEAP1, resulting in stabilization of NRF2 and activation of its cytoprotective transcriptional program. Here, we employed a shRNA-based screen targeting the enzymes of central carbon metabolism to identify additional regulatory nodes bridging metabolism to NRF2 activation. Succinic anhydride, increased by genetic depletion of the TCA cycle enzyme succinyl-CoA synthetase or by direct administration, results in N-succinylation of lysine 131 of KEAP1 to activate NRF2 signaling. This study identifies KEAP1 as capable of sensing reactive metabolites not only by several cysteine residues but also by a conserved lysine residue, indicating its potential to sense an expanded repertoire of reactive metabolic messengers.

DOI: https://doi.org/10.1016/j.chembiol.2023.07.014
JCI Insight. 2023 Aug 22. pii: e163403. [Epub ahead of print]8(16):

Alveolar type II epithelial cell FASN maintains lipid homeostasis in experimental COPD.

Li-Chao Fan, Keith McConn, Maria Plataki, Sarah Kenny, Niamh C Williams, Kihwan Kim, Jennifer A Quirke, Yan Chen, Maor Sauler, Matthias E Möbius, Kuei-Pin Chung, Estela Area Gomez, Augustine Mk Choi, Jin-Fu Xu, Suzanne M Cloonan.

  Alveolar epithelial type II (AEC2) cells strictly regulate lipid metabolism to maintain surfactant synthesis. Loss of AEC2 cell function and surfactant production are implicated in the pathogenesis of the smoking-related lung disease chronic obstructive pulmonary disease (COPD). Whether smoking alters lipid synthesis in AEC2 cells and whether altering lipid metabolism in AEC2 cells contributes to COPD development are unclear. In this study, high-throughput lipidomic analysis revealed increased lipid biosynthesis in AEC2 cells isolated from mice chronically exposed to cigarette smoke (CS). Mice with a targeted deletion of the de novo lipogenesis enzyme, fatty acid synthase (FASN), in AEC2 cells (FasniΔAEC2) exposed to CS exhibited higher bronchoalveolar lavage fluid (BALF) neutrophils, higher BALF protein, and more severe airspace enlargement. FasniΔAEC2 mice exposed to CS had lower levels of key surfactant phospholipids but higher levels of BALF ether phospholipids, sphingomyelins, and polyunsaturated fatty acid-containing phospholipids, as well as increased BALF surface tension. FasniΔAEC2 mice exposed to CS also had higher levels of protective ferroptosis markers in the lung. These data suggest that AEC2 cell FASN modulates the response of the lung to smoke by regulating the composition of the surfactant phospholipidome.

Keywords:  COPD; Intermediary metabolism; Metabolism; Pulmonary surfactants; Pulmonology

DOI:  https://doi.org/10.1172/jci.insight.163403
J Clin Invest. 2023 Aug 24. pii: e166644. [Epub ahead of print]

Peroxisome disruption alters lipid metabolism and potentiates anti-tumor response with MAPK-targeted therapy in melanoma.

Fan Huang, Feiyang Cai, Michael S Dahabieh, Kshemaka Gunawardena, Ali Talebi, Jonas Dehairs, Farah El-Turk, Jae Yeon Park, Mengqi Li, Christophe Goncalves, Natascha Gagnon, Jie Su, Judith H LaPierre, Perrine Gaub, Jean-Sébastien Joyal, John J Mitchell, Johannes V Swinnen, Wilson H Miller, Sonia V Del Rincón.

  Melanomas reprogram their metabolism to rapidly adapt to therapy-induced stress conditions, allowing them to persist and ultimately develop resistance. We report that a subpopulation of melanoma cells tolerate MAPK-pathway inhibitors (MAPKi) through a concerted metabolic reprogramming mediated by peroxisomes and UDP-glucose ceramide glycosyltransferase (UGCG). Compromising peroxisome biogenesis, by repressing PEX3 expression, potentiated the pro-apoptotic effects of MAPKi via an induction of ceramides, an effect limited by UGCG-mediated ceramide metabolism. Co-targeting PEX3 and UGCG selectively eliminated a subset of metabolically active, drug-tolerant CD36+ melanoma persister cells, thereby sensitizing melanoma to MAPKi and delaying resistance. Increased levels of peroxisomal genes and UGCG were found in patient-derived MAPKi-relapsed melanomas, and simultaneously inhibiting PEX3 and UGCG restored MAPKi sensitivity in multiple models of therapy resistance. Finally, combination therapy comprised of a newly identified inhibitor of the PEX3-PEX19 interaction, a UGCG inhibitor and MAPKi demonstrated potent anti-tumor activity in pre-clinical melanoma models, thus representing a promising approach for melanoma treatment.

Keywords:  Melanoma; Oncology

DOI:  https://doi.org/10.1172/JCI166644
Blood. 2023 Aug 25. pii: blood.2023020504. [Epub ahead of print]

The iron chaperone poly C binding protein 1 regulates iron efflux through intestinal ferroportin in mice.

Yubo Wang, Olga Protchenko, Kari Duck Huber, Minoo Shakoury-Elizeh, Manik C Ghosh, Caroline C Philpott.

Iron is an essential nutrient required by all cells but used primarily for red blood cell production. Because humans have no effective mechanism for ridding the body of excess iron, the absorption of dietary iron must be precisely regulated. The critical site of regulation is the transfer of iron from the absorptive enterocyte to the portal circulation via the sole iron efflux transporter, ferroportin. Here we report that poly(rC)-binding protein 1 (PCBP1), the major cytosolic iron chaperone, is necessary for the regulation of iron flux through ferroportin in the intestine of mice. Mice lacking PCBP1 in the intestinal epithelium exhibit low levels of enterocyte iron, poor retention of dietary iron in enterocyte ferritin, and excess efflux of iron through ferroportin. Excess iron efflux occurred despite lower levels of ferroportin protein in enterocytes and upregulation of the iron regulatory hormone hepcidin. PCBP1 deletion and the resulting unregulated dietary iron absorption led to poor growth, severe anemia on a low iron diet and liver oxidative stress with iron loading on a high iron diet. Ex vivo culture of PCBP1-depleted enteroids demonstrated no defects in hepcidin-mediated ferroportin turnover. However, measurement of kinetically labile iron pools in enteroids competent or blocked for iron efflux indicated that PCBP1 functioned to bind and retain cytosolic iron and to limit its availability for ferroportin-mediated efflux. Thus, PCBP1 coordinates enterocyte iron and reduces the concentration of unchaperoned, "free" iron to a low level that is necessary for hepcidin-mediated regulation of ferroportin activity.

DOI: https://doi.org/10.1182/blood.2023020504