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Microorganisms Mar 2024In , pH homeostasis is reliant on ATP due to the use of proton-translocating ATPase (H-ATPase) which constitutes a major drain within cellular ATP supply. Here, an...
In , pH homeostasis is reliant on ATP due to the use of proton-translocating ATPase (H-ATPase) which constitutes a major drain within cellular ATP supply. Here, an exogenous proton-translocating pyrophosphatase (H-PPase) from which uses inorganic pyrophosphate (PP) rather than ATP, was evaluated for its effect on reducing the ATP burden. The H-Ppase was localized to the vacuolar membrane or to the cell membrane, and their impact was studied under acetate stress at a low pH. Biosensors (pHluorin and mQueen-2m) were used to observe changes in intracellular pH (pH) and ATP levels during growth on either glucose or xylose. A significant improvement of 35% in the growth rate at a pH of 3.7 and 6 g·L acetic acid stress was observed in the vacuolar membrane H-PPase strain compared to the parent strain. ATP levels were elevated in the same strain during anaerobic glucose and xylose fermentations. During anaerobic xylose fermentations, co-expression of pHluorin and a vacuolar membrane H-PPase improved the growth characteristics by means of an improved growth rate (11.4%) and elongated logarithmic growth duration. Our study identified a potential method for improving productivity in the use of as a cell factory under the harsh conditions present in industry.
PubMed: 38543676
DOI: 10.3390/microorganisms12030625 -
International Journal of Molecular... Mar 202414-3-3 proteins are widely distributed in eukaryotic cells and play an important role in plant growth, development, and stress tolerance. This study revealed nine genes...
14-3-3 proteins are widely distributed in eukaryotic cells and play an important role in plant growth, development, and stress tolerance. This study revealed nine genes from the genome of Pall., a halophyte with strong salt tolerance. The physicochemical properties, multiple sequence alignment, gene structure and motif analysis, and chromosomal distributions were analyzed, and phylogenetic analysis, cis-regulatory elements analysis, and gene transcription and expression analysis of were conducted. The results revealed that the gene family consists of nine members, which are divided into two groups: ε (four members) and non-ε (five members). These members are acidic hydrophilic proteins. The genes are distributed randomly on chromosomes, and the number of introns varies widely among the two groups. However, all genes have similar conserved domains and three-dimensional protein structures. The main differences are found at the N-terminus and C-terminus. The promoter region of contains multiple cis-acting elements related to light, plant hormones, and abiotic stress responses. Transcriptional profiling and gene expression pattern analysis revealed that were expressed in all tissues, although with varying patterns. Under salt stress conditions, , , , and showed significant changes in gene expression. expression decreased in all tissues, expression decreased by 60% to 71% in roots, and expression increased by 209% to 251% in stems. The most significant change was observed in , with its expression in stems increasing by 213% to 681%. The yeast two-hybrid experiments demonstrated that Ns14-3-3 5a interacts with NsVP1 (vacuolar H-pyrophosphatase). This result indicates that Ns14-3-3 5a may respond to salt stress by promoting ionic vacuole compartmentalization in stems and leaves through interactions with NsVP1. In addition, has a high number of stems, allowing it to compartmentalize more ions through its stem and leaf. This may be a contributing factor to its superior salt tolerance compared to other plants.
Topics: Phylogeny; Salt Stress; Salt Tolerance; Introns; 14-3-3 Proteins; Inorganic Pyrophosphatase; Magnoliopsida; Plant Proteins; Gene Expression Regulation, Plant; Stress, Physiological
PubMed: 38542405
DOI: 10.3390/ijms25063432 -
International Journal of Biological... May 2024An extensive range of new biologically active morpholine based thiosemicarbazones derivatives 3a-r were synthesized, characterized by spectral techniques and evaluated...
An extensive range of new biologically active morpholine based thiosemicarbazones derivatives 3a-r were synthesized, characterized by spectral techniques and evaluated as inhibitors of ENPP isozymes. Most of the novel thiosemicarbazones exhibit potent inhibition towards NPP1 and NPP3 isozymes. Compound 3 h was potent inhibitor of NPP1 with IC value of 0.55 ± 0.02. However, the most powerful inhibitor of NPP3 was 3e with an IC value of 0.24 ± 0.02. Furthermore, Lineweaver-Burk plot for compound 3 h against NPP1 and for compound 3e against NPP3 was devised through enzymes kinetics studies. Molecular docking and in silico studies was also done for analysis of interaction pattern of all newly synthesized compounds. The results were further validated by molecular dynamic (MD) simulation where the stability of conformational transformation of the best protein-ligand complex (3e) were justified on the basis of RMSD and RMSF analysis.
Topics: Morpholines; Phosphoric Diester Hydrolases; Molecular Docking Simulation; Pyrophosphatases; Molecular Dynamics Simulation; Thiosemicarbazones; Humans; Kinetics; Phosphodiesterase Inhibitors; Computer Simulation; Structure-Activity Relationship; Ligands
PubMed: 38531526
DOI: 10.1016/j.ijbiomac.2024.131068 -
Journal of Controlled Release :... May 2024Recent studies in colorectal cancer patients (CRC) have shown that increased resistance to thymidylate synthase (TS) inhibitors such as 5-fluorouracil (5-FU), reduce the...
Recent studies in colorectal cancer patients (CRC) have shown that increased resistance to thymidylate synthase (TS) inhibitors such as 5-fluorouracil (5-FU), reduce the efficacy of standard of care (SoC) treatment regimens. The nucleotide pool cleanser dUTPase is highly expressed in CRC and is an attractive target for potentiating anticancer activity of chemotherapy. The purpose of the current work was to investigate the activity of P, P-di(2',5'-dideoxy-5'-selenouridinyl)-tetraphosphate (P-SedU), a selenium-modified symmetrically capped dinucleoside with prodrug capabilities that is specifically activated by dUTPase. Using mechanochemistry, P-SedU and the corresponding selenothymidine analogue P-SeT were prepared with a yield of 19% and 30% respectively. The phosphate functionality facilitated complexation with the amphipathic cell-penetrating peptide RALA to produce nanoparticles (NPs). These NPs were designed to deliver P-SedU intracellularly and thereby maximise in vivo activity. The NPs demonstrated effective anti-cancer activity and selectivity in the HCT116 CRC cell line, a cell line that overexpresses dUTPase; compared to HT29 CRC cells and NCTC-929 fibroblast cells which have reduced levels of dUTPase expression. In vivo studies in BALB/c SCID mice revealed no significant toxicity with respect to weight or organ histology. Pharmacokinetic analysis of blood serum showed that RALA facilitates effective delivery and rapid internalisation into surrounding tissues with NPs eliciting lower plasma C than the equivalent injection of free P-SedU, translating the in vitro findings. Tumour growth delay studies have demonstrated significant inhibition of growth dynamics with the tumour doubling time extended by >2weeks. These studies demonstrate the functionality and action of a new pro-drug nucleotide for CRC.
Topics: Animals; Colorectal Neoplasms; Prodrugs; Humans; Nanoparticles; Antineoplastic Agents; Pyrophosphatases; Female; Cell Line, Tumor; Peptides; Mice, Inbred BALB C; Mice; Nucleotides; HCT116 Cells
PubMed: 38513729
DOI: 10.1016/j.jconrel.2024.03.036 -
Protein Science : a Publication of the... Apr 2024Mycobacterium tuberculosis (Mtb) adapt to various host environments and utilize a variety of sugars and lipids as carbon sources. Among these sugars, maltose and...
Mycobacterium tuberculosis (Mtb) adapt to various host environments and utilize a variety of sugars and lipids as carbon sources. Among these sugars, maltose and trehalose, also play crucial role in bacterial physiology and virulence. However, some key enzymes involved in trehalose and maltose metabolism in Mtb are not yet known. Here we structurally and functionally characterized a conserved hypothetical gene Rv3400. We determined the crystal structure of Rv3400 at 1.7 Å resolution. The crystal structure revealed that Rv3400 adopts Rossmann fold and shares high structural similarity with haloacid dehalogenase family of proteins. Our comparative structural analysis suggested that Rv3400 could perform either phosphatase or pyrophosphatase or β-phosphoglucomutase (β-PGM) activity. Using biochemical studies, we further confirmed that Rv3400 performs β-PGM activity and hence, Rv3400 encodes for β-PGM in Mtb. Our data also confirm that Mtb β-PGM is a metal dependent enzyme having broad specificity for divalent metal ions. β-PGM converts β-D-glucose-1-phosphate to β-D-glucose-6-phosphate which is required for the generation of ATP and NADPH through glycolysis and pentose phosphate pathway, respectively. Using site directed mutagenesis followed by biochemical studies, we show that two Asp residues in the highly conserved DxD motif, D29 and D31, are crucial for enzyme activity. While D29A, D31A, D29E, D31E and D29N mutants lost complete activity, D31N mutant retained about 30% activity. This study further helps in understanding the role of β-PGM in the physiology of Mtb.
Topics: Phosphoglucomutase; Maltose; Mycobacterium tuberculosis; Trehalose; Phosphates; Glucose
PubMed: 38501428
DOI: 10.1002/pro.4943 -
Advanced Science (Weinheim,... May 2024To evade immune surveillance, tumor cells express ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) on the surface of their membrane, which degrades...
To evade immune surveillance, tumor cells express ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) on the surface of their membrane, which degrades extracellular cyclic GMP-AMP (cGAMP), thereby inhibiting the cyclic GMP-AMP synthase (cGAS) stimulator of interferon gene (STING) DNA-sensing pathway. To fully understand this tumor stealth mechanism, it is essential to determine whether other forms of ENPP1 with hydrolytic cGAMP activity also are present in the tumor microenvironment to regulate this innate immune pathway. Herein, it is reported that various tumor-derived exosomes carry ENPP1, and can hydrolyze synthetic 2'3'-cGAMP and endogenous 2'3'-cGAMP produced by cells to inhibit cGAS-STING pathway in immune cells. Moreover, tumor exosomal ENPP1 also can hydrolyze 2'3'-cGAMP bound to LL-37 (an effective transporter of 2'3'-cGAMP) to inhibit STING signaling. Furthermore, high expression of ENPP1 in exosomes is observed isolated from human breast and lung cancer tissue, and tumor exosomal ENPP1 inhibited the immune infiltration of CD8+ T cells and CD4+ T cells. The results elucidate the essential function of tumor exosomal ENPP1 in the cGAS-STING pathway, furthering understanding of the crosstalk between the tumor cells and immune system.
Topics: Nucleotides, Cyclic; Pyrophosphatases; Signal Transduction; Phosphoric Diester Hydrolases; Membrane Proteins; Humans; Nucleotidyltransferases; Exosomes; Mice; Animals; Neoplasms; Cell Line, Tumor; Tumor Microenvironment
PubMed: 38498770
DOI: 10.1002/advs.202308131 -
Nature Communications Mar 2024Tumor cells must rewire nucleotide synthesis to satisfy the demands of unbridled proliferation. Meanwhile, they exhibit augmented reactive oxygen species (ROS)...
Tumor cells must rewire nucleotide synthesis to satisfy the demands of unbridled proliferation. Meanwhile, they exhibit augmented reactive oxygen species (ROS) production which paradoxically damages DNA and free deoxy-ribonucleoside triphosphates (dNTPs). How these metabolic processes are integrated to fuel tumorigenesis remains to be investigated. MYC family oncoproteins coordinate nucleotide synthesis and ROS generation to drive the development of numerous cancers. We herein perform a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based functional screen targeting metabolic genes and identified nudix hydrolase 1 (NUDT1) as a MYC-driven dependency. Mechanistically, MYC orchestrates the balance of two metabolic pathways that act in parallel, the NADPH oxidase 4 (NOX4)-ROS pathway and the Polo like kinase 1 (PLK1)-NUDT1 nucleotide-sanitizing pathway. We describe LC-1-40 as a potent, on-target degrader that depletes NUDT1 in vivo. Administration of LC-1-40 elicits excessive nucleotide oxidation, cytotoxicity and therapeutic responses in patient-derived xenografts. Thus, pharmacological targeting of NUDT1 represents an actionable MYC-driven metabolic liability.
Topics: Humans; Nudix Hydrolases; Reactive Oxygen Species; Oxidation-Reduction; Nucleotides
PubMed: 38493213
DOI: 10.1038/s41467-024-46572-6 -
Environmental Science & Technology Mar 2024Long-term phosphorus (P) fertilization results in P accumulation in agricultural soil and increases the risk of P leaching into water bodies. However, evaluating P...
Long-term phosphorus (P) fertilization results in P accumulation in agricultural soil and increases the risk of P leaching into water bodies. However, evaluating P leaching into groundwater is challenging, especially in clay soil with a high P sorption capacity. This study examined whether the combination of PO oxygen isotope (δO) analysis and the P saturation ratio (PSR) was useful to identify P enrichment mechanisms in groundwater. We investigated the groundwater and possible P sources in Kubi, western Japan, with intensive citrus cultivation. Shallow groundwater had oxic conditions with high PO concentrations, and orchard soil P accumulation was high compared with forest soil. Although the soil had a high P sorption capacity, the PSR was above the threshold, indicating a high risk of P leaching from the surface orchard soil. The shallow groundwater δO values were higher than the expected isotopic equilibrium with pyrophosphatase. The high PSR and δO orchard soil values indicated that P leaching from orchard soil was the major P enrichment mechanism. The Bayesian mixing model estimated that 76.6% of the P supplied from the orchard soil was recycled by microorganisms. This demonstrates the utility of δO and the PSR to evaluate the P source and biological recycling in groundwater.
Topics: Phosphorus; Phosphates; Soil; Oxygen Isotopes; Adsorption; Bayes Theorem; Groundwater
PubMed: 38488121
DOI: 10.1021/acs.est.3c07170 -
BMC Infectious Diseases Mar 2024Globally, 80 million people are suffering from chronic Hepatitis C virus (HCV) infection. Sofosbuvir ribavirin-based anti-HCV therapy is associated with anemia and...
BACKGROUND
Globally, 80 million people are suffering from chronic Hepatitis C virus (HCV) infection. Sofosbuvir ribavirin-based anti-HCV therapy is associated with anemia and other adverse effects. Polymorphisms of Inosine triphosphatase (ITPA) gene may cause functional impairment in the Inosine triphosphate pyrophosphatase enzyme, resulting in enhanced sustained viral response (SVR) and protection from ribavirin-associated anemia in patients on therapy. The study objective was to investigate the effect of Inosine triphosphatase gene polymorphism on SVR achievement, hemoglobin decline and ribavirin dose reduction in patients on therapy.
METHODS
This prospective cohort study was of 170 hepatitis C infected patients received 6-month sofosbuvir ribavirin therapy. Patient viral load, reduction in ribavirin amount, liver function test, and complete blood count were noted monthly. Inosine triphosphatase variants rs1127354 and rs7270101 were assessed through the restriction fragment length polymorphism and confirmed using Sanger sequencing. The impact of polymorphism on cumulative reduction of ribavirin, and anti-HCV therapy outcome were studied.
RESULTS
A total of 74.3% of patients had ITPA rs1127354 CC genotype, 25.7% were CA and AA 0%. The frequency of ITPA genotype rs7270101-AA was 95%, AC 5%, and CC was 0%. ITPA rs1127354-CA had a notably positive impact on SVR achievement with a zero-relapse rate. ITPA rs1127354-CA genotype was significantly (P ˂0.05) protective against ≥ 2 g/dl Hb reduction from baseline to 1st, 2nd and 6th months of therapy. During treatment, Hb reduction ≥ 10 g/dl was frequently observed in rs1127354-CC genotype and rs7270101-AA genotype patients. Ribavirin dose reduction was significantly (P ˂0.05) high in rs1127354-CC genotype as compared to genotype CA whereas no significant difference was observed in ribavirin dose reduction in rs7270101 AA and non-AA genotype. Patient baseline characteristics such as age, body mass index, rs1127354-CC genotype, and baseline Hb were significantly associated with significant Hb reduction.
CONCLUSION
Pretreatment evaluation of ITPA polymorphism can be a diagnostic tool to find out patients at risk of anemia and improve treatment adherence. ITPA genotype rs1127354-CA contributes to improved compliance with ribavirin dose and protects against hemoglobin decline in HCV patients while taking ribavirin-based therapy. However, ITPA rs1127354, rs7270101 polymorphism have no significant impact on SVR achievement.
Topics: Humans; Ribavirin; Sofosbuvir; Hepatitis C, Chronic; Antiviral Agents; Inosine Triphosphatase; Hepacivirus; Prospective Studies; Polymorphism, Single Nucleotide; Pyrophosphatases; Anemia; Hepatitis C; Genotype; Hemoglobins; Treatment Outcome
PubMed: 38468199
DOI: 10.1186/s12879-024-09188-1 -
Bioorganic & Medicinal Chemistry Letters May 2024Autotaxin is a secreted lysophospholipase D which is a member of the ectonucleotide pyrophosphatase/phosphodiesterase family converting extracellular...
Autotaxin is a secreted lysophospholipase D which is a member of the ectonucleotide pyrophosphatase/phosphodiesterase family converting extracellular lysophosphatidylcholine and other non-choline lysophospholipids, such as lysophosphatidylethanolamine and lysophosphatidylserine, to the lipid mediator lysophosphatidic acid. Autotaxin is implicated in various fibroproliferative diseases including interstitial lung diseases, such as idiopathic pulmonary fibrosis and hepatic fibrosis, as well as in cancer. In this study, we present an effort of identifying ATX inhibitors that bind to allosteric ATX binding sites using the Enalos Asclepios KNIME Node. All the available PDB crystal structures of ATX were collected, prepared, and aligned. Visual examination of these structures led to the identification of four crystal structures of human ATX co-crystallized with four known inhibitors. These inhibitors bind to five binding sites with five different binding modes. These five binding sites were thereafter used to virtually screen a compound library of 14,000 compounds to identify molecules that bind to allosteric sites. Based on the binding mode and interactions, the docking score, and the frequency that a compound comes up as a top-ranked among the five binding sites, 24 compounds were selected for in vitro testing. Finally, two compounds emerged with inhibitory activity against ATX in the low micromolar range, while their mode of inhibition and binding pattern were also studied. The two derivatives identified herein can serve as "hits" towards developing novel classes of ATX allosteric inhibitors.
Topics: Humans; Lysophospholipids; Phosphoric Diester Hydrolases; Neoplasms; Binding Sites; Allosteric Site
PubMed: 38447786
DOI: 10.1016/j.bmcl.2024.129690