Metabolic rewiring promotes anti-inflammatory effects of glucocorticoids.

Glucocorticoids represent the mainstay of therapy for a broad spectrum of immune-mediated inflammatory diseases. However, the molecular mechanisms underlying their anti-inflammatory mode of action have remained incompletely understood1. Here we show that the anti-inflammatory properties of glucocorticoids involve reprogramming of the mitochondrial metabolism of macrophages, resulting in increased and sustained production of the anti-inflammatory metabolite itaconate and consequent inhibition of the inflammatory response. The glucocorticoid receptor interacts with parts of the pyruvate dehydrogenase complex whereby glucocorticoids provoke an increase in activity and enable an accelerated and paradoxical flux of the tricarboxylic acid (TCA) cycle in otherwise pro-inflammatory macrophages. This glucocorticoid-mediated rewiring of mitochondrial metabolism potentiates TCA-cycle-dependent production of itaconate throughout the inflammatory response, thereby interfering with the production of pro-inflammatory cytokines. By contrast, artificial blocking of the TCA cycle or genetic deficiency in aconitate decarboxylase 1, the rate-limiting enzyme of itaconate synthesis, interferes with the anti-inflammatory effects of glucocorticoids and, accordingly, abrogates their beneficial effects during a diverse range of preclinical models of immune-mediated inflammatory diseases. Our findings provide important insights into the anti-inflammatory properties of glucocorticoids and have substantial implications for the design of new classes of anti-inflammatory drugs.

Targeted macrophage phagocytosis by Irg1/itaconate axis improves the prognosis of intracerebral hemorrhagic stroke and peritonitis.

BACKGROUND: Macrophages are innate immune cells whose phagocytosis function is critical to the prognosis of stroke and peritonitis. cis-aconitic decarboxylase immune-responsive gene 1 (Irg1) and its metabolic product itaconate inhibit bacterial infection, intracellular viral replication, and inflammation in macrophages. Here we explore whether itaconate regulates phagocytosis. METHODS: Phagocytosis of macrophages was investigated by time-lapse video recording, flow cytometry, and immunofluorescence staining in macrophage/microglia cultures isolated from mouse tissue. Unbiased RNA-sequencing and ChIP-sequencing assays were used to explore the underlying mechanisms. The effects of Irg1/itaconate axis on the prognosis of intracerebral hemorrhagic stroke (ICH) and peritonitis was observed in transgenic (Irg1flox/flox; Cx3cr1creERT/+, cKO) mice or control mice in vivo. FINDINGS: In a mouse model of ICH, depletion of Irg1 in macrophage/microglia decreased its phagocytosis of erythrocytes, thereby exacerbating outcomes (n = 10 animals/group, p < 0.05). Administration of sodium itaconate/4-octyl itaconate (4-OI) promoted macrophage phagocytosis (n = 7 animals/group, p < 0.05). In addition, in a mouse model of peritonitis, Irg1 deficiency in macrophages also inhibited phagocytosis of Staphylococcus aureus (n = 5 animals/group, p < 0.05) and aggravated outcomes (n = 9 animals/group, p < 0.05). Mechanistically, 4-OI alkylated cysteine 155 on the Kelch-like ECH-associated protein 1 (Keap1), consequent in nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and transcriptional activation of Cd36 gene. Blocking the function of CD36 completely abolished the phagocytosis-promoting effects of Irg1/itaconate axis in vitro and in vivo. INTERPRETATION: Our findings provide a potential therapeutic target for phagocytosis-deficiency disorders, supporting further development towards clinical application for the benefit of stroke and peritonitis patients. FUNDING: The National Natural Science Foundation of China (32070735, 82371321 to Q. Li, 82271240 to F. Yang) and the Beijing Natural Science Foundation Program and Scientific Research Key Program of Beijing Municipal Commission of Education (KZ202010025033 to Q. Li).

Hypoxia-induced circ-CDYL-EEF1A2 transcriptional complex drives lung metastasis of cancer stem cells from hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is often associated with poor outcomes due to lung metastasis. ICAM-1+ circulating tumor cells, termed circulating cancer stem cells (CCSCs), possess stem cell-like characteristics. However, it is still unexplored how their presence indicates lung metastasis tendency, and particularly, what mechanism drives their lung metastasis. Here, we demonstrated that a preoperative CCSC count in 5 mL of blood (CCSC5) of >3 was a risk factor for lung metastasis in clinical HCC patients. The CSCs overexpressed with circ-CDYL entered the bloodstream and developed lung metastases in mice. Mechanistically, circ-CDYL promoted COL14A1 expression and thus ERK signaling to facilitate epithelial-mesenchymal transition. Furthermore, we uncovered that an RNA-binding protein, EEF1A2, acted as a novel transcriptional (co-) factor to cooperate with circ-CDYL and initiate COL14A1 transcription. A high circ-CDYL level is caused by HIF-1⍺-mediated transcriptional upregulation of its parental gene CDYL and splicing factor EIF4A3 under a hypoxia microenvironment. Hence, the hypoxia microenvironment enables the high-tendency lung metastasis of ICAM-1+ CCSCs through the HIF-1⍺/circ-CDYL-EEF1A2/COL14A1 axis, potentially allowing clinicians to preoperatively detect ICAM-1+ CCSCs as a real-time biomarker for precisely deciding HCC treatment strategies.

Deciphering the Biosynthesis of Novel Class I Lanthipeptides from Marine Pseudoalteromonas Reveals a Dehydratase PsfB with Dethiolation Activity.

Lanthipeptides are a representative class of RiPPs that possess characteristic lanthionine and/or methyllanthionine thioether cross-links. The biosynthetic potentials of marine-derived lanthipeptides remain largely unexplored. In this study, we characterized three novel lanthipeptides pseudorosin A-C by heterologous expression of a class I lanthipeptide biosynthetic gene cluster from marine Pseudoalteromonas flavipulchra S16. Interestingly, pseudorosin C contains a large loop spanning 18 amino acid residues, which is rare in lanthipeptides. Unexpectedly, the dehydratase PsfB could catalyze the dethiolation of specific Cys residues in all three core peptides, thereby generating dehydroalanines in the absence of LanC cyclase. To the best of our knowledge, we identified the first member of the LanB dehydratase family to perform glutamylation and subsequent elimination on Cys thiol groups, which likely represents a new bypass for class I lanthipeptide biosynthesis. Furthermore, we employed mutagenesis to determine the important motif of the core peptide for dethiolation activity. Moreover, sequence analysis revealed that PsfB exhibited a distinct phylogenetic distance from the characterized LanBs from Gram-positive bacteria. Our findings, therefore, pave the way for further genome mining of lanthipeptides, novel post-translational modification enzymes from marine Gram-negative bacteria, and bioengineering applications.

Targeting IRG1 reverses the immunosuppressive function of tumor-associated macrophages and enhances cancer immunotherapy.

Immune-responsive gene 1 (IRG1) encodes aconitate decarboxylase (ACOD1) that catalyzes the production of itaconic acids (ITAs). The anti-inflammatory function of IRG1/ITA has been established in multiple pathogen models, but very little is known in cancer. Here, we show that IRG1 is expressed in tumor-associated macrophages (TAMs) in both human and mouse tumors. Mechanistically, tumor cells induce Irg1 expression in macrophages by activating NF-κB pathway, and ITA produced by ACOD1 inhibits TET DNA dioxygenases to dampen the expression of inflammatory genes and the infiltration of CD8+ T cells into tumor sites. Deletion of Irg1 in mice suppresses the growth of multiple tumor types and enhances the efficacy of anti-PD-(L)1 immunotherapy. Our study provides a proof of concept that ACOD1 is a potential target for immune-oncology drugs and IRG1-deficient macrophages represent a potent cell therapy strategy for cancer treatment even in pancreatic tumors that are resistant to T cell-based immunotherapy.

Database-driven in silico-identification and characterization of novel aldoxime dehydratases.

Aldoxime dehydratases (Oxds) are a unique class of enzymes, which catalyzes the dehydration of aldoximes to nitriles in an aqueous environment. Recently, they gained attention as a catalyst for a green and cyanide-free alternative to established nitrile syntheses, which often require the use of toxic cyanides and harsh reaction conditions. Up to now only thirteen aldoxime dehydratases have been discovered and biochemically characterized. This raised the interest for identifying further Oxds with, e.g., complementary properties in terms of substrate scope. In this study, 16 novel genes, presumably encoding aldoxime dehydratases, were selected by using a commercially available 3DM database based on OxdB, an Oxd from Bacillus sp. OxB-1. Out of 16 proteins, six enzymes with aldoxime dehydratases activity were identified, which differ in their substrate scope and activity. While some novel Oxds showed better performance for aliphatic substrate such as n-octanaloxime compared to the well characterized OxdRE from Rhodococcus sp. N-771, some showed activity for aromatic aldoximes, leading to an overall high usability of these enzymes in organic chemistry. The applicability for organic synthesis was underlined by converting 100 mM n-octanaloxime at a 10 mL scale within 5 h with the novel aldoxime dehydratase OxdHR as whole-cell catalyst (33 mgbww/mL).

Essential mitochondrial role in iron-sulfur cluster assembly of the cytoplasmic isopropylmalate isomerase Leu1 in Saccharomyces cerevisiae.

Iron-sulfur (Fe-S) cluster assembly in mitochondria and cytoplasm is essential for cell viability. In the yeast S. cerevisiae, Leu1 [4Fe-4S] is the cytoplasmic isopropylmalate isomerase involved in leucine biosynthesis. Using permeabilized Δleu1 cells and recombinant apo-Leu1R, here we show that the [4Fe-4S] cluster assembly on Leu1R can be reconstituted in a physiologic manner requiring both mitochondria and cytoplasm, as judged by conversion of the inactive enzyme to an active form. The mitochondrial contribution to this reconstitution assay is abrogated by inactivating mutations in the mitochondrial ISC (iron-sulfur cluster assembly) machinery components (such as Nfs1 cysteine desulfurase and Ssq1 chaperone) or the mitochondrial exporter Atm1. Likewise, depletion of a CIA (cytoplasmic iron-sulfur protein assembly) component Dre2 leads to impaired Leu1R reconstitution. Mitochondria likely make and export an intermediate, called X-S or (Fe-S)int, that is needed for cytoplasmic Fe-S cluster biosynthesis. Here we show that once exported, the same intermediate can be used for both [2Fe-2S] and [4Fe-4S] cluster biogenesis in the cytoplasm, with no further requirement of mitochondria. Our data also suggest that the exported intermediate can activate defective/latent CIA components in cytoplasm isolated from nfs1 or Δatm1 mutant cells. These findings may provide a way to isolate X-S or (Fe-S)int.

Immune response gene 1 deficiency aggravates high fat diet-induced nonalcoholic fatty liver disease via promotion of redox-sensitive AKT suppression.

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disorder worldwide. Immune response gene 1 (IRG1) catalyzes the production of bio-active itaconate, which is actively involved in the regulation of signal transduction. A recent study has found that the expression of IRG1 was significantly down-regulated in obesity-associated fatty liver, but the potential roles of IRG1 in the development NAFLD remain unclear. The present study found that genetic deletion of IRG1 aggravated high fat diet (HFD)-induced metabolic disturbance, including obesity, dyslipidemia and insulin resistance. In addition, HFD induced more severe liver steatosis and higher serum ALT and AST level in IRG1 KO mice, which were accompanied with altered expression of genes involved in lipid uptake, synthesis and catabolism. RNA-seq and immunoblot analysis indicated that deficiency of IRG1 is associated with suppressed activation of AKT, a master metabolic regulator. Mechanistically, IRG1/itaconate enhanced the antioxidative NRF2 pathway and prevented redox-sensitive suppression of AKT. Interestingly, supplementation with 4-octyl itaconate (4-OI), a cell-permeable derivate of itaconate, alleviated HFD-induced oxidative stress, AKT suppression and liver steatosis. Therefore, IRG1 probably functions as a protective regulator in the development of NAFLD and the cell-permeable 4-OI might have potential value for the pharmacological intervention of NAFLD.

Evaluation of TS and ENOSF1 Variants as a Biomarker in Response to Neoadjuvant Chemotherapy based on 5FU in Gastric Cancer Patients.

OBJECTIVE: Neoadjuvant chemotherapy with 5-fluorouracil (5FU) is one of the most effective treatment options for gastric cancer patients.  However, treatment response varies significantly between patients based on their genetic profile. The purpose of this study was to determine the association between thymidylate synthase (TS) and enolase superfamily member 1 (ENOSF1) polymorphisms, treatment response, and overall survival in patients with gastric cancer. METHODS: The TS and ENOSF1 variants were analyzed in formalin-fixed paraffin-embedded (FFPE) tissue from 100 gastric cancer patients receiving neoadjuvant 5FU-based chemotherapy. Polymerase chain reaction (PCR) amplification and restriction fragment length polymorphism (RFLP) were used to determine TS polymorphisms' genotypes, and the Tetra Arms PCR method was used to identify ENOSF1 polymorphisms. Patients were followed for up to five years, and the association between variants, treatment response, and overall survival (OS) was examined. RESULTS: There was a significant association between the TS 5' UTR polymorphism and response to treatment in patients with gastric cancer who received neoadjuvant 5FU therapy (P=0.032). Patients with the 2R3R genotype responded better to treatment, whereas those with the 3R3R genotype did not respond to treatment. Patients with the 2R2R and 3R3R genotypes had the longest and shortest median survival times, respectively, and the observed differences were significant (p=0.003). There was a statistically significant relationship between rs2612091 and chemotherapy response (P=0.017). Patients with genotype AG did not respond to treatment. CONCLUSION: This study established that the TS 5' UTR and ENOSF1 rs2612091 polymorphisms could be used to predict treatment response and overall survival in patients with gastric cancer who received neoadjuvant chemotherapy based on 5FU.

circCDYL2 promotes trastuzumab resistance via sustaining HER2 downstream signaling in breast cancer.

BACKGROUND: Approximate 25% HER2-positive (HER2+) breast cancer (BC) patients treated with trastuzumab recurred rapidly. However, the mechanisms underlying trastuzumab resistance remained largely unclear. METHODS: Trastuzumab-resistant associated circRNAs were identified by circRNAs high-throughput screen and qRT-PCR in HER2+ breast cancer tissues with different trastuzumab response. The biological roles of trastuzumab-resistant associated circRNAs were detected by cell vitality assay, colony formation assay, Edu assay, patient-derived xenograft (PDX) models and orthotopic animal models. For mechanisms research, the co-immunoprecipitation, Western blot, immunofluorescence, and pull down assays confirmed the relevant mechanisms of circRNA and binding proteins. RESULTS: We identified a circRNA circCDYL2, which was overexpressed in trastuzumab-resistant patients, which conferred trastuzumab resistance in breast cancer cells in vitro and in vivo. Mechanically, circCDYL2 stabilized GRB7 by preventing its ubiquitination degradation and enhanced its interaction with FAK, which thus sustained the activities of downstream AKT and ERK1/2. Trastuzumab-resistance of HER2+ BC cells with high circCDYL2 could be reversed by FAK or GRB7 inhibitor. Clinically, HER2+ BC patients with high levels of circCDYL2 developed rapid recurrence and had shorter disease-free survival (DFS) and overall survival (OS) following anti-HER2 therapy compared to those with low circCDYL2. CONCLUSIONS: circCDYL2-GRB7-FAK complex plays a critical role in maintaining HER2 signaling, which contributes to trastuzumab resistance and circCDYL2 is a potential biomarker for trastuzumab-resistance in HER2+ BC patients.

lncRNA GMDS­AS1 upregulates IL­6, TNF­α and IL­1ß, and induces apoptosis in human monocytic THP­1 cells via miR­96­5p/caspase 2 signaling.

Long non­coding RNA (lncRNA) is considered a crucial modulator of the initiation and progression of several diseases. However, the roles of lncRNA in sepsis have yet to be fully elucidated. Thus, the aim of the present study was to investigate the effects of the lncRNA GDP­mannose 4,6­dehydratase antisense 1 (GMDS­AS1) and its target in order to understand its role in the pathogenesis of sepsis. An in vitro sepsis model was established by lipopolysaccharide (LPS) induction. Reverse transcription­quantitative PCR analysis was applied to detect the expression of inflammatory cytokines and the levels of GMDS­AS1, microRNA (miR)­96­5p and caspase­2 (CASP2). Flow cytometry was used to quantify the rate of apoptosis. In addition, the interaction between miR­96­5p and CASP2 was verified using a luciferase reporter assay. Western blot analysis was performed to assess the protein levels of CASP2 following alterations in GMDS­AS1 and miR­96­5p expression using transfection. The levels of interleukin (IL)­6, tumor necrosis factor­α and IL­1ß were increased by LPS treatment in THP­1 cells, whereas miR­96­5p expression was downregulated. miR­96­5p overexpression inhibited LPS­induced inflammatory responses and apoptosis. In addition, GMDS­AS1 expression increased, and upregulation of GMDS­AS1 inhibited, the expression of miR­96­5p in the in vitro sepsis model. Moreover, CASP2 was confirmed to be a direct target of miR­96­5p. Therefore, the lncRNA GMDS­AS1 regulated inflammatory responses and apoptosis by modulating CASP2 and sponging miR­96­5p in LPS­induced THP­1 cells. In summary, the findings of the present study demonstrated that lncRNA GMDS­AS1 could promote the development of sepsis by targeting miR­96­5p/CASP2, indicating that the GMDS­AS1/miR­96­5p/CASP2 axis may be a new therapeutic target and potential research direction for sepsis therapy.

Deregulation of phenylalanine biosynthesis evolved with the emergence of vascular plants.

Phenylalanine (Phe) is the precursor of essential secondary products in plants. Here we show that a key, rate-limiting step in Phe biosynthesis, which is catalyzed by arogenate dehydratase, experienced feedback de-regulation during evolution. Enzymes from microorganisms and type-I ADTs from plants are strongly feedback-inhibited by Phe, while type-II isoforms remain active at high levels of Phe. We have found that type-II ADTs are widespread across seed plants and their overproduction resulted in a dramatic accumulation of Phe in planta, reaching levels up to 40 times higher than those observed following the expression of type-I enzymes. Punctual changes in the allosteric binding site of Phe and adjacent region are responsible for the observed relaxed regulation. The phylogeny of plant ADTs evidences that the emergence of type-II isoforms with relaxed regulation occurred at some point in the transition between nonvascular plants and tracheophytes, enabling the massive production of Phe-derived compounds, primarily lignin, a hallmark of vascular plants.

Substrate plasticity of dehydratase SpaKC from the biosynthesis of thiosparsoamide.

Thioamitides are a group of ribosomally synthesized and post-translationally modified peptides that possess diverse bioactivities and are usually featured by thioamide and 2-aminovinyl-cysteine (AviCys) motifs. In natural product thiosparsoamide, the AviCys motif is formed by an enzyme cascade formed by the flavin-dependent decarboxylase SpaD and dehydratase SpaKC. SpaKC is a lanthipeptide synthetase homolog located outside the thiosparsoamide biosynthetic gene cluster. In this study, we show that SpaKC does not strictly require the N-terminal leader peptide of precursor peptide SpaA for substrate recognition and dehydration. The C-terminal seven residues serve as a minimal structural element for enzyme recognition. Through a systematic mutagenesis experiments, our study demonstrates the relaxed substrate specificity of SpaKC as a dehydratase and potentially as an enzymatic tool to install dehydroalanine or dehydrobutyrine motifs in peptides.

Fluorinated rhamnosides inhibit cellular fucosylation.

The sugar fucose is expressed on mammalian cell membranes as part of glycoconjugates and mediates essential physiological processes. The aberrant expression of fucosylated glycans has been linked to pathologies such as cancer, inflammation, infection, and genetic disorders. Tools to modulate fucose expression on living cells are needed to elucidate the biological role of fucose sugars and the development of potential therapeutics. Herein, we report a class of fucosylation inhibitors directly targeting de novo GDP-fucose biosynthesis via competitive GMDS inhibition. We demonstrate that cell permeable fluorinated rhamnose 1-phosphate derivatives (Fucotrim I & II) are metabolic prodrugs that are metabolized to their respective GDP-mannose derivatives and efficiently inhibit cellular fucosylation.

A20 deficiency in myeloid cells protects mice from diet-induced obesity and insulin resistance due to increased fatty acid metabolism.

Obesity-induced inflammation is a major driving force in the development of insulin resistance, type 2 diabetes (T2D), and related metabolic disorders. During obesity, macrophages accumulate in the visceral adipose tissue, creating a low-grade inflammatory environment. Nuclear factor κB (NF-κB) signaling is a central coordinator of inflammatory responses and is tightly regulated by the anti-inflammatory protein A20. Here, we find that myeloid-specific A20-deficient mice are protected from diet-induced obesity and insulin resistance despite an inflammatory environment in their metabolic tissues. Macrophages lacking A20 show impaired mitochondrial respiratory function and metabolize more palmitate both in vitro and in vivo. We hypothesize that A20-deficient macrophages rely more on palmitate oxidation and metabolize the fat present in the diet, resulting in a lean phenotype and protection from metabolic disease. These findings reveal a role for A20 in regulating macrophage immunometabolism.

Biosynthesis of the nucleoside antibiotic angustmycins: identification and characterization of the biosynthetic gene cluster reveal unprecedented dehydratase required for exo-glycal formation.

The nucleoside antibiotic angustmycin, produced by some Streptomyces strains, is composed of adenine and C6 sugar and shows antibiotic and antitumor activities. In this study, we propose a biosynthetic pathway for angustmycin using a heterologous expression experiment coupled with in silico analysis of the angustmycin biosynthetic gene (agm) cluster. The biochemical characterization of Agm6 demonstrated its role in angustmycin biosynthesis as an unprecedented dehydratase.

Hepatic miR-144 Drives Fumarase Activity Preventing NRF2 Activation During Obesity.

BACKGROUND AND AIMS: Oxidative stress plays a key role in the development of metabolic complications associated with obesity, including insulin resistance and the most common chronic liver disease worldwide, nonalcoholic fatty liver disease. We have recently discovered that the microRNA miR-144 regulates protein levels of the master mediator of the antioxidant response, nuclear factor erythroid 2-related factor 2 (NRF2). On miR-144 silencing, the expression of NRF2 target genes was significantly upregulated, suggesting that miR-144 controls NRF2 at the level of both protein expression and activity. Here we explored a mechanism whereby hepatic miR-144 inhibited NRF2 activity upon obesity via the regulation of the tricarboxylic acid (TCA) metabolite, fumarate, a potent activator of NRF2. METHODS: We performed transcriptomic analysis in liver macrophages (LMs) of obese mice and identified the immuno-responsive gene 1 (Irg1) as a target of miR-144. IRG1 catalyzes the production of a TCA derivative, itaconate, an inhibitor of succinate dehydrogenase (SDH). TCA enzyme activities and kinetics were analyzed after miR-144 silencing in obese mice and human liver organoids using single-cell activity assays in situ and molecular dynamic simulations. RESULTS: Increased levels of miR-144 in obesity were associated with reduced expression of Irg1, which was restored on miR-144 silencing in vitro and in vivo. Furthermore, miR-144 overexpression reduces Irg1 expression and the production of itaconate in vitro. In alignment with the reduction in IRG1 levels and itaconate production, we observed an upregulation of SDH activity during obesity. Surprisingly, however, fumarate hydratase (FH) activity was also upregulated in obese livers, leading to the depletion of its substrate fumarate. miR-144 silencing selectively reduced the activities of both SDH and FH resulting in the accumulation of their related substrates succinate and fumarate. Moreover, molecular dynamics analyses revealed the potential role of itaconate as a competitive inhibitor of not only SDH but also FH. Combined, these results demonstrate that silencing of miR-144 inhibits the activity of NRF2 through decreased fumarate production in obesity. CONCLUSIONS: Herein we unravel a novel mechanism whereby miR-144 inhibits NRF2 activity through the consumption of fumarate by activation of FH. Our study demonstrates that hepatic miR-144 triggers a hyperactive FH in the TCA cycle leading to an impaired antioxidant response in obesity.

In-situ muconic acid extraction reveals sugar consumption bottleneck in a xylose-utilizing Saccharomyces cerevisiae strain.

BACKGROUND: The current shift from a fossil-resource based economy to a more sustainable, bio-based economy requires development of alternative production routes based on utilization of biomass for the many chemicals that are currently produced from petroleum. Muconic acid is an attractive platform chemical for the bio-based economy because it can be converted in chemicals with wide industrial applicability, such as adipic and terephthalic acid, and because its two double bonds offer great versatility for chemical modification. RESULTS: We have constructed a yeast cell factory converting glucose and xylose into muconic acid without formation of ethanol. We consecutively eliminated feedback inhibition in the shikimate pathway, inserted the heterologous pathway for muconic acid biosynthesis from 3-dehydroshikimate (DHS) by co-expression of DHS dehydratase from P. anserina, protocatechuic acid (PCA) decarboxylase (PCAD) from K. pneumoniae and oxygen-consuming catechol 1,2-dioxygenase (CDO) from C. albicans, eliminated ethanol production by deletion of the three PDC genes and minimized PCA production by enhancing PCAD overexpression and production of its co-factor. The yeast pitching rate was increased to lower high biomass formation caused by the compulsory aerobic conditions. Maximal titers of 4 g/L, 4.5 g/L and 3.8 g/L muconic acid were reached with glucose, xylose, and a mixture, respectively. The use of an elevated initial sugar level, resulting in muconic acid titers above 2.5 g/L, caused stuck fermentations with incomplete utilization of the sugar. Application of polypropylene glycol 4000 (PPG) as solvent for in situ product removal during the fermentation shows that this is not due to toxicity by the muconic acid produced. CONCLUSIONS: This work has developed an industrial yeast strain able to produce muconic acid from glucose and also with great efficiency from xylose, without any ethanol production, minimal production of PCA and reaching the highest titers in batch fermentation reported up to now. Utilization of higher sugar levels remained conspicuously incomplete. Since this was not due to product inhibition by muconic acid or to loss of viability, an unknown, possibly metabolic bottleneck apparently arises during muconic acid fermentation with high sugar levels and blocks further sugar utilization.

Itaconate confers tolerance to late NLRP3 inflammasome activation.

Itaconate is a unique regulatory metabolite that is induced upon Toll-like receptor (TLR) stimulation in myeloid cells. Here, we demonstrate major inflammatory tolerance and cell death phenotypes associated with itaconate production in activated macrophages. We show that endogenous itaconate is a key regulator of the signal 2 of NLR family pyrin domain containing 3 (NLRP3) inflammasome activation after long lipopolysaccharide (LPS) priming, which establishes tolerance to late NLRP3 inflammasome activation. We show that itaconate acts synergistically with inducible nitric oxide synthase (iNOS) and that the ability of various TLR ligands to establish NLRP3 inflammasome tolerance depends on the pattern of co-expression of IRG1 and iNOS. Mechanistically, itaconate accumulation upon prolonged inflammatory stimulation prevents full caspase-1 activation and processing of gasdermin D, which we demonstrate to be post-translationally modified by endogenous itaconate. Altogether, our data demonstrate that metabolic rewiring in inflammatory macrophages establishes tolerance to NLRP3 inflammasome activation that, if uncontrolled, can result in pyroptotic cell death and tissue damage.

Notch-Regulated Dendritic Cells Restrain Inflammation-Associated Colorectal Carcinogenesis.

Conventional dendritic cells (cDC) play a central role in T-cell antitumor responses. We studied the significance of Notch-regulated DC immune responses in a mouse model of colitis-associated colorectal cancer in which there is epithelial downregulation of Notch/Hes1 signaling. This defect phenocopies that caused by GMDS (GDP-mannose 4,6-dehydratase) mutation in human colorectal cancers. We found that, although wild-type immune cells restrained dysplasia progression and decreased the incidence of adenocarcinoma in chimeric mice, the immune system with Notch2 deleted in all blood lineages or in only DCs promoted inflammation-associated transformation. Notch2 signaling deficiency not only impaired cDC terminal differentiation, but also downregulated CCR7 expression, reduced DC migration, and suppressed antigen cross-presentation to CD8+ T cells. Transfer of Notch-primed DCs restrained inflammation-associated dysplasia progression. Consistent with the mouse data, we observed a correlation between infiltrating cDC1 and Notch2 signaling in human colorectal cancers and found that GMDS-mutant colorectal cancers showed decreased CCR7 expression and suppressed cDC1 signature gene expression. Suppressed cDC1 gene signature expression in human colorectal cancer was associated with a poor prognosis. In summary, our study supports an important role for Notch2 signaling in cDC1-mediated antitumor immunity and indicates that Notch2-controlled DCs restrain inflammation-associated colon cancer development in mice.