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1.
Nat Commun ; 15(1): 2999, 2024 Apr 08.
Artículo en Inglés | MEDLINE | ID: mdl-38589375

RESUMEN

Ribose-5-phosphate (R5P) is a precursor for nucleic acid biogenesis; however, the importance and homeostasis of R5P in the intracellular parasite Toxoplasma gondii remain enigmatic. Here, we show that the cytoplasmic sedoheptulose-1,7-bisphosphatase (SBPase) is dispensable. Still, its co-deletion with transaldolase (TAL) impairs the double mutant's growth and increases 13C-glucose-derived flux into pentose sugars via the transketolase (TKT) enzyme. Deletion of the latter protein affects the parasite's fitness but is not lethal and is correlated with an increased carbon flux via the oxidative pentose phosphate pathway. Further, loss of TKT leads to a decline in 13C incorporation into glycolysis and the TCA cycle, resulting in a decrease in ATP levels and the inability of phosphoribosyl-pyrophosphate synthetase (PRPS) to convert R5P into 5'-phosphoribosyl-pyrophosphate and thereby contribute to the production of AMP and IMP. Likewise, PRPS is essential for the lytic cycle. Not least, we show that RuPE-mediated metabolic compensation is imperative for the survival of the ΔsbpaseΔtal strain. In conclusion, we demonstrate that multiple routes can flexibly supply R5P to enable parasite growth and identify catalysis by TKT and PRPS as critical enzymatic steps. Our work provides novel biological and therapeutic insights into the network design principles of intracellular parasitism in a clinically-relevant pathogen.


Asunto(s)
Toxoplasma , Toxoplasma/metabolismo , Difosfatos/metabolismo , Ribosamonofosfatos/metabolismo , Glucólisis , Vía de Pentosa Fosfato
2.
Mol Biol Rep ; 51(1): 578, 2024 Apr 26.
Artículo en Inglés | MEDLINE | ID: mdl-38668789

RESUMEN

Mg2+-independent phosphatidic acid phosphatase (PAP2), diacylglycerol pyrophosphate phosphatase 1 (Dpp1) is a membrane-associated enzyme in Saccharomyces cerevisiae. The enzyme is responsible for inducing the breakdown of ß-phosphate from diacylglycerol pyrophosphate (DGPP) into phosphatidate (PA) and then removes the phosphate from PA to give diacylglycerol (DAG). In this study through RNAi suppression, we have demonstrated that Trypanosoma brucei diacylglycerol pyrophosphate phosphatase 1 (TbDpp1) procyclic form production is not required for parasite survival in culture. The steady-state levels of triacylglycerol (TAG), the number of lipid droplets, and the PA content are all maintained constant through the inducible down-regulation of TbDpp1. Furthermore, the localization of C-terminally tagged variants of TbDpp1 in the lysosome was demonstrated by immunofluorescence microscopy.


Asunto(s)
Glicerol/análogos & derivados , Lisosomas , Trypanosoma brucei brucei , Trypanosoma brucei brucei/enzimología , Trypanosoma brucei brucei/genética , Lisosomas/metabolismo , Lisosomas/enzimología , Triglicéridos/metabolismo , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Fosfatidato Fosfatasa/metabolismo , Fosfatidato Fosfatasa/genética , Interferencia de ARN , Difosfatos/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Monoéster Fosfórico Hidrolasas/genética , Diglicéridos/metabolismo , Ácidos Fosfatidicos/metabolismo
3.
Arch Biochem Biophys ; 756: 109995, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38621448

RESUMEN

T4 polynucleotide kinase (T4 PNK) phosphorylates the 5'-terminus of DNA and RNA substrates. It is widely used in molecular biology. Single nucleotides can serve as substrates if a 3'-phosphate group is present. In this study, the T4 PNK-catalyzed conversion of adenosine 3'-monophosphate (3'-AMP) to adenosine-3',5'-bisphosphate was characterized using isothermal titration calorimetry (ITC). Although ITC is typically used to study ligand binding, in this case the instrument was used to evaluate enzyme kinetics by monitoring the heat production due to reaction enthalpy. The reaction was initiated with a single injection of 3'-AMP substrate into the sample cell containing T4 PNK and ATP at pH 7.6 and 30 °C, and Michaelis-Menten analysis was performed on the reaction rates derived from the plot of differential power versus time. The Michaelis-Menten constant, KM, was 13 µM, and the turnover number, kcat, was 8 s-1. The effect of inhibitors was investigated using pyrophosphate (PPi). PPi caused a dose-dependent decrease in the apparent kcat and increase in the apparent KM under the conditions tested. Additionally, the intrinsic reaction enthalpy and the activation energy of the T4 PNK-catalyzed phosphorylation of 3'-AMP were determined to be -25 kJ/mol and 43 kJ/mol, respectively. ITC is seldom used as a tool to study enzyme kinetics, particularly for technically-challenging enzymes such as kinases. This study demonstrates that quantitative analysis of kinase activity can be amenable to the ITC single injection approach.


Asunto(s)
Calorimetría , Polinucleótido 5'-Hidroxil-Quinasa , Cinética , Calorimetría/métodos , Polinucleótido 5'-Hidroxil-Quinasa/metabolismo , Polinucleótido 5'-Hidroxil-Quinasa/química , Adenosina Monofosfato/química , Adenosina Monofosfato/metabolismo , Termodinámica , Bacteriófago T4/enzimología , Difosfatos/química , Difosfatos/metabolismo , Fosforilación
4.
Colloids Surf B Biointerfaces ; 238: 113872, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38555762

RESUMEN

Elucidation on the emulsifying behaviors of goose liver protein (GLP) from interfacial perspective was scarce when protein charging was altered. This work aimed to elucidate the role of phosphorylation on the interfacial associative interaction and then emulsion stabilizing properties of GLP using three structurally relevant phosphates of sodium trimetaphosphate (STMP), sodium tripolyphosphate (STPP) and sodium pyrophosphate (TSPP). A monotonic increment of protein charging treated from STMP, STPP to TSPP caused progressively increased particle de-aggregation, surface hydrophobicity and structural flexibility of GLP. Compared with STMP and TSPP, STPP phosphorylation rendered the most strengthened interfacial equilibrium pressure (11.98 ± 0.24 mN/m) due to sufficient unfolding but moderated charging character conveyed. Desorption curve and interfacial protein microstructure indicated that STPP phosphorylation caused the highest interfacial connectivity between proteins adsorbed onto the same droplet, as was also verified by interfacial elastic modulus (10.3 ± 0.21 mN/m). STPP treated GLP also yielded lowest droplet size (8.16 ± 0.10 µm), flocculation (8.18%) and Turbiscan stability index (8.78 ± 0.36) of emulsion but most improved microrheological properties. Overall, phosphorylation functioned itself in fortifying the intradroplet protein-protein interaction but restraining the interdroplet aggregation, and STPP phosphorylation endowed the protein with most enhanced interfacial stabilization and emulsifying efficiency.


Asunto(s)
Emulsiones , Gansos , Interacciones Hidrofóbicas e Hidrofílicas , Hígado , Polifosfatos , Animales , Fosforilación , Emulsiones/química , Polifosfatos/química , Hígado/metabolismo , Difosfatos/química , Difosfatos/metabolismo , Propiedades de Superficie , Fosfatos/química , Tamaño de la Partícula , Adsorción
5.
Biosystems ; 237: 105152, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38346553

RESUMEN

Alanyl-tRNA synthetase (AlaRS) incorrectly recognizes both a slightly smaller glycine and a slightly larger serine in addition to alanine, and the probability of incorrect identification is extremely low at 1/300 and 1/170, respectively. Alanine is the second smallest amino acid after glycine; however, the mechanism by which AlaRS specifically identifies small differences in side chains with high accuracy remains unknown. In this study, using a malachite green assay, we aimed to elucidate the alanine recognition mechanism of a fragment (AlaRS368N) containing only the amino acid activation domain of Escherichia coli AlaRS. This method quantifies monophosphate by decomposing pyrophosphate generated during aminoacyl-AMP production. AlaRS368N produced far more pyrophosphate when glycine or serine was used as a substrate than when alanine was used. Among several mutants tested, an AlaRS mutant in which the widely conserved aspartic acid at the 235th position (D235) near the active center was replaced with glutamic acid (D235E) increased pyrophosphate release for the alanine substrate, compared to that from glycine and serine. These results suggested that D235 is optimal for AlaRS to specifically recognize alanine. Alanylation activities of an RNA minihelix by the mutants of valine at the 214th position (V214) of another fragment (AlaRS442N), which is the smallest AlaRS with alanine charging activity, suggest the existence of the van der Waals-like interaction between the side chain of V214 and the methyl group of the alanine substrate.


Asunto(s)
Alanina-ARNt Ligasa , Alanina-ARNt Ligasa/genética , Alanina-ARNt Ligasa/química , Alanina-ARNt Ligasa/metabolismo , Alanina/genética , Alanina/metabolismo , Difosfatos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Aminoácidos/metabolismo , Glicina , Serina/genética , Serina/metabolismo
6.
Biomolecules ; 14(2)2024 Jan 26.
Artículo en Inglés | MEDLINE | ID: mdl-38397389

RESUMEN

The inositol pyrophosphate pathway, a complex cell signaling network, plays a pivotal role in orchestrating vital cellular processes in the budding yeast, where it regulates cell cycle progression, growth, endocytosis, exocytosis, apoptosis, telomere elongation, ribosome biogenesis, and stress responses. This pathway has gained significant attention in pharmacology and medicine due to its role in generating inositol pyrophosphates, which serve as crucial signaling molecules not only in yeast, but also in higher eukaryotes. As targets for therapeutic development, genetic modifications within this pathway hold promise for disease treatment strategies, offering practical applications in biotechnology. The model organism Saccharomyces cerevisiae, renowned for its genetic tractability, has been instrumental in various studies related to the inositol pyrophosphate pathway. This review is focused on the Kcs1 and Vip1, the two enzymes involved in the biosynthesis of inositol pyrophosphate in S. cerevisiae, highlighting their roles in various cell processes, and providing an up-to-date overview of their relationship with phosphate homeostasis. Moreover, the review underscores the potential applications of these findings in the realms of medicine and biotechnology, highlighting the profound implications of comprehending this intricate signaling network.


Asunto(s)
Difosfatos , Fosfatos de Inositol , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Difosfatos/metabolismo , Fosfatos de Inositol/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal
7.
EMBO J ; 43(3): 462-480, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-38216735

RESUMEN

Kinases that synthesize inositol phosphates (IPs) and pyrophosphates (PP-IPs) control numerous biological processes in eukaryotic cells. Herein, we extend this cellular signaling repertoire to viruses. We have biochemically and structurally characterized a minimalist inositol phosphate kinase (i.e., TvIPK) encoded by Terrestrivirus, a nucleocytoplasmic large ("giant") DNA virus (NCLDV). We show that TvIPK can synthesize inositol pyrophosphates from a range of scyllo- and myo-IPs, both in vitro and when expressed in yeast cells. We present multiple crystal structures of enzyme/substrate/nucleotide complexes with individual resolutions from 1.95 to 2.6 Å. We find a heart-shaped ligand binding pocket comprising an array of positively charged and flexible side chains, underlying the observed substrate diversity. A crucial arginine residue in a conserved "G-loop" orients the γ-phosphate of ATP to allow substrate pyrophosphorylation. We highlight additional conserved catalytic and architectural features in TvIPK, and support their importance through site-directed mutagenesis. We propose that NCLDV inositol phosphate kinases may have assisted evolution of inositol pyrophosphate signaling, and we discuss the potential biogeochemical significance of TvIPK in soil niches.


Asunto(s)
Difosfatos , Virus Gigantes , Difosfatos/metabolismo , Virus Gigantes/metabolismo , Fosfatos de Inositol/química , Fosfatos de Inositol/metabolismo , Fosfatos/metabolismo , Saccharomyces cerevisiae/metabolismo
8.
J Histochem Cytochem ; 72(1): 41-60, 2024 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-38158780

RESUMEN

The present study investigated the localization and the adenosine 5'-triphosphate (ATP)-degrading function of the plasma membrane-bound ecto-nucleotidase, ectonucleoside triphosphate diphosphohydrolase 2 (NTPDase2), in the rat adrenal medulla. The effect of ATP degradation product, adenosine 5'-diphosphate (ADP), on carbachol (CCh)-induced intracellular Ca2+ ([Ca2+]i) responses in adrenal chromaffin cells was examined using calcium imaging. NTPDase2-immunoreactive cells were distributed between chromaffin cells. NTPDase2-immunoreactive cells were immunoreactive for glial fibrillary acidic protein and S100B, suggesting that they were sustentacular cells. NTPDase2-immunoreactive cells surrounded chromaffin cells immunoreactive for vesicular nucleotide transporter and P2Y12 ADP-selective purinoceptors. In ATP bioluminescence assays using adrenal medullary slices, ATP was rapidly degraded and its degradation was attenuated by the NTPDase inhibitors sodium polyoxotungstate (POM-1) and 6-N, N-diethyl-d-ß,γ-dibromomethylene ATP (ARL67156). ADP inhibited CCh-induced [Ca2+]i increases of chromaffin cells in adrenal medullary slices. The inhibition of CCh-induced [Ca2+]i increases by ADP was blocked by the P2Y12 purinoceptor antagonist AZD1283. CCh-induced [Ca2+]i increases were also inhibited by the P2Y1, P2Y12, and P2Y13 purinoceptor agonist 2-methylthioadenosine diphosphate trisodium (2MeSADP), in combination with the P2Y1 purinoceptor antagonist MRS2179. These results suggest that sustentacular cells express NTPDase2 to degrade ATP released from adrenal chromaffin cells, and ADP modulates the excitability of chromaffin cells via P2Y12 purinoceptors to regulate catecholamine release during preganglionic sympathetic stimuli. (J Histochem Cytochem 72: 41-60, 2024).


Asunto(s)
Adenosina Trifosfatasas , Médula Suprarrenal , Células Cromafines , Animales , Ratas , Adenosina/metabolismo , Adenosina Trifosfato/metabolismo , Médula Suprarrenal/metabolismo , Calcio/metabolismo , Células Cromafines/metabolismo , Difosfatos/metabolismo , Adenosina Trifosfatasas/metabolismo
9.
mBio ; 15(2): e0306223, 2024 Feb 14.
Artículo en Inglés | MEDLINE | ID: mdl-38133430

RESUMEN

The inositol pyrophosphate signaling molecule 1,5-IP8 is an agonist of RNA 3'-processing and transcription termination in fission yeast that regulates the expression of phosphate acquisition genes pho1, pho84, and tgp1. IP8 is synthesized from 5-IP7 by the Asp1 N-terminal kinase domain and catabolized by the Asp1 C-terminal pyrophosphatase domain. asp1-STF mutations that delete or inactivate the Asp1 pyrophosphatase domain elicit growth defects in yeast extract with supplements (YES) medium ranging from severe sickness to lethality. We now find that the toxicity of asp1-STF mutants is caused by a titratable constituent of yeast extract. Via a genetic screen for spontaneous suppressors, we identified a null mutation of glycerophosphodiester transporter tgp1 that abolishes asp1-STF toxicity in YES medium. This result, and the fact that tgp1 mRNA expression is increased by >40-fold in asp1-STF cells, prompted discovery that: (i) glycerophosphocholine (GPC) recapitulates the toxicity of yeast extract to asp1-STF cells in a Tgp1-dependent manner, and (ii) induced overexpression of tgp1 in asp1+ cells also elicits toxicity dependent on GPC. asp1-STF suppressor screens yielded a suite of single missense mutations in the essential IP6 kinase Kcs1 that generates 5-IP7, the immediate precursor to IP8. Transcription profiling of the kcs1 mutants in an asp1+ background revealed the downregulation of the same phosphate acquisition genes that were upregulated in asp1-STF cells. The suppressor screen also returned single missense mutations in Plc1, the fission yeast phospholipase C enzyme that generates IP3, an upstream precursor for the synthesis of inositol pyrophosphates.IMPORTANCEThe inositol pyrophosphate metabolite 1,5-IP8 governs repression of fission yeast phosphate homeostasis genes pho1, pho84, and tgp1 by lncRNA-mediated transcriptional interference. Asp1 pyrophosphatase mutations that increase IP8 levels elicit precocious lncRNA termination, leading to derepression of the PHO genes. Deletions of the Asp1 pyrophosphatase domain result in growth impairment or lethality via IP8 agonism of transcription termination. It was assumed that IP8 toxicity ensues from dysregulation of essential genes. In this study, a suppressor screen revealed that IP8 toxicosis of Asp1 pyrophosphatase mutants is caused by: (i) a >40-fold increase in the expression of the inessential tgp1 gene encoding a glycerophosphodiester transporter and (ii) the presence of glycerophosphocholine in the growth medium. The suppressor screen yielded missense mutations in two upstream enzymes of inositol polyphosphate metabolism: the phospholipase C enzyme Plc1 that generates IP3 and the essential Kcs1 kinase that converts IP6 to 5-IP7, the immediate precursor of IP8.


Asunto(s)
Fragmentos de Péptidos , Fosfotransferasas (Aceptor del Grupo Fosfato) , ARN Largo no Codificante , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Tiroglobulina , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Inositol/metabolismo , Difosfatos/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , ARN Largo no Codificante/genética , Proteínas de Transporte de Membrana/metabolismo , Pirofosfatasas/genética , Pirofosfatasas/metabolismo , Fosfatos de Inositol/metabolismo
10.
Br J Pharmacol ; 181(1): 21-35, 2024 01.
Artículo en Inglés | MEDLINE | ID: mdl-37530222

RESUMEN

BACKGROUND AND PURPOSE: Ticagrelor is labelled as a reversible, direct-acting platelet P2Y12 receptor (P2Y12 R) antagonist that is indicated clinically for the prevention of thrombotic events in patients with acute coronary syndrome (ACS). As with many antiplatelet drugs, ticagrelor therapy increases bleeding risk in patients, which may require platelet transfusion in emergency situations. The aim of this study was to further examine the reversibility of ticagrelor at the P2Y12 R. EXPERIMENTAL APPROACH: Studies were performed in human platelets, with P2Y12 R-stimulated GTPase activity and platelet aggregation assessed. Cell-based bioluminescence resonance energy transfer (BRET) assays were undertaken to assess G protein-subunit activation downstream of P2Y12 R activation. KEY RESULTS: Initial studies revealed that a range of P2Y12 R ligands, including ticagrelor, displayed inverse agonist activity at P2Y12 R. Only ticagrelor was resistant to washout and, in human platelet and cell-based assays, washing failed to reverse ticagrelor-dependent inhibition of ADP-stimulated P2Y12 R function. The P2Y12 R agonist 2MeSADP, which was also resistant to washout, was able to effectively compete with ticagrelor. In silico docking revealed that ticagrelor and 2MeSADP penetrated more deeply into the orthosteric binding pocket of the P2Y12 R than other P2Y12 R ligands. CONCLUSION AND IMPLICATIONS: Ticagrelor binding to P2Y12 R is prolonged and more akin to that of an irreversible antagonist, especially versus the endogenous P2Y12 R agonist ADP. This study highlights the potential clinical need for novel ticagrelor reversal strategies in patients with spontaneous major bleeding, and for bleeding associated with urgent invasive procedures.


Asunto(s)
Síndrome Coronario Agudo , Difosfatos , Humanos , Ticagrelor/farmacología , Ticagrelor/metabolismo , Ticagrelor/uso terapéutico , Difosfatos/metabolismo , Difosfatos/farmacología , Difosfatos/uso terapéutico , Adenosina/farmacología , Agonismo Inverso de Drogas , Antagonistas del Receptor Purinérgico P2Y/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Adenosina Difosfato/farmacología , Adenosina Difosfato/metabolismo , Plaquetas , Síndrome Coronario Agudo/tratamiento farmacológico , Síndrome Coronario Agudo/complicaciones , Receptores Purinérgicos P2Y12/metabolismo
11.
PLoS Pathog ; 19(12): e1011818, 2023 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-38048362

RESUMEN

During asexual growth and replication cycles inside red blood cells, the malaria parasite Plasmodium falciparum primarily relies on glycolysis for energy supply, as its single mitochondrion performs little or no oxidative phosphorylation. Post merozoite invasion of a host red blood cell, the ring stage lasts approximately 20 hours and was traditionally thought to be metabolically quiescent. However, recent studies have shown that the ring stage is active in several energy-costly processes, including gene transcription, protein translation, protein export, and movement inside the host cell. It has remained unclear whether a low glycolytic flux alone can meet the energy demand of the ring stage over a long period post invasion. Here, we demonstrate that the metabolic by-product pyrophosphate (PPi) is a critical energy source for the development of the ring stage and its transition to the trophozoite stage. During early phases of the asexual development, the parasite utilizes Plasmodium falciparum vacuolar pyrophosphatase 1 (PfVP1), an ancient pyrophosphate-driven proton pump, to export protons across the parasite plasma membrane. Conditional deletion of PfVP1 leads to a delayed ring stage that lasts nearly 48 hours and a complete blockage of the ring-to-trophozoite transition before the onset of parasite death. This developmental arrest can be partially rescued by an orthologous vacuolar pyrophosphatase from Arabidopsis thaliana, but not by the soluble pyrophosphatase from Saccharomyces cerevisiae, which lacks proton pumping activities. Since proton-pumping pyrophosphatases have been evolutionarily lost in human hosts, the essentiality of PfVP1 suggests its potential as an antimalarial drug target. A drug target of the ring stage is highly desired, as current antimalarials have limited efficacy against this stage.


Asunto(s)
Antimaláricos , Malaria Falciparum , Animales , Humanos , Plasmodium falciparum/metabolismo , Bombas de Protones/metabolismo , Trofozoítos/metabolismo , Difosfatos/metabolismo , Protones , Eritrocitos/parasitología , Pirofosfatasas/metabolismo , Malaria Falciparum/parasitología , Antimaláricos/metabolismo
12.
J Membr Biol ; 256(4-6): 443-458, 2023 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-37955797

RESUMEN

Vigna radiata H+-translocating pyrophosphatases (VrH+-PPases, EC 3.6.1.1) are present in various endomembranes of plants, bacteria, archaea, and certain protozoa. They transport H+ into the lumen by hydrolyzing pyrophosphate, which is a by-product of many essential anabolic reactions. Although the crystal structure of H+-PPases has been elucidated, the H+ translocation mechanism of H+-PPases in the solution state remains unclear. In this study, we used hydrogen-deuterium exchange (HDX) coupled with mass spectrometry (MS) to investigate the dynamics of H+-PPases between the previously proposed R state (resting state, Apo form), I state (intermediate state, bound to a substrate analog), and T state (transient state, bound to inorganic phosphate). When hydrogen was replaced by proteins in deuterium oxide solution, the backbone hydrogen atoms, which were exchanged with deuterium, were identified through MS. Accordingly, we used deuterium uptake to examine the structural dynamics and conformational changes of H+-PPases in solution. In the highly conserved substrate binding and proton exit regions, HDX-MS revealed the existence of a compact conformation with deuterium exchange when H+-PPases were bound with a substrate analog and product. Thus, a novel working model was developed to elucidate the in situ catalytic mechanism of pyrophosphate hydrolysis and proton transport. In this model, a proton is released in the I state, and the TM5 inner wall serves as a proton piston.


Asunto(s)
Pirofosfatasa Inorgánica , Vigna , Pirofosfatasa Inorgánica/metabolismo , Vigna/metabolismo , Protones , Deuterio/metabolismo , Difosfatos/metabolismo , Medición de Intercambio de Deuterio , Hidrógeno/metabolismo , Espectrometría de Masas
13.
Proc Natl Acad Sci U S A ; 120(41): e2309536120, 2023 10 10.
Artículo en Inglés | MEDLINE | ID: mdl-37782800

RESUMEN

Isoprene is emitted by some plants and is the most abundant biogenic hydrocarbon entering the atmosphere. Multiple studies have elucidated protective roles of isoprene against several environmental stresses, including high temperature, excessive ozone, and herbivory attack. However, isoprene emission adversely affects atmospheric chemistry by contributing to ozone production and aerosol formation. Thus, understanding the regulation of isoprene emission in response to varying environmental conditions, for example, elevated CO2, is critical to comprehend how plants will respond to climate change. Isoprene emission decreases with increasing CO2 concentration; however, the underlying mechanism of this response is currently unknown. We demonstrated that high-CO2-mediated suppression of isoprene emission is independent of photosynthesis and light intensity, but it is reduced with increasing temperature. Furthermore, we measured methylerythritol 4-phosphate (MEP) pathway metabolites in poplar leaves harvested at ambient and high CO2 to identify why isoprene emission is reduced under high CO2. We found that hydroxymethylbutenyl diphosphate (HMBDP) was increased and dimethylallyl diphosphate (DMADP) decreased at high CO2. This implies that high CO2 impeded the conversion of HMBDP to DMADP, possibly through the inhibition of HMBDP reductase activity, resulting in reduced isoprene emission. We further demonstrated that although this phenomenon appears similar to abscisic acid (ABA)-dependent stomatal regulation, it is unrelated as ABA treatment did not alter the effect of elevated CO2 on the suppression of isoprene emission. Thus, this study provides a comprehensive understanding of the regulation of the MEP pathway and isoprene emission in the face of increasing CO2.


Asunto(s)
Ozono , Populus , Dióxido de Carbono/metabolismo , Difosfatos/metabolismo , Fotosíntesis , Hemiterpenos , Butadienos/farmacología , Butadienos/metabolismo , Plantas/metabolismo , Ozono/metabolismo , Pentanos/metabolismo , Hojas de la Planta/metabolismo , Populus/genética , Populus/metabolismo
14.
mBio ; 14(5): e0193923, 2023 Oct 31.
Artículo en Inglés | MEDLINE | ID: mdl-37754562

RESUMEN

IMPORTANCE: Although most bacteria are quickly killed after phagocytosis by a eukaryotic cell, some pathogenic bacteria escape death after phagocytosis. Pathogenic Mycobacterium species secrete polyP, and the polyP is necessary for the bacteria to prevent their killing after phagocytosis. Conversely, exogenous polyP prevents the killing of ingested bacteria that are normally killed after phagocytosis by human macrophages and the eukaryotic microbe Dictyostelium discoideum. This suggests the possibility that in these cells, a signal transduction pathway is used to sense polyP and prevent killing of ingested bacteria. In this report, we identify key components of the polyP signal transduction pathway in D. discoideum. In cells lacking these components, polyP is unable to inhibit killing of ingested bacteria. The pathway components have orthologs in human cells, and an exciting possibility is that pharmacologically blocking this pathway in human macrophages would cause them to kill ingested pathogens such as Mycobacterium tuberculosis.


Asunto(s)
Dictyostelium , Polifosfatos , Humanos , Polifosfatos/metabolismo , Difosfatos/metabolismo , Dictyostelium/microbiología , Bacterias/metabolismo , Fagocitosis , Serina-Treonina Quinasas TOR
15.
J Agric Food Chem ; 71(35): 13014-13023, 2023 Sep 06.
Artículo en Inglés | MEDLINE | ID: mdl-37566786

RESUMEN

Antrodia cinnamomea is an endemic species found in Taiwan, known for its medicinal properties in treating various discomforts, including inflammation, diarrhea, abdominal pain, and other diseases. A. cinnamomea contains terpenoids that exhibit numerous bioactivities, making them potential food additives. This discovery piqued our interest in uncovering their biosynthetic pathway. Herein, we conducted functional and structural characterization of a sesquiterpene synthase Cop4 from A. cinnamomea (AcCop4). Through gas chromatography-mass spectrometry analysis, we observed that AcCop4 catalyzes the cyclization of farnesyl pyrophosphate (FPP), primarily producing cubebol. Cubebol is widely used as a long-lasting cooling and refreshing agent in the food industry. The structure of AcCop4, complexed with pyrophosphate and magnesium ions, revealed the closure of the active site facilitated by R311. Interestingly, binding of pyrophosphate and magnesium ions did not cause any significant conformational change in the G1/2 helix of AcCop4, indicating that the apo form is not fully open. This high-resolution structure serves as a solid basis for understanding the biosynthetic mechanism of AcCop4 and supports further production and modification of cubebol for its applications in the food industry.


Asunto(s)
Antrodia , Sesquiterpenos , Difosfatos/metabolismo , Magnesio/metabolismo , Sesquiterpenos/metabolismo , Antrodia/metabolismo
16.
Am J Physiol Gastrointest Liver Physiol ; 325(4): G347-G355, 2023 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-37529835

RESUMEN

Thiamin (vitamin B1) plays a vital role in cellular energy metabolism/ATP production. Pancreatic acinar cells (PACs) obtain thiamin from circulation and convert it to thiamin pyrophosphate (TPP) in the cytoplasm. TPP is then taken up by the mitochondria via a carrier-mediated process that involves the mitochondrial TPP transporter (MTPPT; encoded by the gene SLC25A19). We have previously characterized different aspects of the mitochondrial carrier-mediated TPP uptake process, but nothing is known about its possible regulation at the posttranscriptional level. We address this issue in the current investigations focusing on the role of miRNAs in this regulation. First, we subjected the human (and rat) 3'-untranslated region (3'-UTR) of the SLC25A19 to three in-silico programs, and all have identified putative binding sites for miR-122-5p. Transfecting pmirGLO-hSLC25A19 3'-UTR into rat PAC AR42J resulted in a significant reduction in luciferase activity compared with cells transfected with pmirGLO-empty vector. Mutating as well as truncating the putative miR-122-5p binding sites in the hSLC25A19 3'-UTR led to abrogation of inhibition in luciferase activity in PAC AR42J. Furthermore, transfecting/transducing PAC AR42J and human primary PACs with mimic of miR-122-5p led to a significant inhibition in the level of expression of the MTPPT mRNA and protein as well as in mitochondrial carrier-mediated TPP uptake. Conversely, transfecting PAC AR42J with an inhibitor of miR-122-5p increased MTPPT expression and function. These findings show, for the first time, that expression and function of the MTPPT in PACs are subject to posttranscriptional regulation by miR-122-5p.NEW & NOTEWORTHY This study shows that the expression and function of mitochondrial TPP transporter (MTPPT) are subject to posttranscriptional regulation by miRNA-122-5p in pancreatic acinar cells.


Asunto(s)
Células Acinares , MicroARNs , Humanos , Ratas , Animales , Células Acinares/metabolismo , Difosfatos/metabolismo , Tiamina/metabolismo , Tiamina Pirofosfato/metabolismo , Mitocondrias/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Luciferasas/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas de Transporte de Membrana Mitocondrial/metabolismo
17.
Cell Death Dis ; 14(7): 447, 2023 07 19.
Artículo en Inglés | MEDLINE | ID: mdl-37468461

RESUMEN

Pathological mineralization of intervertebral disc is debilitating and painful and linked to disc degeneration in a subset of human patients. An adenosine triphosphate efflux transporter, progressive ankylosis (ANK) is a regulator of extracellular inorganic pyrophosphate levels and plays an important role in tissue mineralization. However, the function of ANK in intervertebral disc has not been fully explored. Herein we analyzed the spinal phenotype of Ank mutant mice (ank/ank) with attenuated ANK function. Micro-computed tomography and histological analysis showed that loss of ANK function results in the aberrant annulus fibrosus mineralization and peripheral disc fusions with cranial to caudal progression in the spine. Vertebrae in ank mice exhibit elevated cortical bone mass and increased tissue non-specific alkaline phosphatase-positive endplate chondrocytes with decreased subchondral endplate porosity. The acellular dystrophic mineral inclusions in the annulus fibrosus were localized adjacent to apoptotic cells and cells that acquired osteoblast-like phenotype. Fourier transform infrared spectral imaging showed that the apatite mineral in the outer annulus fibrosus had similar chemical composition to that of vertebral bone. Transcriptomic analysis of annulus fibrosus and nucleus pulposus tissues showed changes in several biological themes with a prominent dysregulation of BMAL1/CLOCK circadian regulation. The present study provides new insights into the role of ANK in the disc tissue compartments and highlights the importance of local inorganic pyrophosphate metabolism in inhibiting the mineralization of this important connective tissue.


Asunto(s)
Calcinosis , Degeneración del Disco Intervertebral , Disco Intervertebral , Animales , Humanos , Ratones , Calcinosis/patología , Difosfatos/metabolismo , Disco Intervertebral/metabolismo , Degeneración del Disco Intervertebral/patología , Mutación con Pérdida de Función , Fenotipo , Microtomografía por Rayos X
18.
Nat Chem ; 15(8): 1188-1195, 2023 08.
Artículo en Inglés | MEDLINE | ID: mdl-37308711

RESUMEN

Terpenoids account for more than 60% of all natural products, and their carbon skeletons originate from common isoprenoid units of different lengths such as geranyl pyrophosphate and farnesyl pyrophosphate. Here we characterize a metal-dependent, bifunctional isoprenyl diphosphate synthase from the leaf beetle Phaedon cochleariae by structural and functional analyses. Inter- and intramolecular cooperative effects in the homodimer strongly depend on the provided metal ions and regulate the biosynthetic flux of terpene precursors to either biological defence or physiological development. Strikingly, a unique chain length determination domain adapts to form geranyl or farnesyl pyrophosphate by altering enzyme symmetry and ligand affinity between both subunits. In addition, we identify an allosteric geranyl-pyrophosphate-specific binding site that shares similarity with end-product inhibition in human farnesyl pyrophosphate synthase. Our combined findings elucidate a deeply intertwined reaction mechanism in the P. cochleariae isoprenyl diphosphate synthase that integrates substrate, product and metal-ion concentrations to harness its dynamic potential.


Asunto(s)
Difosfatos , Terpenos , Humanos , Terpenos/metabolismo , Difosfatos/química , Difosfatos/metabolismo , Fosfatos de Poliisoprenilo/química , Fosfatos de Poliisoprenilo/metabolismo
19.
J Mol Biol ; 435(15): 168186, 2023 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-37355033

RESUMEN

RNA polymerase I (Pol I) synthesizes ribosomal RNA (rRNA), which is the first and rate-limiting step in ribosome biosynthesis. A12.2 (A12) is a critical subunit of Pol I that is responsible for activating Pol I's exonuclease activity. We previously reported a kinetic mechanism for single-nucleotide incorporation catalyzed by Pol I lacking the A12 subunit (ΔA12 Pol I) purified from S. cerevisae and revealed that ΔA12 Pol I exhibited much slower incorporation compared to Pol I. However, it is unknown if A12 influences each nucleotide incorporation in the context of transcription elongation. Here, we show that A12 contributes to every repeating cycle of nucleotide addition and that deletion of A12 results in an entirely different kinetic mechanism compared to WT Pol I. We found that instead of one irreversible step between each nucleotide addition cycle, as reported for wild type (WT) Pol I, the ΔA12 variant requires one reversible step to describe each nucleotide addition. Reversibility fundamentally requires slow PPi release. Consistently, we show that Pol I is more pyrophosphate (PPi) concentration dependent than ΔA12 Pol I. This observation supports the model that PPi is retained in the active site of ΔA12 Pol I longer than WT Pol I. These results suggest that A12 promotes PPi release, revealing a larger role for the A12.2 subunit in the nucleotide addition cycle beyond merely activating exonuclease activity.


Asunto(s)
Difosfatos , ARN Polimerasa I , Difosfatos/metabolismo , Exonucleasas , Nucleótidos/metabolismo , ARN Polimerasa I/química , ARN Polimerasa I/genética , ARN Polimerasa I/metabolismo
20.
Nat Commun ; 14(1): 2645, 2023 05 08.
Artículo en Inglés | MEDLINE | ID: mdl-37156835

RESUMEN

Many proteins involved in eukaryotic phosphate homeostasis are regulated by SPX domains. In yeast, the vacuolar transporter chaperone (VTC) complex contains two such domains, but mechanistic details of its regulation are not well understood. Here, we show at the atomic level how inositol pyrophosphates interact with SPX domains of subunits Vtc2 and Vtc3 to control the activity of the VTC complex. Vtc2 inhibits the catalytically active VTC subunit Vtc4 by homotypic SPX-SPX interactions via the conserved helix α1 and the previously undescribed helix α7. Binding of inositol pyrophosphates to Vtc2 abrogates this interaction, thus activating the VTC complex. Accordingly, VTC activation is also achieved by site-specific point mutations that disrupt the SPX-SPX interface. Structural data suggest that ligand binding induces reorientation of helix α1 and exposes the modifiable helix α7, which might facilitate its post-translational modification in vivo. The variable composition of these regions within the SPX domain family might contribute to the diversified SPX functions in eukaryotic phosphate homeostasis.


Asunto(s)
Difosfatos , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Difosfatos/metabolismo , Transporte Biológico , Homeostasis , Fosfatos de Inositol/metabolismo
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