Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 2.468
Filtrar
1.
Mol Cell ; 84(19): 3576-3577, 2024 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-39366345

RESUMO

In a recent publication in Cell, Xie et al.1 report a sensitive and scalable method for the detection and characterization of native glycoRNAs and identify acp3U, an abundant modified nucleoside discovered 50 years ago in tRNAPhe, as one of the primary attachment sites for N-glycans.


Assuntos
Polissacarídeos , Polissacarídeos/metabolismo , Polissacarídeos/química , Humanos , RNA Nucleolar Pequeno/metabolismo , RNA Nucleolar Pequeno/genética , RNA Nucleolar Pequeno/química , Nucleosídeos/química , Nucleosídeos/metabolismo , Aminoácidos/química , Aminoácidos/metabolismo
2.
Nature ; 633(8030): 686-694, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39198647

RESUMO

Faithful DNA replication is essential for genome integrity1-4. Under-replicated DNA leads to defects in chromosome segregation, which are common during embryogenesis5-8. However, the regulation of DNA replication remains poorly understood in early mammalian embryos. Here we constructed a single-cell genome-wide DNA replication atlas of pre-implantation mouse embryos and identified an abrupt replication program switch accompanied by a transient period of genomic instability. In 1- and 2-cell embryos, we observed the complete absence of a replication timing program, and the entire genome replicated gradually and uniformly using extremely slow-moving replication forks. In 4-cell embryos, a somatic-cell-like replication timing program commenced abruptly. However, the fork speed was still slow, S phase was extended, and markers of replication stress, DNA damage and repair increased. This was followed by an increase in break-type chromosome segregation errors specifically during the 4-to-8-cell division with breakpoints enriched in late-replicating regions. These errors were rescued by nucleoside supplementation, which accelerated fork speed and reduced the replication stress. By the 8-cell stage, forks gained speed, S phase was no longer extended and chromosome aberrations decreased. Thus, a transient period of genomic instability exists during normal mouse development, preceded by an S phase lacking coordination between replisome-level regulation and megabase-scale replication timing regulation, implicating a link between their coordination and genome stability.


Assuntos
Período de Replicação do DNA , Embrião de Mamíferos , Desenvolvimento Embrionário , Instabilidade Genômica , Animais , Feminino , Masculino , Camundongos , Blastocisto/citologia , Blastocisto/metabolismo , Aberrações Cromossômicas/efeitos dos fármacos , Segregação de Cromossomos , Dano ao DNA/efeitos dos fármacos , Reparo do DNA , Período de Replicação do DNA/efeitos dos fármacos , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/embriologia , Desenvolvimento Embrionário/genética , Instabilidade Genômica/efeitos dos fármacos , Instabilidade Genômica/genética , Fase S/efeitos dos fármacos , Fase S/genética , Análise de Célula Única , Pontos de Quebra do Cromossomo , Divisão Celular , Nucleosídeos/metabolismo , Nucleosídeos/farmacologia , DNA Polimerase Dirigida por DNA/metabolismo , Complexos Multienzimáticos/metabolismo
3.
Nature ; 614(7949): 781-787, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36725929

RESUMO

The SARS-CoV-2 RNA-dependent RNA polymerase coordinates viral RNA synthesis as part of an assembly known as the replication-transcription complex (RTC)1. Accordingly, the RTC is a target for clinically approved antiviral nucleoside analogues, including remdesivir2. Faithful synthesis of viral RNAs by the RTC requires recognition of the correct nucleotide triphosphate (NTP) for incorporation into the nascent RNA. To be effective inhibitors, antiviral nucleoside analogues must compete with the natural NTPs for incorporation. How the SARS-CoV-2 RTC discriminates between the natural NTPs, and how antiviral nucleoside analogues compete, has not been discerned in detail. Here, we use cryogenic-electron microscopy to visualize the RTC bound to each of the natural NTPs in states poised for incorporation. Furthermore, we investigate the RTC with the active metabolite of remdesivir, remdesivir triphosphate (RDV-TP), highlighting the structural basis for the selective incorporation of RDV-TP over its natural counterpart adenosine triphosphate3,4. Our results explain the suite of interactions required for NTP recognition, informing the rational design of antivirals. Our analysis also yields insights into nucleotide recognition by the nsp12 NiRAN (nidovirus RdRp-associated nucleotidyltransferase), an enigmatic catalytic domain essential for viral propagation5. The NiRAN selectively binds guanosine triphosphate, strengthening proposals for the role of this domain in the formation of the 5' RNA cap6.


Assuntos
RNA-Polimerase RNA-Dependente de Coronavírus , Microscopia Crioeletrônica , SARS-CoV-2 , Humanos , Antivirais/química , Antivirais/metabolismo , Antivirais/farmacologia , RNA-Polimerase RNA-Dependente de Coronavírus/química , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , RNA-Polimerase RNA-Dependente de Coronavírus/ultraestrutura , COVID-19/virologia , Nucleosídeos/metabolismo , Nucleosídeos/farmacologia , RNA Viral/biossíntese , RNA Viral/química , RNA Viral/metabolismo , SARS-CoV-2/enzimologia , Especificidade por Substrato , Guanosina Trifosfato/metabolismo , Capuzes de RNA
4.
PLoS Biol ; 22(9): e3002813, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39348416

RESUMO

Mycobacterium tuberculosis (Mtb) releases the unusual terpene nucleoside 1-tuberculosinyladenosine (1-TbAd) to block lysosomal function and promote survival in human macrophages. Using conventional approaches, we found that genes Rv3377c and Rv3378c, but not Rv3376, were necessary for 1-TbAd biosynthesis. Here, we introduce linear models for mass spectrometry (limms) software as a next-generation lipidomics tool to study the essential functions of lipid biosynthetic enzymes on a whole-cell basis. Using limms, whole-cell lipid profiles deepened the phenotypic landscape of comparative mass spectrometry experiments and identified a large family of approximately 100 terpene nucleoside metabolites downstream of Rv3378c. We validated the identity of previously unknown adenine-, adenosine-, and lipid-modified tuberculosinol-containing molecules using synthetic chemistry and collisional mass spectrometry, including comprehensive profiling of bacterial lipids that fragment to adenine. We tracked terpene nucleoside genotypes and lipid phenotypes among Mycobacterium tuberculosis complex (MTC) species that did or did not evolve to productively infect either human or nonhuman mammals. Although 1-TbAd biosynthesis genes were thought to be restricted to the MTC, we identified the locus in unexpected species outside the MTC. Sequence analysis of the locus showed nucleotide usage characteristic of plasmids from plant-associated bacteria, clarifying the origin and timing of horizontal gene transfer to a pre-MTC progenitor. The data demonstrated correlation between high level terpene nucleoside biosynthesis and mycobacterial competence for human infection, and 2 mechanisms of 1-TbAd biosynthesis loss. Overall, the selective gain and evolutionary retention of tuberculosinyl metabolites in modern species that cause human TB suggest a role in human TB disease, and the newly discovered molecules represent candidate disease-specific biomarkers.


Assuntos
Mycobacterium tuberculosis , Nucleosídeos , Terpenos , Tuberculose , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/genética , Tuberculose/microbiologia , Terpenos/metabolismo , Humanos , Nucleosídeos/metabolismo , Adenosina/metabolismo , Adenosina/análogos & derivados , Lipidômica/métodos , Espectrometria de Massas , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Genes Bacterianos , Lipídeos
5.
Nature ; 600(7890): 720-726, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34880500

RESUMO

The liberation of energy stores from adipocytes is critical to support survival in times of energy deficit; however, uncontrolled or chronic lipolysis associated with insulin resistance and/or insulin insufficiency disrupts metabolic homeostasis1,2. Coupled to lipolysis is the release of a recently identified hormone, fatty-acid-binding protein 4 (FABP4)3. Although circulating FABP4 levels have been strongly associated with cardiometabolic diseases in both preclinical models and humans4-7, no mechanism of action has yet been described8-10. Here we show that hormonal FABP4 forms a functional hormone complex with adenosine kinase (ADK) and nucleoside diphosphate kinase (NDPK) to regulate extracellular ATP and ADP levels. We identify a substantial effect of this hormone on beta cells and given the central role of beta-cell function in both the control of lipolysis and development of diabetes, postulate that hormonal FABP4 is a key regulator of an adipose-beta-cell endocrine axis. Antibody-mediated targeting of this hormone complex improves metabolic outcomes, enhances beta-cell function and preserves beta-cell integrity to prevent both type 1 and type 2 diabetes. Thus, the FABP4-ADK-NDPK complex, Fabkin, represents a previously unknown hormone and mechanism of action that integrates energy status with the function of metabolic organs, and represents a promising target against metabolic disease.


Assuntos
Proteínas de Ligação a Ácido Graxo , Ilhotas Pancreáticas , Fosfotransferases , Adipócitos/metabolismo , Diabetes Mellitus/metabolismo , Proteínas de Ligação a Ácido Graxo/metabolismo , Humanos , Insulina/metabolismo , Ilhotas Pancreáticas/enzimologia , Ilhotas Pancreáticas/fisiologia , Lipólise , Nucleosídeos/metabolismo , Fosfotransferases/metabolismo
6.
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
7.
Nucleic Acids Res ; 52(17): 10102-10118, 2024 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-39162218

RESUMO

RNA and its building blocks play central roles in biology and have become increasingly important as therapeutic agents and targets. Hence, probing and understanding their dynamics in cells is important. Fluorescence microscopy offers live-cell spatiotemporal monitoring but requires labels. We present two fluorescent adenine analogue nucleoside phosphates which show spontaneous uptake and accumulation in cultured human cells, likely via nucleoside transporters, and show their potential utilization as cellular RNA labels. Upon uptake, one nucleotide analogue, 2CNqAXP, localizes to the cytosol and the nucleus. We show that it could then be incorporated into de novo synthesized cellular RNA, i.e. it was possible to achieve metabolic fluorescence RNA labeling without using genetic engineering to enhance incorporation, uptake-promoting strategies, or post-labeling through bio-orthogonal chemistries. By contrast, another nucleotide analogue, pAXP, only accumulated outside of the nucleus and was rapidly excreted. Consequently, this analogue did not incorporate into RNA. This difference in subcellular accumulation and retention results from a minor change in nucleobase chemical structure. This demonstrates the importance of careful design of nucleoside-based drugs, e.g. antivirals to direct their subcellular localization, and shows the potential of fine-tuning fluorescent base analogue structures to enhance the understanding of the function of such drugs.


Assuntos
Corantes Fluorescentes , RNA , Humanos , RNA/metabolismo , RNA/química , Corantes Fluorescentes/química , Núcleo Celular/metabolismo , Nucleosídeos/metabolismo , Nucleosídeos/química , Microscopia de Fluorescência , Fosfatos/metabolismo , Fosfatos/química , Células HeLa , Citosol/metabolismo
8.
Nucleic Acids Res ; 52(12): 6733-6747, 2024 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-38828787

RESUMO

Adenosine Deaminases Acting on RNA (ADARs) are enzymes that catalyze the conversion of adenosine to inosine in RNA duplexes. These enzymes can be harnessed to correct disease-causing G-to-A mutations in the transcriptome because inosine is translated as guanosine. Guide RNAs (gRNAs) can be used to direct the ADAR reaction to specific sites. Chemical modification of ADAR guide strands is required to facilitate delivery, increase metabolic stability, and increase the efficiency and selectivity of the editing reaction. Here, we show the ADAR reaction is highly sensitive to ribose modifications (e.g. 4'-C-methylation and Locked Nucleic Acid (LNA) substitution) at specific positions within the guide strand. Our studies were enabled by the synthesis of RNA containing a new, ribose-modified nucleoside analog (4'-C-methyladenosine). Importantly, the ADAR reaction is potently inhibited by LNA or 4'-C-methylation at different positions in the ADAR guide. While LNA at guide strand positions -1 and -2 block the ADAR reaction, 4'-C-methylation only inhibits at the -2 position. These effects are rationalized using high-resolution structures of ADAR-RNA complexes. This work sheds additional light on the mechanism of ADAR deamination and aids in the design of highly selective ADAR guide strands for therapeutic editing using chemically modified RNA.


Assuntos
Adenosina Desaminase , Edição de RNA , Ribose , Adenosina Desaminase/metabolismo , Adenosina Desaminase/genética , Adenosina Desaminase/química , Ribose/química , Ribose/metabolismo , Humanos , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/química , Metilação , Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina/química , Nucleosídeos/química , Nucleosídeos/metabolismo , RNA/metabolismo , RNA/química , Inosina/metabolismo , Inosina/química
9.
J Biol Chem ; 300(8): 107505, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38944122

RESUMO

Archaeosine (G+) is an archaea-specific tRNA modification synthesized via multiple steps. In the first step, archaeosine tRNA guanine transglucosylase (ArcTGT) exchanges the G15 base in tRNA with 7-cyano-7-deazaguanine (preQ0). In Euryarchaea, preQ015 in tRNA is further modified by archaeosine synthase (ArcS). Thermococcus kodakarensis ArcS catalyzes a lysine-transfer reaction to produce preQ0-lysine (preQ0-Lys) as an intermediate. The resulting preQ0-Lys15 in tRNA is converted to G+15 by a radical S-adenosyl-L-methionine enzyme for archaeosine formation (RaSEA), which forms a complex with ArcS. Here, we focus on the substrate tRNA recognition mechanism of ArcS. Kinetic parameters of ArcS for lysine and tRNA-preQ0 were determined using a purified enzyme. RNA fragments containing preQ0 were prepared from Saccharomyces cerevisiae tRNAPhe-preQ015. ArcS transferred 14C-labeled lysine to RNA fragments. Furthermore, ArcS transferred lysine to preQ0 nucleoside and preQ0 nucleoside 5'-monophosphate. Thus, the L-shaped structure and the sequence of tRNA are not essential for the lysine-transfer reaction by ArcS. However, the presence of D-arm structure accelerates the lysine-transfer reaction. Because ArcTGT from thermophilic archaea recognizes the common D-arm structure, we expected the combination of T. kodakarensis ArcTGT and ArcS and RaSEA complex would result in the formation of preQ0-Lys15 in all tRNAs. This hypothesis was confirmed using 46 T. kodakarensis tRNA transcripts and three Haloferax volcanii tRNA transcripts. In addition, ArcTGT did not exchange the preQ0-Lys15 in tRNA with guanine or preQ0 base, showing that formation of tRNA-preQ0-Lys by ArcS plays a role in preventing the reverse reaction in G+ biosynthesis.


Assuntos
Proteínas Arqueais , Lisina , Thermococcus , Thermococcus/metabolismo , Thermococcus/genética , Thermococcus/enzimologia , Lisina/metabolismo , Lisina/química , Proteínas Arqueais/metabolismo , Proteínas Arqueais/genética , Proteínas Arqueais/química , RNA de Transferência/metabolismo , RNA de Transferência/genética , RNA de Transferência/química , RNA Arqueal/metabolismo , RNA Arqueal/genética , RNA Arqueal/química , Guanina/metabolismo , Guanina/química , Guanina/análogos & derivados , Especificidade por Substrato , Cinética , Nucleosídeos/metabolismo , Nucleosídeos/química , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Guanosina/análogos & derivados
10.
Plant J ; 117(5): 1432-1452, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38044809

RESUMO

Cells save their energy during nitrogen starvation by selective autophagy of ribosomes and degradation of RNA to ribonucleotides and nucleosides. Nucleosides are hydrolyzed by nucleoside N-ribohydrolases (nucleosidases, NRHs). Subclass I of NRHs preferentially hydrolyzes the purine ribosides while subclass II is more active towards uridine and xanthosine. Here, we performed a crystallographic and kinetic study to shed light on nucleoside preferences among plant NRHs followed by in vivo metabolomic and phenotyping analyses to reveal the consequences of enhanced nucleoside breakdown. We report the crystal structure of Zea mays NRH2b (subclass II) and NRH3 (subclass I) in complexes with the substrate analog forodesine. Purine and pyrimidine catabolism are inseparable because nucleobase binding in the active site of ZmNRH is mediated via a water network and is thus unspecific. Dexamethasone-inducible ZmNRH overexpressor lines of Arabidopsis thaliana, as well as double nrh knockout lines of moss Physcomitrium patents, reveal a fine control of adenosine in contrast to other ribosides. ZmNRH overexpressor lines display an accelerated early vegetative phase including faster root and rosette growth upon nitrogen starvation or osmotic stress. Moreover, the lines enter the bolting and flowering phase much earlier. We observe changes in the pathways related to nitrogen-containing compounds such as ß-alanine and several polyamines, which allow plants to reprogram their metabolism to escape stress. Taken together, crop plant breeding targeting enhanced NRH-mediated nitrogen recycling could therefore be a strategy to enhance plant growth tolerance and productivity under adverse growth conditions.


Assuntos
Arabidopsis , Nucleosídeos , Nucleosídeos/metabolismo , Nitrogênio/metabolismo , Melhoramento Vegetal , Plantas/metabolismo , Uridina/metabolismo , Arabidopsis/genética
11.
Cell ; 141(1): 129-41, 2010 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-20371350

RESUMO

RNA transcripts are subject to posttranscriptional gene regulation involving hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) expressed in a cell-type dependent fashion. We developed a cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs. The crosslinked sites are revealed by thymidine to cytidine transitions in the cDNAs prepared from immunopurified RNPs of 4-thiouridine-treated cells. We determined the binding sites and regulatory consequences for several intensely studied RBPs and miRNPs, including PUM2, QKI, IGF2BP1-3, AGO/EIF2C1-4 and TNRC6A-C. Our study revealed that these factors bind thousands of sites containing defined sequence motifs and have distinct preferences for exonic versus intronic or coding versus untranslated transcript regions. The precise mapping of binding sites across the transcriptome will be critical to the interpretation of the rapidly emerging data on genetic variation between individuals and how these variations contribute to complex genetic diseases.


Assuntos
Técnicas Genéticas , MicroRNAs/metabolismo , RNA não Traduzido/genética , Proteínas de Ligação a RNA/metabolismo , Sequências Reguladoras de Ácido Ribonucleico , Sequência de Bases , Reagentes de Ligações Cruzadas/metabolismo , Humanos , Dados de Sequência Molecular , Nucleosídeos/metabolismo , Mutação Puntual , Alinhamento de Sequência
12.
Nature ; 569(7757): 581-585, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31043749

RESUMO

Methylation of cytosine to 5-methylcytosine (5mC) is a prevalent DNA modification found in many organisms. Sequential oxidation of 5mC by ten-eleven translocation (TET) dioxygenases results in a cascade of additional epigenetic marks and promotes demethylation of DNA in mammals1,2. However, the enzymatic activity and function of TET homologues in other eukaryotes remains largely unexplored. Here we show that the green alga Chlamydomonas reinhardtii contains a 5mC-modifying enzyme (CMD1) that is a TET homologue and catalyses the conjugation of a glyceryl moiety to the methyl group of 5mC through a carbon-carbon bond, resulting in two stereoisomeric nucleobase products. The catalytic activity of CMD1 requires Fe(II) and the integrity of its binding motif His-X-Asp, which is conserved in Fe-dependent dioxygenases3. However, unlike previously described TET enzymes, which use 2-oxoglutarate as a co-substrate4, CMD1 uses L-ascorbic acid (vitamin C) as an essential co-substrate. Vitamin C donates the glyceryl moiety to 5mC with concurrent formation of glyoxylic acid and CO2. The vitamin-C-derived DNA modification is present in the genome of wild-type C. reinhardtii but at a substantially lower level in a CMD1 mutant strain. The fitness of CMD1 mutant cells during exposure to high light levels is reduced. LHCSR3, a gene that is critical for the protection of C. reinhardtii from photo-oxidative damage under high light conditions, is hypermethylated and downregulated in CMD1 mutant cells compared to wild-type cells, causing a reduced capacity for photoprotective non-photochemical quenching. Our study thus identifies a eukaryotic DNA base modification that is catalysed by a divergent TET homologue and unexpectedly derived from vitamin C, and describes its role as a potential epigenetic mark that may counteract DNA methylation in the regulation of photosynthesis.


Assuntos
5-Metilcitosina/metabolismo , Proteínas de Algas/metabolismo , Ácido Ascórbico/metabolismo , Biocatálise , Chlamydomonas reinhardtii/enzimologia , DNA/química , DNA/metabolismo , 5-Metilcitosina/química , Dióxido de Carbono/metabolismo , Metilação de DNA , Glioxilatos/metabolismo , Nucleosídeos/química , Nucleosídeos/metabolismo , Fotossíntese
13.
J Biol Chem ; 299(4): 104573, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36870685

RESUMO

Sideromycins are a unique subset of siderophores comprising of a siderophore conjugated to an antimicrobial agent. The "Trojan horse" antibiotic albomycins are unique sideromycins consisting of a ferrichrome-type siderophore conjugated to a peptidyl nucleoside antibiotic. They exhibit potent antibacterial activities against many model bacteria and a number of clinical pathogens. Earlier studies have provided significant insight into the biosynthetic pathway of the peptidyl nucleoside moiety. We herein decipher the biosynthetic pathway of the ferrichrome-type siderophore in Streptomyces sp. ATCC 700974. Our genetic studies suggested that abmA, abmB, and abmQ are involved in the formation of the ferrichrome-type siderophore. Additionally, we performed biochemical studies to demonstrate that a flavin-dependent monooxygenase AbmB and an N-acyltransferase AbmA catalyze sequential modifications of L-ornithine to generate N5-acetyl-N5-hydroxyornithine. Three molecules of N5-acetyl-N5-hydroxyornithine are then assembled to generate the tripeptide ferrichrome through the action of a nonribosomal peptide synthetase AbmQ. Of special note, we found out that orf05026 and orf03299, two genes scattered elsewhere in the chromosome of Streptomyces sp. ATCC 700974, have functional redundancy for abmA and abmB, respectively. Interestingly, both orf05026 and orf03299 are situated within gene clusters encoding putative siderophores. In summary, this study provided new insight into the siderophore moiety of albomycin biosynthesis and shed light on the contingency of multiple siderophores in albomycin-producing Streptomyces sp. ATCC 700974.


Assuntos
Sideróforos , Streptomyces , Sideróforos/metabolismo , Ferricromo/química , Ferricromo/metabolismo , Streptomyces/genética , Streptomyces/metabolismo , Vias Biossintéticas , Nucleosídeos/metabolismo , Antibacterianos/metabolismo
14.
Am J Physiol Renal Physiol ; 326(1): F30-F38, 2024 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-37916286

RESUMO

Plasma nucleosides-pseudouridine (PU) and N2N2-dimethyl guanosine (DMG) predict the progression of type 2 diabetic kidney disease (DKD) to end-stage renal disease, but the mechanisms underlying this relationship are not well understood. We used a well-characterized model of type 2 diabetes (db/db mice) and control nondiabetic mice (db/m mice) to characterize the production and excretion of PU and DMG levels using liquid chromatography-mass spectrometry. The fractional excretion of PU and DMG was decreased in db/db mice compared with control mice at 24 wk before any changes to renal function. We then examined the dynamic changes in nucleoside metabolism using in vivo metabolic flux analysis with the injection of labeled nucleoside precursors. Metabolic flux analysis revealed significant decreases in the ratio of urine-to-plasma labeling of PU and DMG in db/db mice compared with db/m mice, indicating significant tubular dysfunction in diabetic kidney disease. We observed that the gene and protein expression of the renal tubular transporters involved with nucleoside transport in diabetic kidneys in mice and humans was reduced. In conclusion, this study strongly suggests that tubular handling of nucleosides is altered in early DKD, in part explaining the association of PU and DMG with human DKD progression observed in previous studies.NEW & NOTEWORTHY Tubular dysfunction explains the association between the nucleosides pseudouridine and N2N2-dimethyl guanosine and diabetic kidney disease.


Assuntos
Diabetes Mellitus Tipo 2 , Nefropatias Diabéticas , Humanos , Camundongos , Animais , Nefropatias Diabéticas/metabolismo , Pseudouridina/metabolismo , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Nucleosídeos/metabolismo , Eliminação Renal , Rim/metabolismo , Guanosina/metabolismo
15.
RNA ; 28(5): 697-710, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35168996

RESUMO

The human immunodeficiency virus type 1 (HIV-1) encodes multiple RNA molecules. Transcripts that originate from the proviral 5' long terminal repeat (LTR) function as messenger RNAs for the expression of 16 different mature viral proteins. In addition, HIV-1 expresses an antisense transcript (Ast) from the 3'LTR, which has both protein-coding and noncoding properties. While the mechanisms that regulate the coding and noncoding activities of Ast remain unknown, post-transcriptional modifications are known to influence RNA stability, interaction with protein partners, and translation capacity. Here, we report the nucleoside modification profile of Ast obtained through liquid chromatography coupled with mass spectrometry (LC-MS) analysis. The epitranscriptome includes a limited set of modified nucleosides but predominantly ribose methylations. A number of these modifications were mapped to specific positions of the sequence through RNA modification mapping procedures. The presence of modifications on Ast is consistent with the RNA-modifying enzymes interacting with Ast The identification and mapping of Ast post-transcriptional modifications is expected to elucidate the mechanisms through which this versatile molecule can carry out diverse activities in different cell compartments. Manipulation of post-transcriptional modifications on the Ast RNA may have therapeutic implications.


Assuntos
HIV-1 , Cromatografia Líquida , HIV-1/genética , HIV-1/metabolismo , Humanos , Nucleosídeos/metabolismo , Processamento Pós-Transcricional do RNA , RNA Antissenso/genética , Sequências Repetidas Terminais
16.
J Transl Med ; 22(1): 449, 2024 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-38741129

RESUMO

Inherited deficiency of thymidine phosphorylase (TP), encoded by TYMP, leads to a rare disease with multiple mitochondrial DNA (mtDNA) abnormalities, mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). However, the impact of TP deficiency on lysosomes remains unclear, which are important for mitochondrial quality control and nucleic acid metabolism. Muscle biopsy tissue and skin fibroblasts from MNGIE patients, patients with m.3243 A > G mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS) and healthy controls (HC) were collected to perform mitochondrial and lysosomal functional analyses. In addition to mtDNA abnormalities, compared to controls distinctively reduced expression of LAMP1 and increased mitochondrial content were detected in the muscle tissue of MNGIE patients. Skin fibroblasts from MNGIE patients showed decreased expression of LAMP2, lowered lysosomal acidity, reduced enzyme activity and impaired protein degradation ability. TYMP knockout or TP inhibition in cells can also induce the similar lysosomal dysfunction. Using lysosome immunoprecipitation (Lyso- IP), increased mitochondrial proteins, decreased vesicular proteins and V-ATPase enzymes, and accumulation of various nucleosides were detected in lysosomes with TP deficiency. Treatment of cells with high concentrations of dThd and dUrd also triggers lysosomal dysfunction and disruption of mitochondrial homeostasis. Therefore, the results provided evidence that TP deficiency leads to nucleoside accumulation in lysosomes and lysosomal dysfunction, revealing the widespread disruption of organelles underlying MNGIE.


Assuntos
DNA Mitocondrial , Fibroblastos , Lisossomos , Mitocôndrias , Encefalomiopatias Mitocondriais , Nucleosídeos , Timidina Fosforilase , Humanos , Lisossomos/metabolismo , Timidina Fosforilase/metabolismo , Timidina Fosforilase/deficiência , Timidina Fosforilase/genética , Encefalomiopatias Mitocondriais/metabolismo , Encefalomiopatias Mitocondriais/patologia , Encefalomiopatias Mitocondriais/genética , Fibroblastos/metabolismo , Fibroblastos/patologia , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Mitocôndrias/metabolismo , Nucleosídeos/metabolismo , Pseudo-Obstrução Intestinal/metabolismo , Pseudo-Obstrução Intestinal/patologia , Pseudo-Obstrução Intestinal/enzimologia , Pseudo-Obstrução Intestinal/genética , Oftalmoplegia/metabolismo , Oftalmoplegia/patologia , Oftalmoplegia/congênito , Distrofia Muscular Oculofaríngea/metabolismo , Distrofia Muscular Oculofaríngea/patologia , Masculino , Feminino , Pele/patologia , Pele/metabolismo , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo
17.
PLoS Pathog ; 18(9): e1010743, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36067236

RESUMO

The tripartite motif (TRIM) family of E3 ubiquitin ligases is well known for its roles in antiviral restriction and innate immunity regulation, in addition to many other cellular pathways. In particular, TRIM25-mediated ubiquitination affects both carcinogenesis and antiviral response. While individual substrates have been identified for TRIM25, it remains unclear how it regulates diverse processes. Here we characterized a mutation, R54P, critical for TRIM25 catalytic activity, which we successfully utilized to "trap" substrates. We demonstrated that TRIM25 targets proteins implicated in stress granule formation (G3BP1/2), nonsense-mediated mRNA decay (UPF1), nucleoside synthesis (NME1), and mRNA translation and stability (PABPC4). The R54P mutation abolishes TRIM25 inhibition of alphaviruses independently of the host interferon response, suggesting that this antiviral effect is a direct consequence of ubiquitination. Consistent with that, we observed diminished antiviral activity upon knockdown of several TRIM25-R54P specific interactors including NME1 and PABPC4. Our findings highlight that multiple substrates mediate the cellular and antiviral activities of TRIM25, illustrating the multi-faceted role of this ubiquitination network in modulating diverse biological processes.


Assuntos
Antivirais , DNA Helicases , Antivirais/metabolismo , DNA Helicases/metabolismo , Interferons/metabolismo , Nucleosídeos/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas com Motivo Tripartido/genética , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Ubiquitinas/metabolismo
18.
Plant Cell ; 33(2): 270-289, 2021 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-33793855

RESUMO

Detecting and quantifying low-abundance (deoxy)ribonucleotides and (deoxy)ribonucleosides in plants remains difficult; this is a major roadblock for the investigation of plant nucleotide (NT) metabolism. Here, we present a method that overcomes this limitation, allowing the detection of all deoxy- and ribonucleotides as well as the corresponding nucleosides from the same plant sample. The method is characterized by high sensitivity and robustness enabling the reproducible detection and absolute quantification of these metabolites even if they are of low abundance. Employing the new method, we analyzed Arabidopsis thaliana null mutants of CYTIDINE DEAMINASE, GUANOSINE DEAMINASE, and NUCLEOSIDE HYDROLASE 1, demonstrating that the deoxyribonucleotide (dNT) metabolism is intricately interwoven with the catabolism of ribonucleosides (rNs). In addition, we discovered a function of rN catabolic enzymes in the degradation of deoxyribonucleosides in vivo. We also determined the concentrations of dNTs in several mono- and dicotyledonous plants, a bryophyte, and three algae, revealing a correlation of GC to AT dNT ratios with genomic GC contents. This suggests a link between the genome and the metabolome previously discussed but not experimentally addressed. Together, these findings demonstrate the potential of this new method to provide insight into plant NT metabolism.


Assuntos
Arabidopsis/metabolismo , Briófitas/metabolismo , Eucariotos/metabolismo , Nucleosídeos/metabolismo , Nucleotídeos/metabolismo , Arabidopsis/genética , Composição de Bases , Calibragem , Genoma de Planta , Mutação/genética , Folhas de Planta/metabolismo , Análise de Regressão , Reprodutibilidade dos Testes , Plântula/metabolismo
19.
Chem Res Toxicol ; 37(2): 248-258, 2024 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-38198686

RESUMO

Pyridone-containing adenine dinucleotides, ox-NAD, are formed by overoxidation of nicotinamide adenine dinucleotide (NAD+) and exist in three distinct isomeric forms. Like the canonical nucleosides, the corresponding pyridone-containing nucleosides (PYR) are chemically stable, biochemically versatile, and easily converted to nucleotides, di- and triphosphates, and dinucleotides. The 4-PYR isomer is often reported with its abundance increasing with the progression of metabolic diseases, age, cancer, and oxidative stress. Yet, the pyridone-derived nucleotides are largely under-represented in the literature. Here, we report the efficient synthesis of the series of ox-NAD and pyridone nucleotides and measure the abundance of ox-NAD in biological specimens using liquid chromatography coupled with mass spectrometry (LC-MS). Overall, we demonstrate that all three forms of PYR and ox-NAD are found in biospecimens at concentrations ranging from nanomolar to midmicromolar and that their presence affects the measurements of NAD(H) concentrations when standard biochemical redox-based assays are applied. Furthermore, we used liver extracts and 1H NMR spectrometry to demonstrate that each ox-NAD isomer can be metabolized to its respective PYR isomer. Together, these results suggest a need for a better understanding of ox-NAD in the context of human physiology since these species are endogenous mimics of NAD+, the key redox cofactor in metabolism and bioenergetics maintenance.


Assuntos
NAD , Nucleotídeos , Humanos , NAD/metabolismo , Nucleotídeos/metabolismo , Nucleosídeos/metabolismo , Metabolismo Energético , Piridonas
20.
RNA Biol ; 21(1): 1-10, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38117089

RESUMO

Pseudouridine is a noncanonical C-nucleoside containing a C-C glycosidic linkage between uracil and ribose. In the two-step degradation of pseudouridine, pseudouridine 5'-monophosphate glycosylase (PUMY) is responsible for the second step and catalyses the cleavage of the C-C glycosidic bond in pseudouridine 5'-monophosphate (ΨMP) into uridine and ribose 5'-phosphate, which are recycled via other metabolic pathways. Structural features of Escherichia coli PUMY have been reported, but the details of the substrate specificity of ΨMP were unknown. Here, we present three crystal structures of Arabidopsis thaliana PUMY in different ligation states and a kinetic analysis of ΨMP degradation. The results indicate that Thr149 and Asn308, which are conserved in the PUMY family, are structural determinants for recognizing the nucleobase of ΨMP. The distinct binding modes of ΨMP and ribose 5'-phosphate also suggest that the nucleobase, rather than the phosphate group, of ΨMP dictates the substrate-binding mode. An open-to-close transition of the active site is essential for catalysis, which is mediated by two α-helices, α11 and α12, near the active site. Mutational analysis validates the proposed roles of the active site residues in catalysis. Our structural and functional analyses provide further insight into the enzymatic features of PUMY towards ΨMP.


Assuntos
Arabidopsis , Pseudouridina , Pseudouridina/metabolismo , Cinética , Ribose/metabolismo , Escherichia coli/metabolismo , Nucleosídeos/metabolismo , Fosfatos , Catálise , Especificidade por Substrato , Cristalografia por Raios X
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA