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1.
Microbiology ; 165(11): 1153-1165, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31535967

RESUMO

Antibiotic producing Streptomyces sense and respond to environmental signals by using nucleotide second messengers, including (p)ppGpp, cAMP, c-di-GMP and c-di-AMP. As summarized in this review, these molecules are important message carriers that coordinate the complex Streptomyces morphological transition from filamentous growth to sporulation along with the secondary metabolite production. Here, we provide an overview of the enzymes that make and break these second messengers and suggest candidates for (p)ppGpp and cAMP enzymes to be studied. We highlight the target molecules that bind these signalling molecules and elaborate individual functions that they control in the context of Streptomyces development. Finally, we discuss open questions in the field, which may guide future studies in this exciting research area.


Assuntos
AMP Cíclico/metabolismo , Fosfatos de Dinucleosídeos/metabolismo , Nucleotídeos de Guanina/metabolismo , Sistemas do Segundo Mensageiro/fisiologia , Streptomyces/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , AMP Cíclico/química , Fosfatos de Dinucleosídeos/química , Regulação Bacteriana da Expressão Gênica , Nucleotídeos de Guanina/química , Ligação Proteica , Esporos Bacterianos/crescimento & desenvolvimento , Esporos Bacterianos/metabolismo , Esporos Bacterianos/fisiologia , Streptomyces/crescimento & desenvolvimento , Streptomyces/fisiologia
2.
Artigo em Inglês | MEDLINE | ID: mdl-31152640

RESUMO

The active metabolite of azathioprine, 6-thioguanine nucleotide (6-TGN) is the main component responsible for the immunosuppressive effect in treatment of inflammatory bowel disease (IBD). The aim of this study was to assess the correlation between the concentration of 6-thioguanine nucleotide and disease activity, azathioprine-related adverse effects and time duration of treatment in patients with inflammatory bowel disease. Thirty-four patients were included in this study. Type of disease, gender, time duration of therapy and adverse effects were recorded. Metabolite concentration was determined by high performance liquid chromatography. Twenty-one percent of patients have experienced an adverse effect, with leucocytopenia most commonly occurring (42.9%). More adverse effects were registered when patients were treated with azathioprine in a period of less than 3 months in comparison to the group of patients that have been under therapy between 3-12 months and more than 12 months (p˂0.05). Most of the patients that presented any adverse effect had high 6-TGN concentration (>450 pmol/8x108 Er). The mean value of 6-TGN metabolite concentration in IBD patients treated with azathioprine was 437.46 pmol/8x108 Er ± 198.82 pmol/8x108. The time duration of azathioprine treatment did not have any significant impact on the achieved 6-TGN concentration (p>0.05).Twenty patients (58.9%) had achieved remission after therapy initiation with azathioprine. More alertness is recommended to clinicians towards patients in the first 3 months of the therapy. Our study demonstrated that higher 6-TGN concentration is associated with azathioprine toxicity.


Assuntos
Azatioprina/efeitos adversos , Nucleotídeos de Guanina/metabolismo , Imunossupressores/efeitos adversos , Doenças Inflamatórias Intestinais/tratamento farmacológico , Tionucleotídeos/metabolismo , Adulto , Azatioprina/uso terapêutico , Monitoramento de Medicamentos/métodos , Feminino , Nucleotídeos de Guanina/sangue , Humanos , Imunossupressores/uso terapêutico , Doenças Inflamatórias Intestinais/sangue , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Tionucleotídeos/sangue , Fatores de Tempo
3.
Biochemistry ; 58(17): 2228-2242, 2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-30945846

RESUMO

The oxidoreductase YdhV in Escherichia coli has been predicted to belong to the family of molybdenum/tungsten cofactor (Moco/Wco)-containing enzymes. In this study, we characterized the YdhV protein in detail, which shares amino acid sequence homology with a tungsten-containing benzoyl-CoA reductase binding the bis-W-MPT (for metal-binding pterin) cofactor. The cofactor was identified to be of a bis-Mo-MPT type with no guanine nucleotides present, which represents a form of Moco that has not been found previously in any molybdoenzyme. Our studies showed that YdhV has a preference for bis-Mo-MPT over bis-W-MPT to be inserted into the enzyme. In-depth characterization of YdhV by X-ray absorption and electron paramagnetic resonance spectroscopies revealed that the bis-Mo-MPT cofactor in YdhV is redox active. The bis-Mo-MPT and bis-W-MPT cofactors include metal centers that bind the four sulfurs from the two dithiolene groups in addition to a cysteine and likely a sulfido ligand. The unexpected presence of a bis-Mo-MPT cofactor opens an additional route for cofactor biosynthesis in E. coli and expands the canon of the structurally highly versatile molybdenum and tungsten cofactors.


Assuntos
Coenzimas/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Ferredoxinas/química , Metaloproteínas/química , Molibdênio/química , Compostos Organometálicos/química , Oxirredutases/química , Pteridinas/química , Pterinas/química , Coenzimas/genética , Coenzimas/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ferredoxinas/genética , Ferredoxinas/metabolismo , Nucleotídeos de Guanina/química , Nucleotídeos de Guanina/genética , Nucleotídeos de Guanina/metabolismo , Metaloproteínas/genética , Metaloproteínas/metabolismo , Estrutura Molecular , Molibdênio/metabolismo , Compostos Organometálicos/metabolismo , Oxirredução , Oxirredutases/genética , Oxirredutases/metabolismo , Pteridinas/metabolismo , Pterinas/metabolismo
4.
Life Sci Alliance ; 2(2)2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30872413

RESUMO

The Rab GTPase family is a major regulator of membrane traffic in eukaryotic cells. The Rab11 subfamily plays important roles in specific trafficking events such as exocytosis, endosomal recycling, and cytokinesis. SH3BP5 and SH3BP5-like (SH3BP5L) proteins have recently been found to serve as guanine nucleotide exchange factors (GEF) for Rab11. Here, we report the crystal structures of the SH3BP5 GEF domain alone and its complex with Rab11a. SH3BP5 exhibits a V-shaped structure comprising two coiled coils. The coiled coil composed of α1, and α4 is solely responsible for the Rab11a binding and GEF activity. SH3BP5 pulls out and deforms switch I of Rab11a so as to facilitate the GDP release from Rab11a. SH3BP5 interacts with the N-terminal region, switch I, interswitch, and switch II of Rab11a. SH3BP5 and SH3BP5L localize to Rab11-positive recycling endosomes and show GEF activity for all of the Rab11 family but not for Rab14. Fluorescence-based GEF assays combined with site-directed mutagenesis reveal the essential interactions between SH3BP5 and Rab11 family proteins for the GEF reaction on recycling endosomes.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Nucleotídeos de Guanina/metabolismo , Proteínas rab de Ligação ao GTP/química , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Cristalização , Cristalografia , Endossomos/metabolismo , Células HeLa , Humanos , Ligação de Hidrogênio , Proteínas Mutantes , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Transporte Proteico , Transfecção
5.
Biophys Chem ; 247: 13-24, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30780079

RESUMO

One of the final maturation steps of the large ribosomal subunit requires the joint action of the elongation factor-like 1 (human EFL1, yeast Efl1) GTPase and the Shwachman-Diamond syndrome protein (human SBDS, yeast Sdo1) to release the eukaryotic translation initiation factor 6 (human eIF6, yeast Tif6) and allow the assembly of mature ribosomes. EFL1 function is driven by conformational changes. However, the nature of such conformational changes or the mechanism by which they are prompted are still largely unknown. In previous studies, it has been established that this GTPase interacts with its cofactor in solution in an inverted orientation with respect to the binding mode derived from 60S ribosome subunit cryo-EM data. To shed new light on this conundrum, we characterized calorimetrically the energetic basis describing the recognition of Efl1 to GT(D)P, Sdo1 and their intercommunication in solution. A structural-based analysis of the binding signatures indicates that Efl1 has a large structural flexibility. The mutual effects of Sdo1 and nucleotides on Efl1 modulate in a very specific and robust way the complex conformational landscape of Efl1, resembling the behavior observed with other GTPases and their cofactors.


Assuntos
GTP Fosfo-Hidrolases/metabolismo , Nucleotídeos de Guanina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/química , Nucleotídeos de Guanina/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química
6.
J Cell Sci ; 131(17)2018 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-30154209

RESUMO

T cell-mediated adaptive immunity requires naïve, unstimulated T cells to transition from a quiescent metabolic state into a highly proliferative state upon T cell receptor engagement. This complex process depends on transcriptional changes mediated by Ca2+-dependent NFAT signaling, mTOR-mediated signaling and increased activity of the guanine nucleotide biosynthetic inosine-5'-monophosphate (IMP) dehydrogenase 1 and 2 enzymes (IMPDH1 and IMPDH2, hereafter IMPDH). Inhibitors of these pathways serve as potent immunosuppressants. Unexpectedly, we discovered that all three pathways converge to promote the assembly of IMPDH protein into micron-scale macromolecular filamentous structures in response to T cell activation. Assembly is post-transcriptionally controlled by mTOR and the Ca2+ influx regulator STIM1. Furthermore, IMPDH assembly and catalytic activity were negatively regulated by guanine nucleotide levels, suggesting a negative feedback loop that limits biosynthesis of guanine nucleotides. Filamentous IMPDH may be more resistant to this inhibition, facilitating accumulation of the higher GTP levels required for T cell proliferation.


Assuntos
IMP Desidrogenase/metabolismo , Linfócitos T/enzimologia , Animais , Células Cultivadas , Nucleotídeos de Guanina/metabolismo , IMP Desidrogenase/genética , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Baço/enzimologia , Baço/imunologia , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismo , Linfócitos T/imunologia , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo
7.
J Neurochem ; 147(3): 409-428, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30091236

RESUMO

The Parkinson's disease (PD)-causative leucine-rich repeat kinase 2 (LRRK2) belongs to the Roco family of G-proteins comprising a Ras-of-complex (Roc) domain followed by a C-terminal of Roc (COR) domain in tandem (called Roc-COR domain). Two prokaryotic Roc-COR domains have been characterized as 'G proteins activated by guanine nucleotide-dependent dimerization' (GADs), which require dimerization for activation of their GTPase activity and bind guanine nucleotides with relatively low affinities. Additionally, LRRK2 Roc domain in isolation binds guanine nucleotides with relatively low affinities. As such, LRRK2 GTPase domain was predicted to be a GAD. Herein, we describe the design and high-level expression of human LRRK2 Roc-COR domain (LRRK2 Roc-COR). Biochemical analyses of LRRK2 Roc-COR reveal that it forms homodimers, with the C-terminal portion of COR mediating its dimerization. Furthermore, it co-purifies and binds Mg2+ GTP/GDP at 1 : 1 stoichiometry, and it hydrolyzes GTP with Km  and kcat  of 22 nM and 4.70 × 10-4  min-1 ,  respectively. Thus, even though LRRK2 Roc-COR forms GAD-like homodimers, it exhibits conventional Ras-like GTPase properties, with high-affinity binding of Mg2+ -GTP/GDP and low intrinsic catalytic activity. The PD-causative Y1699C mutation mapped to the COR domain was previously reported to reduce the GTPase activity of full-length LRRK2. In contrast, this mutation induces no change in the GTPase activity, and only slight perturbations in the secondary structure contents of LRRK2 Roc-COR. As this mutation does not directly affect the GTPase activity of the isolated Roc-COR tandem, it is possible that the effects of this mutation on full-length LRRK2 occur via other functional domains. Open Practices Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/.


Assuntos
GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Genes ras/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Animais , Dimerização , Escherichia coli , Regulação Enzimológica da Expressão Gênica/genética , Nucleotídeos de Guanina/metabolismo , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/química , Magnésio/metabolismo , Camundongos , Mutação/genética , Neuropeptídeos/biossíntese , Neuropeptídeos/genética , Multimerização Proteica , Estrutura Secundária de Proteína/genética , Proteínas Recombinantes , Proteínas rac1 de Ligação ao GTP/biossíntese , Proteínas rac1 de Ligação ao GTP/genética
8.
Nat Struct Mol Biol ; 25(7): 607-615, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29967541

RESUMO

Microtubules form from longitudinally and laterally assembling tubulin α-ß dimers. The assembly induces strain in tubulin, resulting in cycles of microtubule catastrophe and regrowth. This 'dynamic instability' is governed by GTP hydrolysis that renders the microtubule lattice unstable, but it is unclear how. We used a human microtubule nucleating and stabilizing neuronal protein, doublecortin, and high-resolution cryo-EM to capture tubulin's elusive hydrolysis intermediate GDP•Pi state, alongside the prehydrolysis analog GMPCPP state and the posthydrolysis GDP state with and without an anticancer drug, Taxol. GTP hydrolysis to GDP•Pi followed by Pi release constitutes two distinct structural transitions, causing unevenly distributed compressions of tubulin dimers, thereby tightening longitudinal and loosening lateral interdimer contacts. We conclude that microtubule catastrophe is triggered because the lateral contacts can no longer counteract the strain energy stored in the lattice, while reinforcement of the longitudinal contacts may support generation of force.


Assuntos
Microtúbulos/metabolismo , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Fenômenos Biofísicos , Microscopia Crioeletrônica , GTP Fosfo-Hidrolases/metabolismo , Nucleotídeos de Guanina/metabolismo , Humanos , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/ultraestrutura , Microtúbulos/ultraestrutura , Modelos Biológicos , Modelos Moleculares , Neuropeptídeos/química , Neuropeptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estabilidade Proteica , Tubulina (Proteína)/ultraestrutura
9.
Aging Cell ; 17(4): e12798, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29901258

RESUMO

Nicotinamide adenine dinucleotide (NAD) is an important cofactor that regulates various biological processes, including metabolism and gene expression. As a coenzyme, NAD controls mitochondrial respiration through enzymes of the tricarboxylic acid (TCA) cycle, ß-oxidation, and oxidative phosphorylation and also serves as a substrate for posttranslational protein modifications, such as deacetylation and ADP-ribosylation by sirtuins and poly(ADP-ribose) polymerase (PARP), respectively. Many studies have demonstrated that NAD levels decrease with aging and that these declines cause various aging-associated diseases. In contrast, activation of NAD metabolism prevents declines in NAD levels during aging. In particular, dietary supplementation with NAD precursors has been associated with protection against age-associated insulin resistance. However, it remains unclear which NAD synthesis pathway is important and/or efficient at increasing NAD levels in vivo. In this study, Nmnat3 overexpression in mice efficiently increased NAD levels in various tissues and prevented aging-related declines in NAD levels. We also demonstrated that Nmnat3-overexpressing (Nmnat3 Tg) mice were protected against diet-induced and aging-associated insulin resistance. Moreover, in skeletal muscles of Nmnat3 Tg mice, TCA cycle activity was significantly enhanced, and the energy source for oxidative phosphorylation was shifted toward fatty acid oxidation. Furthermore, reactive oxygen species (ROS) generation was significantly suppressed in aged Nmnat3 Tg mice. Interestingly, we also found that concentrations of the NAD analog nicotinamide guanine dinucleotide (NGD) were dramatically increased in Nmnat3 Tg mice. These results suggest that Nmnat3 overexpression improves metabolic health and that Nmnat3 is an attractive therapeutic target for metabolic disorders that are caused by aging.


Assuntos
Senescência Celular , Nucleotídeos de Guanina/metabolismo , Resistência à Insulina , NAD/análogos & derivados , NAD/metabolismo , Nicotinamida-Nucleotídeo Adenililtransferase/biossíntese , Animais , Calorimetria , Nucleotídeos de Guanina/análise , Camundongos , Camundongos Transgênicos , Músculo Esquelético/metabolismo , NAD/análise , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismo , Espécies Reativas de Oxigênio/metabolismo
10.
Mikrochim Acta ; 185(5): 280, 2018 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-29725866

RESUMO

A method is reported for the fluorometric quantitation of microRNA. It is making use of a luminescent probe deribed from terbium(III) ion whose fluorescence is sensitized with a guanine-rich (G-rich) nucleotide. The probe has a large Stokes' shift and strong and sharp emission bands. The assay relies on the wide substrate specificity of terminal deoxynucleotidyl transferase (TdTase), which catalyzes the formation of long G-rich nucleotides when using microRNA primer as a trigger to start the polymerization. The addition of Tb(III) induces the formation of a G-quadruplex from the G-rich nucleotide, and this strongly enhances the green fluorescence of Tb(III) (peaking at 545 nm upon photoexcitation at 290 nm). Specifically, microRNA-21 was chosen as the analyte. The fluorescence intensity of Tb(III) increases linearly in the 1 pM to 1 nM microRNA concentration range, and the detection limit is as low as 0.11 pM. The method can distinguish between family members of microRNA and performs excellently even when applied to extracts of cancer cells. Graphical abstract A fluorometric technique is reported for the determination of microRNA. It is based on signal enhancement based on the sensitization of terbium(III) via a guanine-rich nucleotide sequence. Klenow Fragment exo- (KFexo-) generates DNA sequence at the 3'-OH of microRNA, and terminal deoxynucleotidyl transferase (TdTase) catalyzes the formation of long G-rich nucleotides.


Assuntos
Técnicas Biossensoriais/métodos , DNA Nucleotidilexotransferase/metabolismo , Nucleotídeos de Guanina/química , Nucleotídeos de Guanina/metabolismo , Medições Luminescentes/métodos , MicroRNAs/análise , Térbio/química , Células A549 , Humanos , Células MCF-7
11.
Methods Enzymol ; 599: 157-196, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29746239

RESUMO

[Fe-S] clusters are essential cofactors in all domains of life. They play many biological roles due to their unique abilities for electron transfer and conformational control. Yet, producing and analyzing Fe-S proteins can be difficult and even misleading if not done anaerobically. Due to unique redox properties of [Fe-S] clusters and their oxygen sensitivity, they pose multiple challenges and can lose enzymatic activity or cause their component proteins to be structurally disordered due to [Fe-S] cluster oxidation and loss in air. Here we highlight tested protocols and strategies enabling efficient and stable [Fe-S] protein production, purification, crystallization, X-ray diffraction data collection, and structure determination. From multiple high-resolution anaerobic crystal structures, we furthermore analyze exemplary data defining [Fe-S] clusters, substrate entry, and product exit for the functional oxidation states of type II molybdo-bis(molybdopterin guanine dinucleotide) (Mo-bisMGD) enzymes. Notably, these enzymes perform electron shuttling between quinone pools and specific substrates to catalyze respiratory metabolism. The identified structure-activity relationships for this enzyme class have broad implications germane to perchlorate environments on Earth and Mars extending to an alternative mechanism underlying metabolic origins for the evolution of the oxygen atmosphere. Integrated structural analyses of type II Mo-bisMGD enzymes unveil novel distinctive shared molecular mechanisms for dynamic control of substrate entry and product release gated by hydrophobic residues. Collective findings support a prototypic model for type II Mo-bisMGD enzymes including insights for a fundamental molecular mechanistic understanding of selectivity and regulation by a conformationally gated channel with general implications for [Fe-S] cluster respiratory enzymes.


Assuntos
Nucleotídeos de Guanina/metabolismo , Proteínas com Ferro-Enxofre/química , Proteínas com Ferro-Enxofre/metabolismo , Pterinas/metabolismo , Animais , Cristalização/métodos , Cristalografia por Raios X/métodos , Elétrons , Humanos , Proteínas com Ferro-Enxofre/genética , Modelos Moleculares , Oxirredução , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
12.
Mol Cell ; 69(5): 828-839.e5, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29478808

RESUMO

DksA and ppGpp are the central players in the stringent response and mediate a complete reprogramming of the transcriptome. A major component of the response is a reduction in ribosome synthesis, which is accomplished by the synergistic action of DksA and ppGpp bound to RNA polymerase (RNAP) inhibiting transcription of rRNAs. Here, we report the X-ray crystal structures of Escherichia coli RNAP in complex with DksA alone and with ppGpp. The structures show that DksA accesses the template strand at the active site and the downstream DNA binding site of RNAP simultaneously and reveal that binding of the allosteric effector ppGpp reshapes the RNAP-DksA complex. The structural data support a model for transcriptional inhibition in which ppGpp potentiates the destabilization of open complexes by DksA. This work establishes a structural basis for understanding the pleiotropic effects of DksA and ppGpp on transcriptional regulation in proteobacteria.


Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/química , Nucleotídeos de Guanina/química , Modelos Químicos , Modelos Moleculares , Regulação Alostérica , Domínio Catalítico , Cristalografia por Raios X , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Nucleotídeos de Guanina/metabolismo , Transcriptoma/fisiologia
13.
Cell Signal ; 44: 72-81, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29337043

RESUMO

The acrosome reaction is the regulated exocytosis of mammalian sperm's single secretory granule, essential for fertilization. It relies on small GTPases, the cAMP binding protein Epac, and the SNARE complex, among other components. Here, we describe a novel tool to investigate Rab27-related signaling pathways: a hybrid recombinant protein consisting of human Rab27A fused to TAT, a cell penetrating peptide. With this tool, we aimed to unravel the connection between Rab3, Rab27 and Rap1 in sperm exocytosis and to deepen our understanding about how isoprenylation and guanine nucleotides influence the behaviour of Rab27 in exocytosis. Our results show that TAT-Rab27A-GTP-γ-S permeated into live sperm and triggered acrosomal exocytosis per se when geraylgeranylated but inhibited it when not lipid-modified. Likewise, an impermeant version of Rab27A elicited exocytosis in streptolysin O-permeabilized - but not in non-permeabilized - cells when geranylgeranylated and active. When GDP-ß-S substituted for GTP-γ-S, isoprenylated TAT-Rab27A inhibited the acrosome reaction triggered by progesterone and an Epac-selective cAMP analogue, whereas the non-isoprenylated protein did not. Geranylgeranylated TAT-Rab27A-GTP-γ-S promoted the exchange of GDP for GTP on Rab3 and Rap1 detected by far-immunofluorescence with Rab3-GTP and Rap1-GTP binding cassettes. In contrast, TAT-Rab27A lacking isoprenylation or loaded with GDP-ß-S prevented the activation of Rab3 and Rap1 elicited by progesterone. Challenging streptolysin O-permeabilized human sperm with calcium increased the population of sperm with Rap1-GTP, Rab3-GTP and Rab27-GTP in the acrosomal region; pretreatment with anti-Rab27 antibodies prevented the activation of all three. The novel findings reported here include: the description of membrane permeant TAT-Rab27A as a trustworthy tool to unveil the regulation of the human sperm acrosome reaction by Rab27 under physiological conditions; that the activation of endogenous Rab27 is required for that of Rab3 and Rap1; and the connection between Epac and Rab27 and between Rab27 and the configuration of the SNARE complex. Moreover, we present direct evidence that Rab27A's lipid modification, and activation/inactivation status correlate with its stimulatory or inhibitory roles in exocytosis.


Assuntos
Reação Acrossômica , Exocitose , Nucleotídeos de Guanina/metabolismo , Prenilação , Proteínas de Ligação a Telômeros/metabolismo , Proteínas rab27 de Ligação ao GTP/metabolismo , Proteínas rab3 de Ligação ao GTP/metabolismo , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular , Peptídeos Penetradores de Células/genética , Peptídeos Penetradores de Células/metabolismo , Fatores de Troca do Nucleotídeo Guanina , Humanos , Masculino , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Prenilação de Proteína , Proteínas Recombinantes/genética , Proteínas SNARE/metabolismo , Transdução de Sinais , Proteínas rab27 de Ligação ao GTP/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo
14.
Biochemistry ; 57(7): 1130-1143, 2018 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-29334455

RESUMO

The well-studied enterobacterium Escherichia coli present in the human gut can reduce trimethylamine N-oxide (TMAO) to trimethylamine during anaerobic respiration. The TMAO reductase TorA is a monomeric, bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor-containing enzyme that belongs to the dimethyl sulfoxide reductase family of molybdoenzymes. We report on a system for the in vitro reconstitution of TorA with molybdenum cofactors (Moco) from different sources. Higher TMAO reductase activities for TorA were obtained when using Moco sources containing a sulfido ligand at the molybdenum atom. For the first time, we were able to isolate functional bis-MGD from Rhodobacter capsulatus formate dehydrogenase (FDH), which remained intact in its isolated state and after insertion into apo-TorA yielded a highly active enzyme. Combined characterizations of the reconstituted TorA enzymes by electron paramagnetic resonance spectroscopy and direct electrochemistry emphasize that TorA activity can be modified by changes in the Mo coordination sphere. The combination of these results together with studies of amino acid exchanges at the active site led us to propose a novel model for binding of the substrate to the molybdenum atom of TorA.


Assuntos
Coenzimas/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Metaloproteínas/metabolismo , Oxirredutases N-Desmetilantes/metabolismo , Pteridinas/metabolismo , Nucleotídeos de Guanina/metabolismo , Humanos , Modelos Moleculares , Molibdênio/metabolismo , Pterinas/metabolismo , Sulfetos/metabolismo
15.
Elife ; 62017 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-28968219

RESUMO

Rab GTPases, which are involved in intracellular trafficking pathways, have recently been reported to be ubiquitinated. However, the functions of ubiquitinated Rab proteins remain unexplored. Here we show that Rab5 is monoubiquitinated on K116, K140, and K165. Upon co-transfection with ubiquitin, Rab5 exhibited abnormalities in endosomal localization and EGF-induced EGF receptor degradation. Rab5 K140R and K165R mutants restored these abnormalities, whereas K116R did not. We derived structural models of individual monoubiquitinated Rab5 proteins (mUbRab5s) by solution scattering and observed different conformational flexibilities in a site-specific manner. Structural analysis combined with biochemical data revealed that interactions with downstream effectors were impeded in mUbRab5K140, whereas GDP release and GTP loading activities were altered in mUbRab5K165. By contrast, mUbRab5K116 apparently had no effect. We propose a regulatory mechanism of Rab5 where monoubiquitination downregulates effector recruitment and GDP/GTP conversion in a site-specific manner.


Assuntos
Regulação para Baixo , Nucleotídeos de Guanina/metabolismo , Ubiquitinação , Proteínas rab5 de Ligação ao GTP/metabolismo , Linhagem Celular , Análise Mutacional de DNA , Humanos , Hidrólise , Ligação Proteica , Conformação Proteica , Espalhamento a Baixo Ângulo , Proteínas rab5 de Ligação ao GTP/química , Proteínas rab5 de Ligação ao GTP/genética
16.
BMC Neurol ; 17(1): 130, 2017 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-28679367

RESUMO

BACKGROUND: Neuromyelitis optica spectrum disorders (NMOSD) are demyelinating autoimmune diseases in the central nervous system (CNS) that are characterized by a high relapse rate and the presence of anti-aquaporin 4 antibodies (AQP4-IgG) in the serum. Azathioprine (AZA) is a first-line immunomodulatory drug that is widely used for the treatment of patients with NMOSD. However, the efficacy and safety of AZA vary in different individuals. METHOD: Thirty-two patients with NMOSD who regularly took AZA were enrolled in the study at Beijing Tiantan Hospital, Capital Medical University. The efficacy of AZA was evaluated using the expanded disability status scale (EDSS) and the annual relapse rate (ARR). The erythrocyte concentrations of AZA metabolites were detected using an LC-MS/MS method. RESULTS: The erythrocyte concentrations of 6-thioguanine nucleotides (6-TGNs) and 6-methylmercaptopurine nucleotides (6-MMPNs) were 202.03 ± 63.35 pmol/8*108 RBC and 1618.90 ± 1607.06 pmol/8*108 RBC, respectively. After the patients had received AZA therapy for more than one year, the EDSS score decreased from 5.21 ± 0.24 to 2.57 ± 0.33 (p < 0.0001), and the ARR decreased from 1.41 ± 0.23 to 0.36 ± 0.09 (p < 0.0001). The 6-TGN and 6-MMPN levels were significantly different between the non-relapsed and relapsed groups (p < 0.0001, p = 0.006, respectively). A higher ARR was significantly correlated with higher erythrocyte concentrations of 6-TGNs (p < 0.0001) and 6-MMPNs (p = 0.004). CONCLUSION: AZA can reduce the EDSS score and ARR in NMOSD patients. Additionally, the efficacy of AZA is significantly related to the erythrocyte concentrations of 6-TGNs and 6-MMPNs. Within the safe upper limits, a higher concentration of 6-TGNs is associated with better efficacy of AZA. TRIAL REGISTRATION NUMBER: ISRCTN16551495 , retrospectively registered on May 22, 2017.


Assuntos
Azatioprina/administração & dosagem , Imunossupressores/administração & dosagem , Neuromielite Óptica/tratamento farmacológico , Adolescente , Adulto , Aquaporina 4/imunologia , Grupo com Ancestrais do Continente Asiático , Azatioprina/metabolismo , Feminino , Nucleotídeos de Guanina/metabolismo , Humanos , Imunossupressores/metabolismo , Masculino , Mercaptopurina/análogos & derivados , Mercaptopurina/metabolismo , Pessoa de Meia-Idade , Estudos Prospectivos , Recidiva , Espectrometria de Massas em Tandem , Tionucleotídeos/metabolismo , Adulto Jovem
17.
Sci Rep ; 7(1): 2648, 2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-28572600

RESUMO

Inosine-5'-monophosphate dehydrogenase (IMPDH) is an essential enzyme for nucleotide metabolism and cell proliferation. Despite IMPDH is the target of drugs with antiviral, immunosuppressive and antitumor activities, its physiological mechanisms of regulation remain largely unknown. Using the enzyme from the industrial fungus Ashbya gossypii, we demonstrate that the binding of adenine and guanine nucleotides to the canonical nucleotide binding sites of the regulatory Bateman domain induces different enzyme conformations with significantly distinct catalytic activities. Thereby, the comparison of their high-resolution structures defines the mechanistic and structural details of a nucleotide-controlled conformational switch that allosterically modulates the catalytic activity of eukaryotic IMPDHs. Remarkably, retinopathy-associated mutations lie within the mechanical hinges of the conformational change, highlighting its physiological relevance. Our results expand the mechanistic repertoire of Bateman domains and pave the road to new approaches targeting IMPDHs.


Assuntos
Nucleotídeos de Adenina/metabolismo , Nucleotídeos de Guanina/metabolismo , IMP Desidrogenase/metabolismo , Nucleotídeos de Adenina/química , Sítios de Ligação , Nucleotídeos de Guanina/química , IMP Desidrogenase/química , Modelos Moleculares , Conformação Molecular , Saccharomycetales
18.
Ther Drug Monit ; 39(5): 483-491, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28650902

RESUMO

BACKGROUND: The implication of inosine triphosphate pyrophosphatase (ITPA) on thiopurine drug response variability has been investigated but little data are available on its role on thiopurine metabolites. The ability of ITPA to modify the thiopurine metabolite levels is currently used to optimize azathioprine (AZA) therapy in relation to thiopurine S-methyltransferase (TPMT) activity, the aim of this study is to investigate ITPA phenotype in a large population and to evaluate the relation between ITPA and TPMT activities and thiopurine metabolites. METHODS: ITPA activity was determined in 183 adults and 138 children with or without AZA therapy. 6-thioguanine nucleotides (6-TGN), 6-methylmercaptopurine nucleotides (6-MeMPN) levels, and ITPA as well as TPMT activities were measured in red blood cells. Using the Gaussian mixture model, distribution of ITPA activity was evaluated. Intraindividual variability and influence of age, sex, AZA treatment and associated co-medications on ITPA activity were also assessed. RESULTS: This retrospective study shows a quadrimodal distribution in ITPA activity. No influence of age, sex, AZA therapy, and co-medications was found. In adults, ITPA activity was not significantly associated with 6-TGN or 6-MeMPN concentrations, whereas a weak negative correlation was observed with 6-MeMPN levels in pediatric populations (rs = -0.261; P = 0.024). A weak positive correlation was observed between ITPA and TPMT activities in children (rs = 0.289; P = 0.001). CONCLUSIONS: ITPA activity was poorly influenced by nongenetic parameters and has no influence on 6-TGN and 6-MeMPN concentrations in adults and only a weak correlation with 6-MeMPN and TPMT activity in children. These results demonstrate that ITPA is not a rate-limiting enzyme in the formation of 6-TGN but suggest that a decrease in ITPA activity in children may be a risk factor for accumulation of 6-MeMPN in cells.


Assuntos
Azatioprina/uso terapêutico , Imunossupressores/uso terapêutico , Metiltransferases/metabolismo , Pirofosfatases/metabolismo , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Feminino , Nucleotídeos de Guanina/metabolismo , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Fenótipo , Estudos Retrospectivos , Tioinosina/análogos & derivados , Tioinosina/metabolismo , Tionucleotídeos/metabolismo , Adulto Jovem
19.
Sci Rep ; 7: 45207, 2017 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-28322335

RESUMO

Ribonucleases play an important role in the RNA metabolism which is critical for the localization, stability and function of mature RNA transcripts. More and more ribonucleases were discovered in recent years with the progress of technology. In the present study, we found that the uncharacterized C19orf43, a novel interacting protein of human telomerase RNA (hTR), digested T7 transcribed RNA, total cellular RNA and RNA oligos but not DNA. Thus we named this new RNase as hTRIR (human telomerase RNA interacting RNase). Genetic analysis showed that hTRIR is conserved among eukaryotic species and widely expressed in different cell lines. The RNase activity of hTRIR works in a broad temperature and pH range while divalent cations are not required. The conserved C-terminus of C19orf43 is necessary for its activity. Finally, we found that hTRIR cleaves all four unpaired RNA nucleotides from 5' end or 3' end with higher efficiency for purine bases, which suggested that hTRIR is an exoribonuclease. Taken together, our study showed the first evidence of the novel function of hTRIR in vitro, which provides clue to study the regulatory mechanism of hTR homeostasis in vivo.


Assuntos
RNA/metabolismo , Ribonucleases/metabolismo , Telomerase/metabolismo , Nucleotídeos de Adenina/química , Nucleotídeos de Adenina/metabolismo , Animais , Cricetinae , Cricetulus , Cães , Estabilidade Enzimática , Nucleotídeos de Guanina/química , Nucleotídeos de Guanina/metabolismo , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Células Madin Darby de Rim Canino , Domínios Proteicos , RNA/química , RNA/genética , Ribonucleases/química , Ribonucleases/genética , Especificidade por Substrato , Telomerase/química , Telomerase/genética
20.
J Biol Chem ; 291(44): 22988-22998, 2016 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-27613871

RESUMO

Guanosine-5'-monophosphate reductase (GMPR) catalyzes the reduction of GMP to IMP and ammonia with concomitant oxidation of NADPH. Here we investigated the structure and dynamics of enzyme-bound substrates and cofactors by measuring 31P relaxation rates over a large magnetic field range using high resolution field cycling NMR relaxometry. Surprisingly, these experiments reveal differences in the low field relaxation profiles for the monophosphate of GMP compared with IMP in their respective NADP+ complexes. These complexes undergo partial reactions that mimic different steps in the overall catalytic cycle. The relaxation profiles indicate that the substrate monophosphates have distinct interactions in E·IMP·NADP+ and E·GMP·NADP+ complexes. These findings were not anticipated by x-ray crystal structures, which show identical interactions for the monophosphates of GMP and IMP in several inert complexes. In addition, the motion of the cofactor is enhanced in the E·GMP·NADP+ complex. Last, the motions of the substrate and cofactor are coordinately regulated; the cofactor has faster local motions than GMP in the deamination complex but is more constrained than IMP in that complex, leading to hydride transfer. These results show that field cycling can be used to investigate the dynamics of protein-bound ligands and provide new insights into how portions of the substrate remote from the site of chemical transformation promote catalysis.


Assuntos
Coenzimas/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , GMP Redutase/química , Biocatálise , Coenzimas/metabolismo , Cristalografia por Raios X , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , GMP Redutase/genética , GMP Redutase/metabolismo , Nucleotídeos de Guanina/química , Nucleotídeos de Guanina/metabolismo , Inosina Monofosfato/química , Inosina Monofosfato/metabolismo , Cinética , Espectroscopia de Ressonância Magnética , NADP/química , NADP/metabolismo , Ligação Proteica
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