ABSTRACT
The evolution of eukaryotic genomes has been propelled by a series of gene duplication events, leading to an expansion in new functions and pathways. While duplicate genes may retain some functional redundancy, it is clear that to survive selection they cannot simply serve as a backup but rather must acquire distinct functions required for cellular processes to work accurately and efficiently. Understanding these differences and characterizing gene-specific functions is complex. Here we explore different gene pairs and families within the context of the endoplasmic reticulum (ER), the main cellular hub of lipid biosynthesis and the entry site for the secretory pathway. Focusing on each of the ER functions, we highlight specificities of related proteins and the capabilities conferred to cells through their conservation. More generally, these examples suggest why related genes have been maintained by evolutionary forces and provide a conceptual framework to experimentally determine why they have survived selection.
Subject(s)
Endoplasmic Reticulum/metabolism , Evolution, Molecular , Gene Duplication , Saccharomyces cerevisiae/metabolism , Selection, Genetic , Activating Transcription Factor 6/genetics , Activating Transcription Factor 6/metabolism , Animals , Antiporters/genetics , Antiporters/metabolism , Carboxy-Lyases/genetics , Carboxy-Lyases/metabolism , Endoplasmic Reticulum/genetics , Endoribonucleases/genetics , Endoribonucleases/metabolism , Eukaryotic Cells/cytology , Eukaryotic Cells/metabolism , HSP40 Heat-Shock Proteins/genetics , HSP40 Heat-Shock Proteins/metabolism , Hexosyltransferases/genetics , Hexosyltransferases/metabolism , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Sphingosine N-Acyltransferase/genetics , Sphingosine N-Acyltransferase/metabolism , Vesicular Transport Proteins/genetics , Vesicular Transport Proteins/metabolismABSTRACT
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.
Subject(s)
Anti-Inflammatory Agents , Glucocorticoids , Inflammation , Macrophages , Mitochondria , Succinates , Animals , Female , Humans , Male , Mice , Anti-Inflammatory Agents/pharmacology , Carboxy-Lyases/metabolism , Carboxy-Lyases/antagonists & inhibitors , Citric Acid Cycle/drug effects , Citric Acid Cycle/genetics , Cytokines/immunology , Cytokines/metabolism , Glucocorticoids/pharmacology , Glucocorticoids/metabolism , Hydro-Lyases/deficiency , Hydro-Lyases/genetics , Inflammation/drug therapy , Inflammation/metabolism , Macrophages/cytology , Macrophages/drug effects , Macrophages/immunology , Macrophages/metabolism , Mice, Inbred C57BL , Mitochondria/metabolism , Mitochondria/drug effects , Pyruvate Dehydrogenase Complex/metabolism , Receptors, Glucocorticoid/metabolism , Succinates/metabolism , Enzyme Activation/drug effectsABSTRACT
In this issue of Immunity, Daniels et al. (2019) demonstrate that RIPK3 signaling limits Zika virus (ZIKV) infection in the central nervous system independently of its function in necroptosis by promoting itaconate production in infected neurons, thereby revealing a neuron-specific mechanism of metabolite-mediated ZIKV control.
Subject(s)
Antiviral Agents , Zika Virus Infection , Zika Virus , Apoptosis , Carboxy-Lyases , Humans , Necrosis , Neurons , Nucleotides , Proteins , Receptor-Interacting Protein Serine-Threonine KinasesABSTRACT
Mitochondria depend on the import of phospholipid precursors for the biosynthesis of phosphatidylethanolamine (PE) and cardiolipin, yet the mechanism of their transport remains elusive. A dynamic lipidomics approach revealed that mitochondria preferentially import di-unsaturated phosphatidylserine (PS) for subsequent conversion to PE by the mitochondrial PS decarboxylase Psd1p. Several protein complexes tethering mitochondria to the endomembrane system have been implicated in lipid transport in yeast, including the endoplasmic reticulum (ER)-mitochondrial encounter structure (ERMES), ER-membrane complex (EMC), and the vacuole and mitochondria patch (vCLAMP). By limiting the availability of unsaturated phospholipids, we created conditions to investigate the mechanism of lipid transfer and the contributions of the tethering complexes in vivo. Under these conditions, inactivation of ERMES components or of the vCLAMP component Vps39p exacerbated accumulation of saturated lipid acyl chains, indicating that ERMES and Vps39p contribute to the mitochondrial sink for unsaturated acyl chains by mediating transfer of di-unsaturated phospholipids. These results support the concept that intermembrane lipid flow is rate-limited by molecular species-dependent lipid efflux from the donor membrane and driven by the lipid species' concentration gradient between donor and acceptor membrane.
Subject(s)
Mitochondria/metabolism , Phospholipids/metabolism , Adaptor Proteins, Vesicular Transport/genetics , Adaptor Proteins, Vesicular Transport/metabolism , Biological Transport , Carboxy-Lyases/genetics , Carboxy-Lyases/metabolism , Endoplasmic Reticulum/metabolism , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Saccharomyces cerevisiae , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
The Krebs cycle enzyme aconitate decarboxylase 1 (ACOD1) mediates itaconate synthesis in monocytes and macrophages. Previously, we reported that administration of 4-octyl itaconate to lupus-prone mice abrogated immune dysregulation and clinical features. In this study, we explore the role of the endogenous ACOD1/itaconate pathway in the development of TLR7-induced lupus (imiquimod [IMQ] model). We found that, in vitro, ACOD1 was induced in mouse bone marrow-derived macrophages and human monocyte-derived macrophages following TLR7 stimulation. This induction was partially dependent on type I IFN receptor signaling and on specific intracellular pathways. In the IMQ-induced mouse model of lupus, ACOD1 knockout (Acod1-/-) displayed disruptions of the splenic architecture, increased serum levels of anti-dsDNA and proinflammatory cytokines, and enhanced kidney immune complex deposition and proteinuria, when compared with the IMQ-treated wild-type mice. Consistent with these results, Acod1-/- bone marrow-derived macrophages treated in vitro with IMQ showed higher proinflammatory features. Furthermore, itaconate serum levels in systemic lupus erythematosus patients were decreased compared with healthy individuals, in association with disease activity and specific perturbed cardiometabolic parameters. These findings suggest that the ACOD1/itaconate pathway plays important immunomodulatory and vasculoprotective roles in systemic lupus erythematosus, supporting the potential therapeutic role of itaconate analogs in autoimmune diseases.
Subject(s)
Carboxy-Lyases , Lupus Erythematosus, Systemic , Macrophages , Mice, Knockout , Succinates , Animals , Lupus Erythematosus, Systemic/immunology , Mice , Humans , Female , Macrophages/immunology , Succinates/pharmacology , Cardiovascular Diseases/immunology , Biomarkers , Mice, Inbred C57BL , Signal Transduction/immunology , Adult , Male , Disease Models, Animal , Middle Aged , Cytokines/metabolism , Toll-Like Receptor 7/metabolism , Hydro-LyasesABSTRACT
Enzymes are increasingly explored for use in asymmetric synthesis1-3, but their applications are generally limited by the reactions available to naturally occurring enzymes. Recently, interest in photocatalysis4 has spurred the discovery of novel reactivity from known enzymes5. However, so far photoinduced enzymatic catalysis6 has not been used for the cross-coupling of two molecules. For example, the intermolecular coupling of alkenes with α-halo carbonyl compounds through a visible-light-induced radical hydroalkylation, which could provide access to important γ-chiral carbonyl compounds, has not yet been achieved by enzymes. The major challenges are the inherent poor photoreactivity of enzymes and the difficulty in achieving stereochemical control of the remote prochiral radical intermediate7. Here we report a visible-light-induced intermolecular radical hydroalkylation of terminal alkenes that does not occur naturally, catalysed by an 'ene' reductase using readily available α-halo carbonyl compounds as reactants. This method provides an efficient approach to the synthesis of various carbonyl compounds bearing a γ-stereocentre with excellent yields and enantioselectivities (up to 99 per cent yield with 99 per cent enantiomeric excess), which otherwise are difficult to access using chemocatalysis. Mechanistic studies suggest that the formation of the complex of the substrates (α-halo carbonyl compounds) and the 'ene' reductase triggers the enantioselective photoinduced radical reaction. Our work further expands the reactivity repertoire of biocatalytic, synthetically useful asymmetric transformations by the merger of photocatalysis and enzyme catalysis.
Subject(s)
Alkenes/chemistry , Alkenes/metabolism , Hydrogen/chemistry , Hydrogen/metabolism , Light , Oxidoreductases/metabolism , Photochemical Processes/radiation effects , Alcohols/chemistry , Alcohols/metabolism , Alkylation/radiation effects , Biocatalysis/radiation effects , Biomass , Carboxy-Lyases/metabolism , Flavins/metabolism , Models, Chemical , Models, Molecular , StereoisomerismABSTRACT
Phenazine-1-carboxylic acid decarboxylase (PhdA) is a prenylated-FMN-dependent (prFMN) enzyme belonging to the UbiD family of decarboxylases. Many UbiD-like enzymes catalyze (de)carboxylation reactions on aromatic rings and conjugated double bonds and are potentially valuable industrial catalysts. We have investigated the mechanism of PhdA using a slow turnover substrate, 2,3-dimethylquinoxaline-5-carboxylic acid (DQCA). Detailed analysis of the pH dependence and solvent deuterium isotope effects associated with the reaction uncovered unusual kinetic behavior. At low substrate concentrations, a substantial inverse solvent isotope effect (SIE) is observed on Vmax/KM of â¼ 0.5 when reaction rates of DQCA in H2O and D2O are compared. Under the same conditions, a normal SIE of 4.15 is measured by internal competition for proton transfer to the product. These apparently contradictory results indicate that the SIE values report on different steps in the mechanism. A proton inventory analysis of the reaction under Vmax/KM and Vmax conditions points to a "medium effect" as the source of the inverse SIE. Molecular dynamics simulations of the effect of D2O on PhdA structure support that D2O reduces the conformational lability of the enzyme and results in a more compact structure, akin to the active, "closed" conformer observed in crystal structures of some UbiD-like enzymes. Consistent with the simulations, PhdA was found to be more stable in D2O and to bind DQCA more tightly, leading to the observed rate enhancement under Vmax/KM conditions.
Subject(s)
Carboxy-Lyases , Carboxy-Lyases/chemistry , Isotopes , Kinetics , Phenazines , Protons , Solvents , Mycobacteriaceae/enzymologyABSTRACT
The UbiD enzyme family of prenylated flavin (prFMN)-dependent reversible decarboxylases is near ubiquitously present in microbes. For some UbiD family members, enzyme activation through prFMNH2 binding and subsequent oxidative maturation of the cofactor readily occurs, both in vivo in a heterologous host and through in vitro reconstitution. However, isolation of the active holo-enzyme has proven intractable for others, notably the canonical Escherichia coli UbiD. We show that E. coli heterologous expression of the small protein LpdD-associated with the UbiD-like gallate decarboxylase LpdC from Lactobacillus plantarum-unexpectedly leads to 3,4-dihydroxybenzoic acid decarboxylation whole-cell activity. This activity was shown to be linked to endogenous E. coli ubiD expression levels. The crystal structure of the purified LpdD reveals a dimeric protein with structural similarity to the eukaryotic heterodimeric proteasome assembly chaperone Pba3/4. Solution studies demonstrate that LpdD protein specifically binds to reduced prFMN species only. The addition of the LpdD-prFMNH2 complex supports reconstitution and activation of the purified E. coli apo-UbiD in vitro, leading to modest 3,4-dihydroxybenzoic acid decarboxylation. These observations suggest that LpdD acts as a prFMNH2-binding chaperone, enabling apo-UbiD activation through enhanced prFMNH2 incorporation and subsequent oxidative maturation. Hence, while a single highly conserved flavin prenyltransferase UbiX is found associated with UbiD enzymes, our observations suggest considerable diversity in UbiD maturation, ranging from robust autocatalytic to chaperone-mediated processes. Unlocking the full (de)carboxylation scope of the UbiD-enzyme family will thus require more than UbiX coexpression.
Subject(s)
Carboxy-Lyases , Hydroxybenzoates , Lactobacillaceae , Carboxy-Lyases/genetics , Carboxy-Lyases/chemistry , Escherichia coli/metabolism , Flavins/metabolism , Oxidation-Reduction , Lactobacillaceae/genetics , Lactobacillaceae/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Protein BindingABSTRACT
YisK is an uncharacterized protein in Bacillus subtilis previously shown to interact genetically with the elongasome protein Mbl. YisK overexpression leads to cell widening and lysis, phenotypes that are dependent on mbl and suppressed by mbl mutations. In the present work, we characterize YisK's localization, structure, and enzymatic activity. We show that YisK localizes as puncta that depend on Mbl. YisK belongs to the fumarylacetoacetate hydrolase (FAH) superfamily, and crystal structures revealed close structural similarity to two oxaloacetate (OAA) decarboxylases: human mitochondrial FAHD1 and Corynebacterium glutamicum Cg1458. We demonstrate that YisK can also catalyze the decarboxylation of OAA (K m = 134 µM, K cat = 31 min-1). A catalytic dead variant (YisK E148A, E150A) retains wild-type localization and still widens cells following overexpression, indicating these activities are not dependent on YisK catalysis. Conversely, a non-localizing variant (YisK E30A) retains wild-type enzymatic activity in vitro but localizes diffusely and no longer widens cells following overexpression. Together, these results suggest that YisK may be subject to spatial regulation that depends on the cell envelope synthesis machinery. IMPORTANCE The elongasome is a multiprotein complex that guides lengthwise growth in some bacteria. We previously showed that, in B. subtilis, overexpression of an uncharacterized putative enzyme (YisK) perturbed function of the actin-like elongasome protein Mbl. Here, we show that YisK exhibits Mbl-dependent localization. Through biochemical and structural characterization, we demonstrate that, like its mitochondrial homolog FAHD1, YisK can catalyze the decarboxylation of the oxaloacetate to pyruvate and CO2. YisK is the first example of an enzyme implicated in central carbon metabolism with subcellular localization that depends on Mbl.
Subject(s)
Bacillus subtilis , Carboxy-Lyases , Humans , Bacillus subtilis/metabolism , Carboxy-Lyases/genetics , Pyruvic Acid , Oxaloacetates , Hydrolases/geneticsABSTRACT
Glycerophospholipids have emerged as a significant contributor to the intracellular growth of pathogenic protist Toxoplasma gondii. Phosphatidylserine (PtdSer) is one such lipid, attributed to the locomotion and motility-dependent invasion and egress events in its acutely infectious tachyzoite stage. However, the de novo synthesis of PtdSer and the importance of the pathway in tachyzoites remain poorly understood. We show that a base-exchange-type PtdSer synthase (PSS) located in the parasite's endoplasmic reticulum produces PtdSer, which is rapidly converted to phosphatidylethanolamine (PtdEtn) by PtdSer decarboxylase (PSD) activity. The PSS-PSD pathway enables the synthesis of several lipid species, including PtdSer (16:0/18:1) and PtdEtn (18:2/20:4, 18:1/18:2 and 18:2/22:5). The PSS-depleted strain exhibited a lower abundance of the major ester-linked PtdEtn species and concurrent accrual of host-derived ether-PtdEtn species. Most phosphatidylthreonine (PtdThr) species-an exclusive natural analog of PtdSer, also made in the endoplasmic reticulum-were repressed. PtdSer species, however, remained largely unaltered, likely due to the serine-exchange reaction of PtdThr synthase in favor of PtdSer upon PSS depletion. Not least, the loss of PSS abrogated the lytic cycle of tachyzoites, impairing the cell division, motility, and egress. In a nutshell, our data demonstrate a critical role of PSS in the biogenesis of PtdSer and PtdEtn species and its physiologically essential repurposing for the asexual reproduction of a clinically relevant intracellular pathogen.
Subject(s)
Endoplasmic Reticulum , Toxoplasma , Toxoplasma/enzymology , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/enzymology , Humans , Phosphatidylserines/metabolism , CDPdiacylglycerol-Serine O-Phosphatidyltransferase/metabolism , CDPdiacylglycerol-Serine O-Phosphatidyltransferase/genetics , Transferases (Other Substituted Phosphate Groups)/metabolism , Transferases (Other Substituted Phosphate Groups)/genetics , Carboxy-LyasesABSTRACT
The everninomicins are bacterially produced antibiotic octasaccharides characterized by the presence of two interglycosidic spirocyclic ortho-δ-lactone (orthoester) moieties. The terminating G- and H-ring sugars, L-lyxose and C-4 branched sugar ß-D-eurekanate, are proposed to be biosynthetically derived from nucleotide diphosphate pentose sugar pyranosides; however, the identity of these precursors and their biosynthetic origin remain to be determined. Herein we identify a new glucuronic acid decarboxylase from Micromonospora belonging to the superfamily of short-chain dehydrogenase/reductase enzymes, EvdS6. Biochemical characterization demonstrated that EvdS6 is an NAD+-dependent bifunctional enzyme that produces a mixture of two products, differing in the sugar C-4 oxidation state. This product distribution is atypical for glucuronic acid decarboxylating enzymes, most of which favor production of the reduced sugar and a minority of which favor release of the oxidized product. Spectroscopic and stereochemical analysis of reaction products revealed that the first product released is the oxidatively produced 4-keto-D-xylose and the second product is the reduced D-xylose. X-ray crystallographic analysis of EvdS6 at 1.51 Å resolution with bound co-factor and TDP demonstrated that the overall geometry of the EvdS6 active site is conserved with other SDR enzymes and enabled studies probing structural determinants for the reductive half of the net neutral catalytic cycle. Critical active site threonine and aspartate residues were unambiguously identified as essential in the reductive step of the reaction and resulted in enzyme variants producing almost exclusively the keto sugar. This work defines potential precursors for the G-ring L-lyxose and resolves likely origins of the H-ring ß-D-eurekanate sugar precursor.
Subject(s)
Aminoglycosides , Bacterial Proteins , Carboxy-Lyases , Micromonospora , Multigene Family , Xylose , Aminoglycosides/genetics , Carboxy-Lyases/genetics , Carboxy-Lyases/metabolism , Crystallography, X-Ray , Micromonospora/enzymology , Micromonospora/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolismABSTRACT
S-adenosylmethionine decarboxylase (AdoMetDC/SpeD) is a key polyamine biosynthetic enzyme required for conversion of putrescine to spermidine. Autocatalytic self-processing of the AdoMetDC/SpeD proenzyme generates a pyruvoyl cofactor from an internal serine. Recently, we discovered that diverse bacteriophages encode AdoMetDC/SpeD homologs that lack AdoMetDC activity and instead decarboxylate L-ornithine or L-arginine. We reasoned that neofunctionalized AdoMetDC/SpeD homologs were unlikely to have emerged in bacteriophages and were probably acquired from ancestral bacterial hosts. To test this hypothesis, we sought to identify candidate AdoMetDC/SpeD homologs encoding L-ornithine and L-arginine decarboxylases in bacteria and archaea. We searched for the anomalous presence of AdoMetDC/SpeD homologs in the absence of its obligatory partner enzyme spermidine synthase, or the presence of two AdoMetDC/SpeD homologs encoded in the same genome. Biochemical characterization of candidate neofunctionalized genes confirmed lack of AdoMetDC activity, and functional presence of L-ornithine or L-arginine decarboxylase activity in proteins from phyla Actinomycetota, Armatimonadota, Planctomycetota, Melainabacteria, Perigrinibacteria, Atribacteria, Chloroflexota, Sumerlaeota, Omnitrophota, Lentisphaerota, and Euryarchaeota, the bacterial candidate phyla radiation and DPANN archaea, and the δ-Proteobacteria class. Phylogenetic analysis indicated that L-arginine decarboxylases emerged at least three times from AdoMetDC/SpeD, whereas L-ornithine decarboxylases arose only once, potentially from the AdoMetDC/SpeD-derived L-arginine decarboxylases, revealing unsuspected polyamine metabolic plasticity. Horizontal transfer of the neofunctionalized genes appears to be the more prevalent mode of dissemination. We identified fusion proteins of bona fide AdoMetDC/SpeD with homologous L-ornithine decarboxylases that possess two, unprecedented internal protein-derived pyruvoyl cofactors. These fusion proteins suggest a plausible model for the evolution of the eukaryotic AdoMetDC.
Subject(s)
Adenosylmethionine Decarboxylase , Carboxy-Lyases , Adenosylmethionine Decarboxylase/genetics , Adenosylmethionine Decarboxylase/metabolism , Archaea/genetics , Archaea/metabolism , Ornithine , Phylogeny , Carboxy-Lyases/genetics , Carboxy-Lyases/metabolism , Polyamines/metabolism , Bacteria/metabolism , Ornithine Decarboxylase/metabolism , Arginine/geneticsABSTRACT
Decarboxylation of phosphatidylserine (PS) to form phosphatidylethanolamine by PS decarboxylases (PSDs) is an essential process in most eukaryotes. Processing of a malarial PSD proenzyme into its active alpha and beta subunits is by an autoendoproteolytic mechanism regulated by anionic phospholipids, with PS serving as an activator and phosphatidylglycerol (PG), phosphatidylinositol, and phosphatidic acid acting as inhibitors. The biophysical mechanism underlying this regulation remains unknown. We used solid phase lipid binding, liposome-binding assays, and surface plasmon resonance to examine the binding specificity of a processing-deficient Plasmodium PSD (PkPSDS308A) mutant enzyme and demonstrated that the PSD proenzyme binds strongly to PS and PG but not to phosphatidylethanolamine and phosphatidylcholine. The equilibrium dissociation constants (Kd) of PkPSD with PS and PG were 80.4 nM and 66.4 nM, respectively. The interaction of PSD with PS is inhibited by calcium, suggesting that the binding mechanism involves ionic interactions. In vitro processing of WT PkPSD proenzyme was also inhibited by calcium, consistent with the conclusion that PS binding to PkPSD through ionic interactions is required for the proenzyme processing. Peptide mapping identified polybasic amino acid motifs in the proenzyme responsible for binding to PS. Altogether, the data demonstrate that malarial PSD maturation is regulated through a strong physical association between PkPSD proenzyme and anionic lipids. Inhibition of the specific interaction between the proenzyme and the lipids can provide a novel mechanism to disrupt PSD enzyme activity, which has been suggested as a target for antimicrobials, and anticancer therapies.
Subject(s)
Carboxy-Lyases , Malaria , Phospholipids , Plasmodium , Amino Acid Motifs , Calcium/metabolism , Calcium/pharmacology , Carboxy-Lyases/antagonists & inhibitors , Carboxy-Lyases/chemistry , Carboxy-Lyases/metabolism , Enzyme Precursors/metabolism , Liposomes , Phosphatidic Acids/metabolism , Phosphatidic Acids/pharmacology , Phosphatidylcholines/metabolism , Phosphatidylcholines/pharmacology , Phosphatidylethanolamines/metabolism , Phosphatidylethanolamines/pharmacology , Phosphatidylglycerols/metabolism , Phosphatidylglycerols/pharmacology , Phosphatidylinositols/metabolism , Phosphatidylinositols/pharmacology , Phosphatidylserines/metabolism , Phosphatidylserines/pharmacology , Phospholipids/chemistry , Phospholipids/metabolism , Phospholipids/pharmacology , Protein Binding , Malaria/parasitology , Proteolysis/drug effects , Surface Plasmon Resonance , Plasmodium/enzymologyABSTRACT
Excessive inflammation is a major cause of morbidity and mortality in many viral infections including influenza. Therefore, there is a need for therapeutic interventions that dampen and redirect inflammatory responses and, ideally, exert antiviral effects. Itaconate is an immunomodulatory metabolite which also reprograms cell metabolism and inflammatory responses when applied exogenously. We evaluated effects of endogenous itaconate and exogenous application of itaconate and its variants dimethyl- and 4-octyl-itaconate (DI, 4OI) on host responses to influenza A virus (IAV). Infection induced expression of ACOD1, the enzyme catalyzing itaconate synthesis, in monocytes and macrophages, which correlated with viral replication and was abrogated by DI and 4OI treatment. In IAV-infected mice, pulmonary inflammation and weight loss were greater in Acod1-/- than in wild-type mice, and DI treatment reduced pulmonary inflammation and mortality. The compounds reversed infection-triggered interferon responses and modulated inflammation in human cells supporting non-productive and productive infection, in peripheral blood mononuclear cells, and in human lung tissue. All three itaconates reduced ROS levels and STAT1 phosphorylation, whereas AKT phosphorylation was reduced by 4OI and DI but increased by itaconate. Single-cell RNA sequencing identified monocytes as the main target of infection and the exclusive source of ACOD1 mRNA in peripheral blood. DI treatment silenced IFN-responses predominantly in monocytes, but also in lymphocytes and natural killer cells. Ectopic synthesis of itaconate in A549 cells, which do not physiologically express ACOD1, reduced infection-driven inflammation, and DI reduced IAV- and IFNγ-induced CXCL10 expression in murine macrophages independent of the presence of endogenous ACOD1. The compounds differed greatly in their effects on cellular gene homeostasis and released cytokines/chemokines, but all three markedly reduced release of the pro-inflammatory chemokines CXCL10 (IP-10) and CCL2 (MCP-1). Viral replication did not increase under treatment despite the dramatically repressed IFN responses. In fact, 4OI strongly inhibited viral transcription in peripheral blood mononuclear cells, and the compounds reduced viral titers (4OI>Ita>DI) in A549 cells whereas viral transcription was unaffected. Taken together, these results reveal itaconates as immunomodulatory and antiviral interventions for influenza virus infection.
Subject(s)
Influenza A virus/immunology , Macrophages/immunology , Orthomyxoviridae Infections/drug therapy , Succinates/pharmacology , A549 Cells , Animals , Carboxy-Lyases/deficiency , Carboxy-Lyases/immunology , Cytokines/genetics , Cytokines/immunology , Humans , Macrophages/virology , Mice , Mice, Knockout , Orthomyxoviridae Infections/genetics , Orthomyxoviridae Infections/immunology , THP-1 CellsABSTRACT
Aspergillus oryzae spores, when sprinkled onto steamed rice and allowed to propagate, are referred to as rice "koji." Agmatine, a natural polyamine derived from arginine through the action of arginine decarboxylase (ADC), is abundantly produced by solid state-cultivated rice koji of A. oryzae RIB40 under low pH conditions, despite the apparent absence of ADC orthologs in its genome. Mass spectrometry imaging revealed that agmatine was accumulated inside rice koji at low pH conditions, where arginine was distributed. ADC activity was predominantly observed in substrate mycelia and minimally in aerial mycelia. Natural ADC was isolated from solid state-cultivated A. oryzae rice koji containing substrate mycelia, using ammonium sulfate fractionation, ion exchange, and gel-filtration chromatography. The purified protein was subjected to sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE), and the detected peptide band was digested for identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The gene AO090102000327 of strain RIB40 was identified, previously annotated as phosphatidylserine decarboxylase (PSD), and encoded a 483-amino acid peptide. Recombinant protein encoded by AO090102000327 was expressed in Escherichia coli cells cultivated at 20°C, resulting in the detection of 49 kDa and 5 kDa peptides. The protein exhibited pyruvoyl-dependent decarboxylase activity, favoring arginine over ornithine and showing no activity with phosphatidylserine. The gene was designated Ao-adc1. Ao-ADC1 expression in rice koji at pH 4-6 was confirmed through western blotting using the anti-Ao-ADC1 serum. These findings indicate that Ao-adc1 encodes arginine decarboxylase involved in agmatine production.IMPORTANCEGene AO090102000327 in A. oryzae RIB40, previously annotated as a PSD, falls into a distinct clade when examining the phylogenetic distribution of PSDs. Contrary to the initial PSD annotation, our analysis indicates that the protein encoded by AO090102000327 is expressed in the substrate mycelia area of solid state-cultivated A. oryzae rice koji and functions as an arginine decarboxylase (ADC). The clade to which Ao-ADC1 belongs includes three other Ao-ADC1 paralogs (AO090103000445, AO090701000800, and AO090701000802) that presumably encode ADC rather than PSDs. Regarding PSD, AO090012000733 and AO090005001124 were speculated to be nonmitochondrial and mitochondrial PSDs in A. oryzae RIB40, respectively.
Subject(s)
Aspergillus oryzae , Carboxy-Lyases , Fungal Proteins , Oryza , Aspergillus oryzae/genetics , Aspergillus oryzae/enzymology , Carboxy-Lyases/genetics , Carboxy-Lyases/metabolism , Carboxy-Lyases/chemistry , Oryza/microbiology , Fungal Proteins/genetics , Fungal Proteins/metabolism , Fungal Proteins/chemistry , Agmatine/metabolismABSTRACT
Ten Gouda cheese wheels with an age of 31 weeks from six different batch productions were affected by a crack defect and displayed an unpleasant off-flavor. To unravel the causes of these defects, the concentrations of free amino acids, other organic acids, volatile organic compounds, and biogenic amines were quantified in zones around the cracks and in zones without cracks, and compared with those of similar Gouda cheeses without crack defect. The Gouda cheeses with cracks had a significantly different metabolome. The production of the non-proteinogenic amino acid γ-aminobutyric acid (GABA) could be unraveled as the key mechanism leading to crack formation, although the production of the biogenic amines cadaverine and putrescine contributed as well. High-throughput amplicon sequencing of the full-length 16S rRNA gene based on whole-community DNA revealed the presence of Loigolactobacillus rennini and Tetragenococcus halophilus as most abundant non-starter lactic acid bacteria in the zones with cracks. Shotgun metagenomic sequencing allowed to obtain a metagenome-assembled genome of both Loil. rennini and T. halophilus. However, only Loil. rennini contained genes necessary for the production of GABA, cadaverine, and putrescine. Metagenetics further revealed the brine and the rennet used during cheese manufacturing as the most plausible inoculation sources of both Loil. rennini and T. halophilus.IMPORTANCECrack defects in Gouda cheeses are still poorly understood, although they can lead to major economic losses in cheese companies. In this study, the bacterial cause of a crack defect in Gouda cheeses was identified, and the pathways involved in the crack formation were unraveled. Moreover, possible contamination sources were identified. The brine bath might be a major source of bacteria with the potential to deteriorate cheese quality, which suggests that cheese producers should regularly investigate the quality and microbial composition of their brines. This study illustrated how a multiphasic approach can understand and mitigate problems in a cheese company.
Subject(s)
Carboxy-Lyases , Cheese , Lactobacillales , Lactobacillus , Salts , Lactobacillales/genetics , Cheese/microbiology , RNA, Ribosomal, 16S/genetics , Cadaverine , Putrescine , Bacteria/genetics , gamma-Aminobutyric Acid , Lactic Acid , Food MicrobiologyABSTRACT
Biallelic variants in PISD cause a phenotypic spectrum ranging from short stature with spondyloepimetaphyseal dysplasia (SEMD) to a multisystem disorder affecting eyes, ears, bones, and brain. PISD encodes the mitochondrial-localized enzyme phosphatidylserine decarboxylase. The PISD precursor is self-cleaved to generate a heteromeric mature enzyme that converts phosphatidylserine to the phospholipid phosphatidylethanolamine. We describe a 17-year-old male patient, born to unrelated healthy parents, with disproportionate short stature and SEMD, featuring platyspondyly, prominent epiphyses, and metaphyseal dysplasia. Trio genome sequencing revealed compound heterozygous PISD variants c.569C>T; p.(Ser190Leu) and c.799C>T; p.(His267Tyr) in the patient. Investigation of fibroblasts showed similar levels of the PISD precursor protein in both patient and control cells. However, patient cells had a significantly higher proportion of fragmented mitochondria compared to control cells cultured under basal condition and after treatment with 2-deoxyglucose that represses glycolysis and stimulates respiration. Structural data from the PISD orthologue in Escherichia coli suggest that the amino acid substitutions Ser190Leu and His267Tyr likely impair PISD's autoprocessing activity and/or phosphatidylethanolamine biosynthesis. Based on the data, we propose that the novel PISD p.(Ser190Leu) and p.(His267Tyr) variants likely act as hypomorphs and underlie the pure skeletal phenotype in the patient.
Subject(s)
Carboxy-Lyases , Mitochondria , Mutation, Missense , Osteochondrodysplasias , Humans , Osteochondrodysplasias/genetics , Osteochondrodysplasias/pathology , Male , Mutation, Missense/genetics , Adolescent , Mitochondria/genetics , Mitochondria/pathology , Carboxy-Lyases/genetics , Alleles , Phenotype , Dwarfism/genetics , Dwarfism/pathologyABSTRACT
Verticillium wilt, a soilborne vascular disease caused by Verticillium dahliae, strongly affects cotton yield and quality. In this study, an isolated rhizosphere bacterium, designated Bacillus velezensis BvZ45-1, exhibited >46% biocontrol efficacy against cotton verticillium wilt under greenhouse and field conditions. Moreover, through crude protein extraction and mass spectrometry analyses, we found many antifungal compounds present in the crude protein extract of BvZ45-1. The purified oxalate decarboxylase Odx_S12 from BvZ45-1 inhibited the growth of V. dahliae Vd080 by reducing the spore yield, causing mycelia to rupture, spore morphology changes, cell membrane rupture, and cell death. Subsequently, overexpression of Odx_S12 in Arabidopsis significantly improved plant resistance to V. dahliae. Through studies of the resistance mechanism of Odx_S12, V. dahliae was shown to produce oxalic acid (OA), which has a toxic effect on Arabidopsis leaves. Odx_S12 overexpression reduced Arabidopsis OA content, enhanced tolerance to OA, and improved resistance to verticillium wilt. Transcriptomics and quantitative real-time PCR analysis revealed that Odx_S12 promoted a reactive oxygen species burst and a salicylic acid- and abscisic acid-mediated defence response in Arabidopsis. In summary, this study not only identified B. velezensis BvZ45-1 as an efficient biological control agent, but also identified the resistance gene Odx_S12 as a candidate for cotton breeding against verticillium wilt.
Subject(s)
Arabidopsis , Ascomycota , Bacillus , Carboxy-Lyases , Gossypium , Plant Diseases , Plant Diseases/microbiology , Plant Diseases/immunology , Bacillus/physiology , Gossypium/genetics , Gossypium/microbiology , Gossypium/metabolism , Carboxy-Lyases/metabolism , Carboxy-Lyases/genetics , Arabidopsis/genetics , Arabidopsis/microbiology , Arabidopsis/metabolism , Arabidopsis/immunology , Ascomycota/physiology , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Disease Resistance/genetics , Verticillium/physiologyABSTRACT
BACKGROUND: Obesity is the main risk factor leading to the development of various respiratory diseases, such as asthma and pulmonary hypertension. Pulmonary microvascular endothelial cells (PMVECs) play a significant role in the development of lung diseases. Aconitate decarboxylase 1 (Acod1) mediates the production of itaconate, and Acod1/itaconate axis has been reported to play a protective role in multiple diseases. However, the roles of Acod1/itaconate axis in the PMVECs of obese mice are still unclear. METHODS: mRNA-seq was performed to identify the differentially expressed genes (DEGs) between high-fat diet (HFD)-induced PMVECs and chow-fed PMVECs in mice (|log2 fold change| ≥ 1, p ≤ 0.05). Free fatty acid (FFA) was used to induce cell injury, inflammation and mitochondrial oxidative stress in mouse PMVECs after transfection with the Acod1 overexpressed plasmid or 4-Octyl Itaconate (4-OI) administration. In addition, we investigated whether the nuclear factor erythroid 2-like 2 (Nrf2) pathway was involved in the effects of Acod1/itaconate in FFA-induced PMVECs. RESULTS: Down-regulated Acod1 was identified in HFD mouse PMVECs by mRNA-seq. Acod1 expression was also reduced in FFA-treated PMVECs. Acod1 overexpression inhibited cell injury, inflammation and mitochondrial oxidative stress induced by FFA in mouse PMVECs. 4-OI administration showed the consistent results in FFA-treated mouse PMVECs. Moreover, silencing Nrf2 reversed the effects of Acod1 overexpression and 4-OI administration in FFA-treated PMVECs, indicating that Nrf2 activation was required for the protective effects of Acod1/itaconate. CONCLUSION: Our results demonstrated that Acod1/Itaconate axis might protect mouse PMVECs from FFA-induced injury, inflammation and mitochondrial oxidative stress via activating Nrf2 pathway. It was meaningful for the treatment of obesity-caused pulmonary microvascular endotheliopathy.
Subject(s)
Carboxy-Lyases , Endothelial Cells , Lung , Mice, Inbred C57BL , NF-E2-Related Factor 2 , Obesity , Succinates , Animals , NF-E2-Related Factor 2/metabolism , NF-E2-Related Factor 2/genetics , Mice , Endothelial Cells/metabolism , Endothelial Cells/drug effects , Endothelial Cells/pathology , Carboxy-Lyases/metabolism , Carboxy-Lyases/genetics , Obesity/metabolism , Obesity/complications , Male , Succinates/pharmacology , Lung/metabolism , Lung/drug effects , Lung/pathology , Lung/blood supply , Cells, Cultured , Microvessels/metabolism , Microvessels/drug effects , Microvessels/pathology , Oxidative Stress/drug effects , Oxidative Stress/physiology , Diet, High-Fat/adverse effects , Endothelium, Vascular/metabolism , Endothelium, Vascular/drug effects , Endothelium, Vascular/pathology , Hydro-LyasesABSTRACT
BACKGROUND: Perinatal depression (including antenatal-, postnatal-, and depression that spans both timepoints) is a prevalent disorder with high morbidity that affects both mother and child. Even though the full biological blueprints of perinatal depression remain incomplete, multiple studies indicate that, at least for antenatal depression, the disorder has an inflammatory component likely linked to a dysregulation of the enzymatic kynurenine pathway. The production of neuroactive metabolites in this pathway, including quinolinic acid (QUIN), is upregulated in the placenta due to the multiple immunological roles of the metabolites during pregnancy. Since neuroactive metabolites produced by the pathway also may affect mood by directly affecting glutamate neurotransmission, we sought to investigate whether the placental expression of kynurenine pathway enzymes controlling QUIN production was associated with both peripheral inflammation and depressive symptoms during pregnancy. METHODS: 68 placentas obtained at birth were analyzed using qPCR to determine the expression of kynurenine pathway enzymes. Cytokines and metabolites were quantified in plasma using high-sensitivity electroluminescence and ultra-performance liquid chromatography, respectively. Maternal depressive symptoms were assessed using the Edinburgh Postnatal Depression Scale (EPDS) throughout pregnancy and the post-partum. Associations between these factors were assessed using robust linear regression with ranked enzymes. RESULTS: Low placental quinolinate phosphoribosyl transferase (QPRT), the enzyme responsible for degrading QUIN, was associated with higher IL-6 and higher QUIN/kynurenic acid ratios at the 3rd trimester. Moreover, women with severe depressive symptoms in the 3rd trimester had significantly lower placental expression of both QPRT and 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase (ACMSD); impaired activity of these two enzymes leads to QUIN accumulation. CONCLUSION: Overall, our data support that a compromised placental environment, featuring low expression of critical kynurenine pathway enzymes is associated with increased levels of plasma cytokines and the dysregulated kynurenine metabolite pattern observed in depressed women during pregnancy.