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1.
Acta Physiol (Oxf) ; 240(9): e14209, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39072954

RESUMO

AIM: Mitochondrial uncoupling protein 1 (UCP1) is a unique protein of brown adipose tissue. Upon activation by free fatty acids, UCP1 facilitates a thermogenic net proton flux across the mitochondrial inner membrane. Non-complexed purine nucleotides inhibit this fatty acid-induced activity of UCP1. The most available data have been generated from rodent model systems. In light of its role as a putative pharmacological target for treating metabolic disease, in-depth analyses of human UCP1 activity, regulation, and structural features are essential. METHODS: In the present study, we established a doxycycline-regulated cell model with inducible human or murine UCP1 expression and conducted functional studies using respirometry comparing wild-type and mutant variants of human UCP1. RESULTS: We demonstrate that human and mouse UCP1 exhibit similar specific fatty acid-induced activity but a different inhibitory potential of purine nucleotides. Mutagenesis of non-conserved residues in human UCP1 revealed structural components in α-helix 56 and α-helix 6 crucial for uncoupling function. CONCLUSION: Comparative studies of human UCP1 with other orthologs can provide new insights into the structure-function relationship for this mitochondrial carrier and will be instrumental in searching for new activators.


Assuntos
Proteína Desacopladora 1 , Proteína Desacopladora 1/metabolismo , Proteína Desacopladora 1/genética , Animais , Humanos , Camundongos , Mitocôndrias/metabolismo , Nucleotídeos de Purina/metabolismo
2.
Mol Cell ; 84(13): 2407-2409, 2024 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-38996457

RESUMO

In two recent studies appearing in Cell1 and Cell Metabolism,2 Tran et al. and Wu et al. describe underappreciated nuance in organismal and cellular purine nucleotide salvage pathways and identify purine salvage as a metabolic limitation for tumor growth.


Assuntos
Purinas , Purinas/metabolismo , Humanos , Animais , Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/patologia , Nucleotídeos de Purina/metabolismo
3.
Cell ; 187(14): 3602-3618.e20, 2024 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-38823389

RESUMO

Purine nucleotides are vital for RNA and DNA synthesis, signaling, metabolism, and energy homeostasis. To synthesize purines, cells use two principal routes: the de novo and salvage pathways. Traditionally, it is believed that proliferating cells predominantly rely on de novo synthesis, whereas differentiated tissues favor the salvage pathway. Unexpectedly, we find that adenine and inosine are the most effective circulating precursors for supplying purine nucleotides to tissues and tumors, while hypoxanthine is rapidly catabolized and poorly salvaged in vivo. Quantitative metabolic analysis demonstrates comparative contribution from de novo synthesis and salvage pathways in maintaining purine nucleotide pools in tumors. Notably, feeding mice nucleotides accelerates tumor growth, while inhibiting purine salvage slows down tumor progression, revealing a crucial role of the salvage pathway in tumor metabolism. These findings provide fundamental insights into how normal tissues and tumors maintain purine nucleotides and highlight the significance of purine salvage in cancer.


Assuntos
Neoplasias , Nucleotídeos de Purina , Purinas , Animais , Camundongos , Purinas/metabolismo , Purinas/biossíntese , Neoplasias/metabolismo , Neoplasias/patologia , Nucleotídeos de Purina/metabolismo , Humanos , Inosina/metabolismo , Hipoxantina/metabolismo , Camundongos Endogâmicos C57BL , Adenina/metabolismo , Linhagem Celular Tumoral , Feminino
4.
Chembiochem ; 25(15): e202400202, 2024 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-38818670

RESUMO

RNA labeling is an invaluable tool for investigation of the function and localization of nucleic acids. Labels are commonly incorporated into 3' end of RNA and the primary enzyme used for this purpose is RNA poly(A) polymerase (PAP), which belongs to the class of terminal nucleotidyltransferases (NTases). However, PAP preferentially adds ATP analogs, thus limiting the number of available substrates. Here, we report the use of another NTase, CutA from the fungus Thielavia terrestris. Using this enzyme, we were able to incorporate into the 3' end of RNA not only purine analogs, but also pyrimidine analogs. We engaged strain-promoted azide-alkyl cycloaddition (SPAAC) to obtain fluorescently labeled or biotinylated transcripts from RNAs extended with azide analogs by CutA. Importantly, modified transcripts retained their biological properties. Furthermore, fluorescently labeled mRNAs were suitable for visualization in cultured mammalian cells. Finally, we demonstrate that either affinity studies or molecular dynamic (MD) simulations allow for rapid screening of NTase substrates, what opens up new avenues in the search for the optimal substrates for this class of enzymes.


Assuntos
Nucleotídeos de Pirimidina , Humanos , Nucleotídeos de Pirimidina/química , Nucleotídeos de Pirimidina/metabolismo , RNA/metabolismo , RNA/química , Nucleotídeos de Purina/metabolismo , Nucleotídeos de Purina/química , Simulação de Dinâmica Molecular , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/genética
5.
Nat Commun ; 15(1): 1931, 2024 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-38431691

RESUMO

Supporting cell proliferation through nucleotide biosynthesis is an essential requirement for cancer cells. Hence, inhibition of folate-mediated one carbon (1C) metabolism, which is required for nucleotide synthesis, has been successfully exploited in anti-cancer therapy. Here, we reveal that mitochondrial folate metabolism is upregulated in patient-derived leukaemic stem cells (LSCs). We demonstrate that inhibition of mitochondrial 1C metabolism through impairment of de novo purine synthesis has a cytostatic effect on chronic myeloid leukaemia (CML) cells. Consequently, changes in purine nucleotide levels lead to activation of AMPK signalling and suppression of mTORC1 activity. Notably, suppression of mitochondrial 1C metabolism increases expression of erythroid differentiation markers. Moreover, we find that increased differentiation occurs independently of AMPK signalling and can be reversed through reconstitution of purine levels and reactivation of mTORC1. Of clinical relevance, we identify that combination of 1C metabolism inhibition with imatinib, a frontline treatment for CML patients, decreases the number of therapy-resistant CML LSCs in a patient-derived xenograft model. Our results highlight a role for folate metabolism and purine sensing in stem cell fate decisions and leukaemogenesis.


Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva , Leucemia Mieloide , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Proteínas Quinases Ativadas por AMP , Purinas/uso terapêutico , Nucleotídeos de Purina , Ácido Fólico/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico
6.
Mol Cancer Res ; 22(1): 82-93, 2024 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-37773022

RESUMO

Small cell lung cancer (SCLC) has a poor prognosis, emphasizing the necessity for developing new therapies. The de novo synthesis pathway of purine nucleotides, which is involved in the malignant growth of SCLC, has emerged as a novel therapeutic target. Purine nucleotides are supplied by two pathways: de novo and salvage. However, the role of the salvage pathway in SCLC and the differences in utilization and crosstalk between the two pathways remain largely unclear. Here, we found that deletion of the HPRT1 gene, which codes for the rate-limiting enzyme of the purine salvage pathway, significantly suppressed tumor growth in vivo in several SCLC cells. We also demonstrated that HPRT1 expression confers resistance to lemetrexol (LMX), an inhibitor of the purine de novo pathway. Interestingly, HPRT1-knockout had less effect on SCLC SBC-5 cells, which are more sensitive to LMX than other SCLC cell lines, suggesting that a preference for either the purine de novo or salvage pathway occurs in SCLC. Furthermore, metabolome analysis of HPRT1-knockout cells revealed increased intermediates in the pentose phosphate pathway and elevated metabolic flux in the purine de novo pathway, indicating compensated metabolism between the de novo and salvage pathways in purine nucleotide biosynthesis. These results suggest that HPRT1 has therapeutic implications in SCLC and provide fundamental insights into the regulation of purine nucleotide biosynthesis. IMPLICATIONS: SCLC tumors preferentially utilize either the de novo or salvage pathway in purine nucleotide biosynthesis, and HPRT1 has therapeutic implications in SCLC.


Assuntos
Neoplasias Pulmonares , Carcinoma de Pequenas Células do Pulmão , Humanos , Carcinoma de Pequenas Células do Pulmão/genética , Purinas/metabolismo , Nucleotídeos de Purina/metabolismo , Hipoxantina Fosforribosiltransferase/metabolismo , Neoplasias Pulmonares/genética
7.
J Biol Chem ; 300(1): 105485, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37992808

RESUMO

EZH2 (Enhancer of Zeste Homolog 2), a subunit of Polycomb Repressive Complex 2 (PRC2), catalyzes the trimethylation of histone H3 at lysine 27 (H3K27me3), which represses expression of genes. It also has PRC2-independent functions, including transcriptional coactivation of oncogenes, and is frequently overexpressed in lung cancers. Clinically, EZH2 inhibition can be achieved with the FDA-approved drug EPZ-6438 (tazemetostat). To realize the full potential of EZH2 blockade, it is critical to understand how cell-cell/cell-matrix interactions present in 3D tissue and cell culture systems influences this blockade in terms of growth-related metabolic functions. Here, we show that EZH2 suppression reduced growth of human lung adenocarcinoma A549 cells in 2D cultures but stimulated growth in 3D cultures. To understand the metabolic underpinnings, we employed [13C6]-glucose stable isotope-resolved metabolomics to determine the effect of EZH2 suppression on metabolic networks in 2D versus 3D A549 cultures. The Krebs cycle, neoribogenesis, γ-aminobutyrate metabolism, and salvage synthesis of purine nucleotides were activated by EZH2 suppression in 3D spheroids but not in 2D cells, consistent with the growth effect. Using simultaneous 2H7-glucose + 13C5,15N2-Gln tracers and EPZ-6438 inhibition of H3 trimethylation, we delineated the effects on the Krebs cycle, γ-aminobutyrate metabolism, gluconeogenesis, and purine salvage to be PRC2-dependent. Furthermore, the growth/metabolic effects differed for mouse Matrigel versus self-produced A549 extracellular matrix. Thus, our findings highlight the importance of the presence and nature of extracellular matrix in studying the function of EZH2 and its inhibitors in cancer cells for modeling the in vivo outcomes.


Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste , Reprogramação Metabólica , Humanos , Linhagem Celular Tumoral , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Reprogramação Metabólica/genética , Complexo Repressor Polycomb 2/antagonistas & inibidores , Complexo Repressor Polycomb 2/genética , Células A549 , Adenocarcinoma de Pulmão/fisiopatologia , Técnicas de Silenciamento de Genes , Glicólise/genética , Ciclo do Ácido Cítrico/genética , Via de Pentose Fosfato/genética , Nucleotídeos de Purina/genética , Regulação Neoplásica da Expressão Gênica
8.
Compr Physiol ; 14(1): 5345-5369, 2023 12 29.
Artigo em Inglês | MEDLINE | ID: mdl-38158366

RESUMO

Purine nucleotides play central roles in energy metabolism in the heart. Most fundamentally, the free energy of hydrolysis of the adenine nucleotide adenosine triphosphate (ATP) provides the thermodynamic driving force for numerous cellular processes including the actin-myosin crossbridge cycle. Perturbations to ATP supply and/or demand in the myocardium lead to changes in the homeostatic balance between purine nucleotide synthesis, degradation, and salvage, potentially affecting myocardial energetics and, consequently, myocardial mechanics. Indeed, both acute myocardial ischemia and decompensatory remodeling of the myocardium in heart failure are associated with depletion of myocardial adenine nucleotides and with impaired myocardial mechanical function. Yet there remain gaps in the understanding of mechanistic links between adenine nucleotide degradation and contractile dysfunction in heart disease. The scope of this article is to: (i) review current knowledge of the pathways of purine nucleotide depletion and salvage in acute ischemia and in chronic heart disease; (ii) review hypothesized mechanisms linking myocardial mechanics and energetics with myocardial adenine nucleotide regulation; and (iii) highlight potential targets for treating myocardial metabolic and mechanical dysfunction associated with these pathways. It is hypothesized that an imbalance in the degradation, salvage, and synthesis of adenine nucleotides leads to a net loss of adenine nucleotides in both acute ischemia and under chronic high-demand conditions associated with the development of heart failure. This reduction in adenine nucleotide levels results in reduced myocardial ATP and increased myocardial inorganic phosphate. Both of these changes have the potential to directly impact tension development and mechanical work at the cellular level. © 2024 American Physiological Society. Compr Physiol 14:5345-5369, 2024.


Assuntos
Cardiopatias , Insuficiência Cardíaca , Humanos , Trifosfato de Adenosina/metabolismo , Miocárdio/metabolismo , Nucleotídeos de Purina/metabolismo , Nucleotídeos/metabolismo , Cardiopatias/metabolismo , Insuficiência Cardíaca/metabolismo , Metabolismo Energético , Isquemia
9.
Int J Mol Sci ; 24(16)2023 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-37628755

RESUMO

Most cancer cells have an increased synthesis of purine nucleotides to fulfil their enhanced division rate. The de novo synthesis of purines requires folic acid in the form of N10-formyltetrahydrofolate (10-formyl-THF). However, regular cell culture media contain very high, non-physiological concentrations of folic acid, which may have an impact on cell metabolism. Using cell culture media with physiological levels of folic acid (25 nM), we uncover purine alterations in several human cell lines. HEK293T, Jurkat, and A549 cells accumulate 5'-aminoimidazole-4-carboxamide ribonucleotide (ZMP), an intermediary of the de novo biosynthetic pathway, at physiological levels of folic acid, but not with the artificially high levels (2200 nM) present in regular media. Interestingly, HEK293T and Jurkat cells do not accumulate high levels of ZMP when AICAr, the precursor of ZMP, is added to medium containing 2200 nM folate; instead, ATP levels are increased, suggesting an enhanced de novo synthesis. On the other hand, HeLa and EHEB cells do not accumulate ZMP at physiological levels of folic acid, but they do accumulate in medium containing AICAr plus 2200 nM folate. Expression of SLC19A1, which encodes the reduced folate carrier (RFC), is increased in HEK293T and Jurkat cells compared with HeLa and EHEB, and it is correlated with the total purine nucleotide content at high levels of folic acid or with ZMP accumulation at physiological levels of folic acid. In conclusion, tumoral cell lines show a heterogenous response to folate changes in the media, some of them accumulating ZMP at physiological levels of folic acid. Further research is needed to clarify the ZMP downstream targets and their impact on cell function.


Assuntos
Ácido Fólico , Nucleotídeos de Purina , Humanos , Células HEK293 , Linhagem Celular Tumoral , Células HeLa
10.
Nat Commun ; 14(1): 5078, 2023 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-37604815

RESUMO

Purine-containing nucleotide second messengers regulate diverse cellular activities. Cyclic di-pyrimidines mediate anti-phage functions in bacteria; however, the synthesis mechanism remains elusive. Here, we determine the high-resolution structures of cyclic di-pyrimidine-synthesizing cGAS/DncV-like nucleotidyltransferases (CD-NTases) in clade E (CdnE) in its apo, substrate-, and intermediate-bound states. A conserved (R/Q)xW motif controlling the pyrimidine specificity of donor nucleotide is identified. Mutation of Trp or Arg from the (R/Q)xW motif to Ala rewires its specificity to purine nucleotides, producing mixed purine-pyrimidine cyclic dinucleotides (CDNs). Preferential binding of uracil over cytosine bases explains the product specificity of cyclic di-pyrimidine-synthesizing CdnE to cyclic di-UMP (cUU). Based on the intermediate-bound structures, a synthetic pathway for cUU containing a unique 2'3'-phosphodiester linkage through intermediate pppU[3'-5']pU is deduced. Our results provide a framework for pyrimidine selection and establish the importance of conserved residues at the C-terminal loop for the specificity determination of CD-NTases.


Assuntos
Nucleotidiltransferases , Pirimidinas , Nucleotidiltransferases/genética , Nucleotídeos , Cromogranina A , Nucleotídeos de Purina
11.
Biochemistry ; 62(17): 2587-2596, 2023 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-37552766

RESUMO

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, N5-CAIR. Toward the development of therapeutics that target these differences, we have solved crystal structures of the N5-CAIR mutase of the human pathogens Legionella pneumophila (LpPurE) and Burkholderia cenocepacia (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 in silico and identified a set of 25 candidates for further analysis. Among these, we identified several new PurE inhibitors with micromolar IC50 values. Several of these compounds, including the α1-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.


Assuntos
Transferases Intramoleculares , Ribonucleotídeos , Humanos , Ribonucleotídeos/química , Escherichia coli/metabolismo , Transferases Intramoleculares/metabolismo , Nucleotídeos de Purina/metabolismo
12.
J Gen Appl Microbiol ; 69(2): 109-116, 2023 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-37302828

RESUMO

Adenylosuccinate synthetase (PurA) is an enzyme responsible for the nitrogen addition to inosine monophosphate (IMP) by aspartate in the purine nucleotide biosynthetic pathway. And after which the fumarate is removed by adenylosuccinate lyase (PurB), leaving an amino group. There are two other enzymes that catalyze aspartate addition reactions similar to PurA, one in the purine nucleotide biosynthetic pathway (SAICAR synthetase, PurC) and the other in the arginine biosynthetic pathway (argininosuccinate sythetase, ArgG). To investigate the origin of these nitrogen-adding enzymes, PurA from Thermus thermophilus HB8 (TtPurA) was purified and crystallized, and crystal structure complexed with IMP was determined with a resolution of 2.10 Å. TtPurA has a homodimeric structure, and at the dimer interface, Arg135 of one subunit interacts with the IMP bound to the other subunit, suggesting that IMP binding contributes to dimer stability. The different conformation of His41 side chain in TtPurA and EcPurA suggests that side chain flipping of the His41 might play an important role in orienting γ-phosphate of GTP close to oxygen at position 6 of IMP, to receive the nucleophilic attack. Moreover, through comparison of the three-dimensional structures and active sites of PurA, PurC, and ArgG, it was suggested that the active sites of PurA and PurC converged to similar structures for performing similar reactions.


Assuntos
Adenilossuccinato Sintase , Ácido Aspártico , Adenilossuccinato Sintase/genética , Adenilossuccinato Sintase/química , Adenilossuccinato Sintase/metabolismo , Ácido Aspártico/metabolismo , Vias Biossintéticas , Nucleotídeos de Purina/metabolismo
13.
Nat Commun ; 14(1): 2594, 2023 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-37147287

RESUMO

Brown adipose tissue expresses uncoupling protein 1 (UCP1), which dissipates energy as heat, making it a target for treating metabolic disorders. Here, we investigate how purine nucleotides inhibit respiration uncoupling by UCP1. Our molecular simulations predict that GDP and GTP bind UCP1 in the common substrate binding site in an upright orientation, where the base moiety interacts with conserved residues R92 and E191. We identify a triplet of uncharged residues, F88/I187/W281, forming hydrophobic contacts with nucleotides. In yeast spheroplast respiration assays, both I187A and W281A mutants increase the fatty acid-induced uncoupling activity of UCP1 and partially suppress the inhibition of UCP1 activity by nucleotides. The F88A/I187A/W281A triple mutant is overactivated by fatty acids even at high concentrations of purine nucleotides. In simulations, E191 and W281 interact with purine but not pyrimidine bases. These results provide a molecular understanding of the selective inhibition of UCP1 by purine nucleotides.


Assuntos
Canais Iônicos , Proteínas de Membrana , Canais Iônicos/genética , Canais Iônicos/metabolismo , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismo , Ácidos Graxos/metabolismo , Nucleotídeos de Purina/metabolismo , Tecido Adiposo Marrom/metabolismo , Saccharomyces cerevisiae/metabolismo
14.
Microbiol Spectr ; 11(3): e0082323, 2023 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-37195232

RESUMO

Haemophilus influenzae is a human-adapted bacterial pathogen that causes airway infections. Bacterial and host elements associated with the fitness of H. influenzae within the host lung are not well understood. Here, we exploited the strength of in vivo-omic analyses to study host-microbe interactions during infection. We used in vivo transcriptome sequencing (RNA-seq) for genome-wide profiling of both host and bacterial gene expression during mouse lung infection. Profiling of murine lung gene expression upon infection showed upregulation of lung inflammatory response and ribosomal organization genes, and downregulation of cell adhesion and cytoskeleton genes. Transcriptomic analysis of bacteria recovered from bronchoalveolar lavage fluid samples from infected mice showed a significant metabolic rewiring during infection, which was highly different from that obtained upon bacterial in vitro growth in an artificial sputum medium suitable for H. influenzae. In vivo RNA-seq revealed upregulation of bacterial de novo purine biosynthesis, genes involved in non-aromatic amino acid biosynthesis, and part of the natural competence machinery. In contrast, the expression of genes involved in fatty acid and cell wall synthesis and lipooligosaccharide decoration was downregulated. Correlations between upregulated gene expression and mutant attenuation in vivo were established, as observed upon purH gene inactivation leading to purine auxotrophy. Likewise, the purine analogs 6-thioguanine and 6-mercaptopurine reduced H. influenzae viability in a dose-dependent manner. These data expand our understanding of H. influenzae requirements during infection. In particular, H. influenzae exploits purine nucleotide synthesis as a fitness determinant, raising the possibility of purine synthesis as an anti-H. influenzae target. IMPORTANCE In vivo-omic strategies offer great opportunities for increased understanding of host-pathogen interplay and for identification of therapeutic targets. Here, using transcriptome sequencing, we profiled host and pathogen gene expression during H. influenzae infection within the murine airways. Lung pro-inflammatory gene expression reprogramming was observed. Moreover, we uncovered bacterial metabolic requirements during infection. In particular, we identified purine synthesis as a key player, highlighting that H. influenzae may face restrictions in purine nucleotide availability within the host airways. Therefore, blocking this biosynthetic process may have therapeutic potential, as supported by the observed inhibitory effect of 6-thioguanine and 6-mercaptopurine on H. influenzae growth. Together, we present key outcomes and challenges for implementing in vivo-omics in bacterial airway pathogenesis. Our findings provide metabolic insights into H. influenzae infection biology, raising the possibility of purine synthesis as an anti-H. influenzae target and of purine analog repurposing as an antimicrobial strategy against this pathogen.


Assuntos
Infecções por Haemophilus , Haemophilus influenzae , Camundongos , Humanos , Animais , Haemophilus influenzae/genética , Mercaptopurina/metabolismo , Mercaptopurina/uso terapêutico , Tioguanina , Pulmão/patologia , Perfilação da Expressão Gênica , Infecções por Haemophilus/tratamento farmacológico , Nucleotídeos de Purina/metabolismo , Nucleotídeos de Purina/uso terapêutico
15.
Sci Adv ; 9(22): eadh4251, 2023 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-37256948

RESUMO

Mitochondrial uncoupling protein 1 (UCP1) gives brown adipose tissue of mammals its specialized ability to burn calories as heat for thermoregulation. When activated by fatty acids, UCP1 catalyzes the leak of protons across the mitochondrial inner membrane, short-circuiting the mitochondrion to generate heat, bypassing ATP synthesis. In contrast, purine nucleotides bind and inhibit UCP1, regulating proton leak by a molecular mechanism that is unclear. We present the cryo-electron microscopy structure of the GTP-inhibited state of UCP1, which is consistent with its nonconducting state. The purine nucleotide cross-links the transmembrane helices of UCP1 with an extensive interaction network. Our results provide a structural basis for understanding the specificity and pH dependency of the regulatory mechanism. UCP1 has retained all of the key functional and structural features required for a mitochondrial carrier-like transport mechanism. The analysis shows that inhibitor binding prevents the conformational changes that UCP1 uses to facilitate proton leak.


Assuntos
Canais Iônicos , Prótons , Humanos , Microscopia Crioeletrônica , Canais Iônicos/química , Proteínas Mitocondriais/metabolismo , Nucleotídeos de Purina , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismo
16.
FEBS J ; 290(12): 3165-3184, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-36748301

RESUMO

In human cells, de novo purine nucleotide biosynthesis is known to be regulated through the formation of a metabolon called purinosome. Here, we employed a bacterial two-hybrid approach to characterize the protein-protein interactions network among the corresponding enzymes of Escherichia coli. Our study revealed a dense network of binary interactions that connect most purine nucleotide biosynthesis enzymes. Notably, PurK, an exclusive prokaryotic enzyme, appears as one of the central hubs of this network. We further showed that modifications in PurK, which disrupted several interactions in the network, affected the purine nucleotide pools and altered the bacterial fitness. Our data suggest that the bacterial de novo purine nucleotide biosynthesis enzymes can assemble in a supramolecular complex and that proper interactions among the components of this complex can contribute to bacterial fitness.


Assuntos
Escherichia coli , Nucleotídeos , Humanos , Escherichia coli/genética , Purinas , Nucleotídeos de Purina
17.
Plant Cell ; 35(1): 510-528, 2023 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-36342213

RESUMO

In nucleotide metabolism, nucleoside kinases recycle nucleosides into nucleotides-a process called nucleoside salvage. Nucleoside kinases for adenosine, uridine, and cytidine have been characterized from many organisms, but kinases for inosine and guanosine salvage are not yet known in eukaryotes and only a few such enzymes have been described from bacteria. Here we identified Arabidopsis thaliana PLASTID NUCLEOSIDE KINASE 1 (PNK1), an enzyme highly conserved in plants and green algae belonging to the Phosphofructokinase B family. We demonstrate that PNK1 from A. thaliana is located in plastids and catalyzes the phosphorylation of inosine, 5-aminoimidazole-4-carboxamide-1-ß-d-ribose (AICA ribonucleoside), and uridine but not guanosine in vitro, and is involved in inosine salvage in vivo. PNK1 mutation leads to increased flux into purine nucleotide catabolism and, especially in the context of defective uridine degradation, to over-accumulation of uridine and UTP as well as growth depression. The data suggest that PNK1 is involved in feedback regulation of purine nucleotide biosynthesis and possibly also pyrimidine nucleotide biosynthesis. We additionally report that cold stress leads to accumulation of purine nucleotides, probably by inducing nucleotide biosynthesis, but that this adjustment of nucleotide homeostasis to environmental conditions is not controlled by PNK1.


Assuntos
Inosina , Nucleosídeos , Inosina/metabolismo , Inosina/farmacologia , Nucleosídeos/metabolismo , Nucleotídeos , Nucleotídeos de Purina/genética , Nucleotídeos de Purina/metabolismo , Uridina
18.
Food Chem ; 401: 134145, 2023 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-36088713

RESUMO

Endogenous hydrocortisone causes detrimental effects on public health and domestic animal products, but the potential mechanisms remain elusive. Hydrocortisone was detected from seventy-two Guanzhong-Black pigs in three replicates (216 samples) (0.00 ± 46.38 µg kg-1), indicating the existence of endogenous hydrocortisone. Herein, we investigated the effects of hydrocortisone on the metabolic signatures in pork via integrative metabolomics and proteomics by UHPLC-Q-Orbitrap HRMS. Animal-derived foods under hydrocortisone-bioaccumulation cause metabolic perturbation by regulating glutamine synthetase expression at the transcriptional level contributed to hydrocortisone-induced toxicity and accelerating the down-regulation of essential amino acids (l-Histidine 10.74-6.48 mg kg-1, l-Phenylalanine 3.70-1.57 mg kg-1), purine nucleotides (GMP 40.29-5.00 µg kg-1, etc) that provide nutritional value in a positive feedback loop. Protein-metabolite interactions suggesting that hydrocortisone enhanced nitric oxide synthase and GMP reductase expression (LOQ 3.24-63.39 µg kg-1), and affected meat flavor perception, eventually lead to the decline of nutritional value and flesh quality of pork.


Assuntos
Histidina , Hidrocortisona , Suínos , Animais , Cromatografia Líquida de Alta Pressão , GMP Redutase , Glutamato-Amônia Ligase , Nucleotídeos de Purina , Fenilalanina
19.
New Phytol ; 237(5): 1759-1775, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36464781

RESUMO

In plants, inosine is enzymatically introduced in some tRNAs, but not in other RNAs or DNA. Nonetheless, our data show that RNA and DNA from Arabidopsis thaliana contain (deoxy)inosine, probably derived from nonenzymatic adenosine deamination in nucleic acids and usage of (deoxy)inosine triphosphate (dITP and ITP) during nucleic acid synthesis. We combined biochemical approaches, LC-MS, as well as RNA-Seq to characterize a plant INOSINE TRIPHOSPHATE PYROPHOSPHATASE (ITPA) from A. thaliana, which is conserved in many organisms, and investigated the sources of deaminated purine nucleotides in plants. Inosine triphosphate pyrophosphatase dephosphorylates deaminated nucleoside di- and triphosphates to the respective monophosphates. ITPA loss-of-function causes inosine di- and triphosphate accumulation in vivo and an elevated inosine and deoxyinosine content in RNA and DNA, respectively, as well as salicylic acid (SA) accumulation, early senescence, and upregulation of transcripts associated with immunity and senescence. Cadmium-induced oxidative stress and biochemical inhibition of the INOSINE MONOPHOSPHATE DEHYDROGENASE leads to more IDP and ITP in the wild-type (WT), and this effect is enhanced in itpa mutants, suggesting that ITP originates from ATP deamination and IMP phosphorylation. Inosine triphosphate pyrophosphatase is part of a molecular protection system in plants, preventing the accumulation of (d)ITP and its usage for nucleic acid synthesis.


Assuntos
Inosina Trifosfato , Nucleotídeos de Purina , Pirofosfatases , Trifosfato de Adenosina , DNA , Inosina Trifosfato/genética , Ácidos Nucleicos , Pirofosfatases/genética , RNA
20.
Neuropharmacology ; 224: 109370, 2023 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-36493858

RESUMO

Purine-based molecules play ancient, fundamental, and evolutionarily-conserved roles across life on Earth, ranging from DNA and RNA, to the universal energy currency, ATP. In mammals, the two primary routes for the synthesis of the adenine nucleotides ATP, ADP and AMP, and, as a consequence, the major bioactive metabolite adenosine, are the de novo purine biosynthesis (DNPB) pathway, and the purine salvage pathway (PSP). Of the two, the PSP dominates in both the mammalian brain and heart. This is because the PSP utilizes the breakdown products of ATP, occasioned by the high energy demands of these organs, to rapidly regenerate adenine nucleotides. This resynthesis route, while efficient and energetically favourable, leaves these organs vulnerable to loss of salvageable metabolites, with the potential for protracted depletion of the means to synthesize ATP, and the ability to deploy neuro- and cardioprotective adenosine. Having previously shown that hippocampal cellular ATP and adenosine release can be increased by supplying substrates for the PSP (d-ribose and adenine), we now explore the expression of DNPB and PSP enzymes in hippocampal neurons and astrocytes based on available transcriptomic data. We find that key enzymes of the PSP are expressed at higher levels than those in the DNPB pathway, and that PSP enzymes are expressed at higher levels in neurons than in astrocytes. These data reflect the importance of the PSP in the mammalian brain and imply that pharmacological targeting of the PSP may be particularly beneficial to neurons at times of metabolic stress. This article is part of the Special Issue on 'Purinergic Signaling: 50 years'.


Assuntos
Lesões Encefálicas , Purinas , Animais , Purinas/metabolismo , Adenosina/metabolismo , Mamíferos/metabolismo , Trifosfato de Adenosina/metabolismo , Metabolismo Energético , Nucleotídeos de Purina/metabolismo
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