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Biochemistry Sep 2023Because purine nucleotides are essential for all life, differences between how microbes and humans metabolize purines can be exploited for the development of...
Because purine nucleotides are essential for all life, differences between how microbes and humans metabolize purines can be exploited for the development of antimicrobial therapies. While humans biosynthesize purine nucleotides in a 10-step pathway, most microbes utilize an additional 11th enzymatic activity. The human enzyme, aminoimidazole ribonucleotide (AIR) carboxylase generates the product 4-carboxy-5-aminoimidazole ribonucleotide (CAIR) directly. Most microbes, however, require two separate enzymes, a synthetase (PurK) and a mutase (PurE), and proceed through the intermediate, N-CAIR. Toward the development of therapeutics that target these differences, we have solved crystal structures of the N-CAIR mutase of the human pathogens (LpPurE) and (BcPurE) and used a structure-guided approach to identify inhibitors. Analysis of the structures reveals a highly conserved fold and active site architecture. Using this data, and three additional structures of PurE enzymes, we screened a library of FDA-approved compounds and identified a set of 25 candidates for further analysis. Among these, we identified several new PurE inhibitors with micromolar IC values. Several of these compounds, including the α-blocker Alfuzosin, inhibit the microbial PurE enzymes much more effectively than the human homologue. These structures and the newly described PurE inhibitors are valuable tools to aid in further studies of this enzyme and provide a foundation for the development of compounds that target differences between human and microbial purine metabolism.
Topics: Humans; Ribonucleotides; Escherichia coli; Intramolecular Transferases; Purine Nucleotides
PubMed: 37552766
DOI: 10.1021/acs.biochem.2c00705 -
Genes & Diseases Nov 2023nucleotide biosynthetic pathway is a highly conserved and essential biochemical pathway in almost all organisms. Both purine nucleotides and pyrimidine nucleotides are... (Review)
Review
nucleotide biosynthetic pathway is a highly conserved and essential biochemical pathway in almost all organisms. Both purine nucleotides and pyrimidine nucleotides are necessary for cell metabolism and proliferation. Thus, the dysregulation of the nucleotide biosynthetic pathway contributes to the development of many human diseases, such as cancer. It has been shown that many enzymes in this pathway are overactivated in different cancers. In this review, we summarize and update the current knowledge on the nucleotide biosynthetic pathway, regulatory mechanisms, its role in tumorigenesis, and potential targeting opportunities.
PubMed: 37554216
DOI: 10.1016/j.gendis.2022.04.018 -
Journal of Plant Physiology Jul 2023
Review
Topics: Nucleotides, Cyclic; Plant Growth Regulators; Cyclic AMP; Cyclic GMP
PubMed: 37186984
DOI: 10.1016/j.jplph.2023.154005 -
Cardiovascular Research Dec 2023Empagliflozin (EMPA), a potent inhibitor of the renal sodium-glucose cotransporter 2 and an effective treatment for Type 2 diabetes, has been shown to have...
AIMS
Empagliflozin (EMPA), a potent inhibitor of the renal sodium-glucose cotransporter 2 and an effective treatment for Type 2 diabetes, has been shown to have cardioprotective effects, independent of improved glycaemic control. Several non-canonical mechanisms have been proposed to explain these cardiac effects, including increasing circulating ketone supply to the heart. This study aims to test whether EMPA directly alters cardiac ketone metabolism independent of supply.
METHODS AND RESULTS
The direct effects of EMPA on cardiac function and metabolomics were investigated in Langendorff rat heart perfused with buffer containing 5 mM glucose, 4 mM β-hydroxybutyrate (βHb) and 0.4 mM intralipid, subject to low flow ischaemia/reperfusion. Cardiac energetics were monitored in situ using 31P NMR spectroscopy. Steady-state 13C labelling was performed by switching 12C substrates for 13C1 glucose or 13C4 βHb and 13C incorporation into metabolites determined using 2D 1H-13C HSQC NMR spectroscopy. EMPA treatment improved left ventricular-developed pressure during ischaemia and reperfusion compared to vehicle-treated hearts. In EMPA-treated hearts, total adenosine triphosphate (ATP) and phosphocreatine (PCr) levels, and Gibbs free energy for ATP hydrolysis were significantly higher during ischaemia and reperfusion. EMPA treatment did not alter the incorporation of 13C from glucose into glycolytic products lactate or alanine neither during ischaemia nor reperfusion. In ischaemia, EMPA led to a decrease in 13C1 glucose incorporation and a concurrent increase in 13C4 βHb incorporation into tricarboxylic acid (TCA) cycle intermediates succinate, citrate, and glutamate. During reperfusion, the concentration of metabolites originating from 13C1 glucose was similar to vehicle but those originating from 13C4 βHb remained elevated in EMPA-treated hearts.
CONCLUSION
Our findings indicate that EMPA causes a switch in metabolism away from glucose oxidation towards increased ketone utilization in the rat heart, thereby improving function and energetics both during ischaemia and recovery during reperfusion. This preference of ketone utilization over glucose was observed under conditions of constant supply of substrate, suggesting that EMPA acts directly by modulating cardiac substrate preference, independent of substrate availability. The mechanisms underlying our findings are currently unknown, warranting further study.
Topics: Rats; Animals; Diabetes Mellitus, Type 2; Glucose; Adenosine Triphosphate; Ischemia; Reperfusion
PubMed: 37819017
DOI: 10.1093/cvr/cvad157 -
Molecular Biology Reports Jan 2024D-ribose, an ubiquitous pentose compound found in all living cells, serves as a vital constituent of numerous essential biomolecules, including RNA, nucleotides, and... (Review)
Review
D-ribose, an ubiquitous pentose compound found in all living cells, serves as a vital constituent of numerous essential biomolecules, including RNA, nucleotides, and riboflavin. It plays a crucial role in various fundamental life processes. Within the cellular milieu, exogenously supplied D-ribose can undergo phosphorylation to yield ribose-5-phosphate (R-5-P). This R-5-P compound serves a dual purpose: it not only contributes to adenosine triphosphate (ATP) production through the nonoxidative phase of the pentose phosphate pathway (PPP) but also participates in nucleotide synthesis. Consequently, D-ribose is employed both as a therapeutic agent for enhancing cardiac function in heart failure patients and as a remedy for post-exercise fatigue. Nevertheless, recent clinical studies have suggested a potential link between D-ribose metabolic disturbances and type 2 diabetes mellitus (T2DM) along with its associated complications. Additionally, certain in vitro experiments have indicated that exogenous D-ribose exposure could trigger apoptosis in specific cell lines. This article comprehensively reviews the current advancements in D-ribose's digestion, absorption, transmembrane transport, intracellular metabolic pathways, impact on cellular behaviour, and elevated levels in diabetes mellitus. It also identifies areas requiring further investigation.
Topics: Humans; Diabetes Mellitus, Type 2; Ribose; Heart Failure; Metabolic Diseases; Adenosine Triphosphate
PubMed: 38281218
DOI: 10.1007/s11033-023-09076-y -
Nature Communications Oct 2023Lower respiratory tract infections caused by Streptococcus pneumoniae (Spn) are a leading cause of death globally. Here we investigate the bronchial epithelial cellular...
Lower respiratory tract infections caused by Streptococcus pneumoniae (Spn) are a leading cause of death globally. Here we investigate the bronchial epithelial cellular response to Spn infection on a transcriptomic, proteomic and metabolic level. We found the NAD salvage pathway to be dysregulated upon infection in a cell line model, primary human lung tissue and in vivo in rodents, leading to a reduced production of NAD. Knockdown of NAD salvage enzymes (NAMPT, NMNAT1) increased bacterial replication. NAD treatment of Spn inhibited its growth while growth of other respiratory pathogens improved. Boosting NAD production increased NAD levels in immortalized and primary cells and decreased bacterial replication upon infection. NAD treatment of Spn dysregulated the bacterial metabolism and reduced intrabacterial ATP. Enhancing the bacterial ATP metabolism abolished the antibacterial effect of NAD. Thus, we identified the NAD salvage pathway as an antibacterial pathway in Spn infections, predicting an antibacterial mechanism of NAD.
Topics: Humans; NAD; Proteomics; Cytokines; Cell Line; Bacterial Infections; Adenosine Triphosphate; Respiratory Tract Infections; Nicotinamide-Nucleotide Adenylyltransferase
PubMed: 37783679
DOI: 10.1038/s41467-023-41372-w -
Frontiers in Immunology 2023Adenosine (Ado) is a well-known immunosuppressive agent that may be released or generated extracellularly by cells, via degrading ATP by the sequential actions of the... (Review)
Review
Adenosine (Ado) is a well-known immunosuppressive agent that may be released or generated extracellularly by cells, via degrading ATP by the sequential actions of the ectonucleotides CD39 and CD73. During inflammation Ado is produced by leukocytes and tissue cells by different means to initiate the healing phase. Ado downregulates the activation and the effector functions of different leukocyte (sub-) populations and stimulates proliferation of fibroblasts for re-establishment of intact tissues. Therefore, the anti-inflammatory actions of Ado are already intrinsically triggered during each episode of inflammation. These tissue-regenerating and inflammation-tempering purposes of Ado can become counterproductive. In chronic inflammation, it is possible that Ado-driven anti-inflammatory actions sustain the inflammation and prevent the final clearance of the tissues from possible pathogens. These chronic infections are characterized by increased tissue damage, remodeling and accumulating DNA damage, and are thus prone for tumor formation. Developing tumors may further enhance immunosuppressive actions by producing Ado by themselves, or by "hijacking" CD39/CD73 cells that had already developed during chronic inflammation. This review describes different and mostly convergent mechanisms of how Ado-induced immune suppression, initially induced in inflammation, can lead to tumor formation and outgrowth.
Topics: Humans; Adenosine; Neoplasms; Adenosine Triphosphate; Inflammation; Anti-Inflammatory Agents
PubMed: 38022572
DOI: 10.3389/fimmu.2023.1258637 -
Cellular and Molecular Neurobiology Nov 2023Hypercholesterolemia affects the neurovascular unit, including the cerebral blood vessel endothelium. Operation of this system, especially in the context of energy...
Hypercholesterolemia affects the neurovascular unit, including the cerebral blood vessel endothelium. Operation of this system, especially in the context of energy metabolism, is controlled by extracellular concentration of purines, regulated by ecto-enzymes, such as e-NTPDase-1/CD39, ecto-5'-NT/CD73, and eADA. We hypothesize that hypercholesterolemia, via modulation of the activity of nucleotide metabolism-regulating ecto-enzymes, deteriorates glycolytic efficiency and energy metabolism of endothelial cells, which may potentially contribute to development of neurodegenerative processes. We aimed to determine the effect of hypercholesterolemia on the concentration of purine nucleotides, glycolytic activity, and activity of ecto-enzymes in the murine brain microvascular endothelial cells (mBMECs). We used 3-month-old male LDLR/Apo E double knockout mice to model hypercholesterolemia and atherosclerosis. The age-matched wild-type C57/BL6 mice were a control group. The intracellular concentration of ATP and NAD and extracellular activity of the ecto-enzymes were measured by HPLC. The glycolytic function of mBMECs was assessed by means of the extracellular acidification rate (ECAR) using the glycolysis stress test. The results showed an increased activity of ecto-5'-NT and eADA in mBMECs of the hypercholesterolemic mice, but no differences in intracellular concentration of ATP, NAD, and ECAR between the hypercholesterolemic and control groups. The changed activity of ecto-5'-NT and eADA leads to increased purine nucleotides turnover and a shift in their concentration balance towards adenosine and inosine in the extracellular space. However, no changes in the energetic metabolism of the mBMECs are reported. Our results confirm the influence of hypercholesterolemia on regulation of purine nucleotides metabolism, which may impair the function of the cerebral vascular endothelium. The effect of hypercholesterolemia on the murine brain microvascular endothelial cells (mBMECs). An increased activity of ecto-5'-NT and eADA in mBMECs of the LDLR/Apo E mice leads to a shift in the concentration balance towards adenosine and inosine in the extracellular space with no differences in intracellular concentration of ATP. Figure was created with Biorender.com.
Topics: Male; Mice; Animals; Hypercholesterolemia; Endothelial Cells; NAD; Adenosine; Adenosine Triphosphate; Brain; Mice, Knockout; Endothelium; Inosine; Apolipoproteins E; 5'-Nucleotidase
PubMed: 37801200
DOI: 10.1007/s10571-023-01415-8 -
The EMBO Journal Sep 2023Cilia are important cellular organelles for signaling and motility and are constructed via intraflagellar transport (IFT). RabL2 is a small GTPase that localizes to the...
Cilia are important cellular organelles for signaling and motility and are constructed via intraflagellar transport (IFT). RabL2 is a small GTPase that localizes to the basal body of cilia via an interaction with the centriolar protein CEP19 before downstream association with the IFT machinery, which is followed by initiation of IFT. We reconstituted and purified RabL2 with CEP19 or IFT proteins to show that a reconstituted pentameric IFT complex containing IFT81/74 enhances the GTP hydrolysis rate of RabL2. The binding site on IFT81/74 that promotes GTP hydrolysis in RabL2 was mapped to a 70-amino-acid-long coiled-coil region of IFT81/74. We present structural models for RabL2-containing IFT complexes that we validate in vitro and in cellulo and demonstrate that Chlamydomonas IFT81/74 enhances GTP hydrolysis of human RabL2, suggesting an ancient evolutionarily conserved activity. Our results provide an architectural understanding of how RabL2 is incorporated into the IFT complex and a molecular rationale for why RabL2 dissociates from anterograde IFT trains soon after departure from the ciliary base.
Topics: Humans; GTPase-Activating Proteins; Signal Transduction; Biological Transport; Amino Acids; Guanosine Triphosphate; Muscle Proteins; Cytoskeletal Proteins
PubMed: 37606072
DOI: 10.15252/embj.2022111807 -
Science Advances Aug 2023Pseudouridine is enriched in ribosomal, spliceosomal, transfer, and messenger RNA and thus integral to the central dogma. The chemical basis for how pseudouridine...
Pseudouridine is enriched in ribosomal, spliceosomal, transfer, and messenger RNA and thus integral to the central dogma. The chemical basis for how pseudouridine affects the molecular apparatus such as ribosome, however, remains elusive owing to the lack of structures without this natural modification. Here, we studied the translation of a hypopseudouridylated ribosome initiated by the internal ribosome entry site (IRES) elements. We analyzed eight cryo-electron microscopy structures of the ribosome bound with the Taura syndrome virus IRES in multiple functional states. We found widespread loss of pseudouridine-mediated interactions through water and long-range base pairings. In the presence of the translocase, eukaryotic elongation factor 2, and guanosine 5'-triphosphate hydrolysis, the hypopseudouridylated ribosome favors a rare unconducive conformation for decoding that is partially recouped in the ribosome population that remains modified at the P-site uridine. The structural principles learned establish the link between functional defects and modification loss and are likely applicable to other pseudouridine-associated processes.
Topics: RNA, Ribosomal; Cryoelectron Microscopy; Pseudouridine; Ribosomes; Eukaryota; Guanosine Triphosphate
PubMed: 37595043
DOI: 10.1126/sciadv.adg8190