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1.
Biochemistry ; 55(18): 2553-66, 2016 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-27064654

RESUMO

p54(nrb)/NonO is a nuclear RNA-binding protein involved in many cellular events such as pre-mRNA processing, transcription, and nuclear retention of hyper-edited RNAs. In particular, it participates in the splicing process by directly binding the 5' splice site of pre-mRNAs. The protein also concentrates in a nuclear body called paraspeckle by binding a G-rich segment of the ncRNA NEAT1. The N-terminal section of p54(nrb)/NonO contains tandem RNA recognition motifs (RRMs) preceded by an HQ-rich region including a threonine residue (Thr15) whose phosphorylation inhibits its RNA binding ability, except for G-rich RNAs. In this work, our goal was to understand the rules that characterize the binding of the p54(nrb)/NonO RRMs to their RNA target. We have done in vitro RNA binding experiments which revealed that only the first RRM of p54(nrb)/NonO binds to the 5' splice site RNA. We have then determined the structure of the p54(nrb)/NonO RRM1 by liquid-state NMR which revealed the presence of a canonical fold (ß1α1ß2ß3α2ß4) and the conservation of aromatic amino acids at the protein surface. We also investigated the dynamics of this domain by NMR. The p54(nrb)/NonO RRM1 displays some motional properties that are typical of a well-folded protein with some regions exhibiting more flexibility (loops and ß-strands). Furthermore, we determined the affinity of p54(nrb)/NonO RRM1 interaction to the 5' splice site RNA by NMR and fluorescence quenching and mapped its binding interface by NMR, concluding in a classical nucleic acid interaction. This study provides an improved understanding of the molecular basis (structure and dynamics) that governs the binding of the p54(nrb)/NonO RRM1 to one of its target RNAs.


Assuntos
Proteínas Associadas à Matriz Nuclear/química , Precursores de RNA/química , Sítios de Splice de RNA , Splicing de RNA , RNA Longo não Codificante/química , Proteínas de Ligação a RNA/química , Ribonucleotídeo Redutases/química , Animais , Camundongos , Proteínas Associadas à Matriz Nuclear/genética , Proteínas Associadas à Matriz Nuclear/metabolismo , Domínios Proteicos , Estrutura Secundária de Proteína , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonucleosídeo Difosfato Redutase , Ribonucleotídeo Redutases/genética , Ribonucleotídeo Redutases/metabolismo
2.
Chem Sci ; 7(8): 4867-4874, 2016 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-30155134

RESUMO

Substrate salvage or recycling is common and important for primary metabolism in cells but is rare in secondary metabolism. Herein we report flavoenzyme CrmK-mediated shunt product recycling in the biosynthesis of caerulomycin A (CRM A 1), a 2,2'-bipyridine-containing natural product that is under development as a potent novel immunosuppressive agent. We demonstrated that the alcohol oxidase CrmK, belonging to the family of bicovalent FAD-binding flavoproteins, catalyzed the conversion of an alcohol into a carboxylate via an aldehyde. The CrmK-mediated reactions were not en route to 1 biosynthesis but played an unexpectedly important role by recycling shunt products back to the main pathway of 1. Crystal structures and site-directed mutagenesis studies uncovered key residues for FAD-binding, substrate binding and catalytic activities, enabling the proposal for the CrmK catalytic mechanism. This study provides the first biochemical and structural evidence for flavoenzyme-mediated substrate recycling in secondary metabolism.

3.
PLoS One ; 9(6): e99211, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24911872

RESUMO

Indoleamine 2,3-dioxygenase (IDO) is found in multiple malignancies and exerts immunosuppressive effects that are central in protecting tumors from host T lymphocyte rejection. IDO is an enzyme involved in the catabolism of tryptophan resulting in inhibition of T lymphocyte function. While inhibition of IDO enzymatic activity results in tumor rejection, it is still unknown how we can directly target IDO expression within tumors using drugs. We have chosen to interfere with IDO expression by targeting the key-signaling event signal transducer and activator of transcription 1 (STAT1). We evaluated the efficacy of fludarabine, previously described to inhibit STAT1 phosphorylation. Interestingly, fludarabine was efficient in suppressing protein expression and consequently IDO activity in two different cell lines derived from breast cancer and melanoma when IDO was activated with interferon-gamma (IFN-γ) or supernatants prepared from activated T lymphocytes. However, fludarabine had no inhibitory effect on STAT1 phosphorylation. Other IFN-γ-responsive genes were only marginally inhibited by fludarabine. The level of IDO transcript was unaffected by this inhibitor, suggesting the involvement of post-transcriptional control. Strikingly, we have found that the inhibition of proteasome partially protected IDO from fludarabine-induced degradation, indicating that fludarabine induces IDO degradation through a proteasome-dependent pathway. Currently used in the clinic to treat some malignancies, fludarabine has the potential for use in the treatment of human tumors through induction of IDO degradation and consequently, for the promotion of T cell-mediated anti-tumor response.


Assuntos
Antineoplásicos/toxicidade , Regulação para Baixo/efeitos dos fármacos , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Vidarabina/análogos & derivados , Antígeno B7-H1/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Interferon gama/farmacologia , Fosforilação/efeitos dos fármacos , Estabilidade Proteica , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Fator de Transcrição STAT1/antagonistas & inibidores , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Células Tumorais Cultivadas , Vidarabina/toxicidade
4.
Can J Microbiol ; 58(4): 475-82, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22452666

RESUMO

Viral assembly is a crucial key step in the life cycle of every virus. In the case of Hepatitis C virus (HCV), the core protein is the only structural protein to interact directly with the viral genomic RNA. Purified recombinant core protein is able to self-assemble in vitro into nucleocapsid-like particles upon addition of a structured RNA, providing a robust assay with which to study HCV assembly. Inhibition of self-assembly of the C170 core protein (first 170 amino acids) was tested using short peptides derived from the HCV core, from HCV NS5A protein, and from diverse proteins (p21 and p73) known to interact with HCV core protein. Interestingly, peptides derived from the core were the best inhibitors. These peptides are derived from regions of the core predicted to be involved in the interaction between core subunits during viral assembly. We also demonstrated that a peptide derived from the C-terminal end of NS5A protein moderately inhibits the assembly process.


Assuntos
Hepacivirus/fisiologia , Peptídeos/metabolismo , Proteínas do Core Viral/metabolismo , Montagem de Vírus/fisiologia , Hepacivirus/genética , Hepacivirus/metabolismo , Nucleocapsídeo/genética , Nucleocapsídeo/metabolismo , RNA Viral/metabolismo , Proteínas não Estruturais Virais/metabolismo
5.
Mol Immunol ; 49(1-2): 253-9, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21937116

RESUMO

The immune system must be under tight control to avoid undesired responses. The enzyme indoleamine 2,3-dioxygenase (IDO) can exert necessary regulating effects by catabolizing tryptophan, leading to the suppression of immune responses in different settings, such as pregnancy and tumor growth. IDO's immuno-suppressive actions are mediated by tryptophan starvation and the accumulation of toxic tryptophan metabolites, resulting in T cell anergy, inhibition of clonal expansion or apoptosis. IDO activity in human macrophages and dendritic cells has been observed after interaction with T lymphocytes, and is triggered by interferon-gamma (IFN-γ) as well as CD40-ligand (CD40L). However, it is unclear whether IDO activity is present in B lymphocytes, which have been identified as having suppressive properties involved in anti-tumor immunity inhibition. In this study, we investigated whether IDO expression is induced in human B cells after exposure to T lymphocyte stimuli and TLR ligands. We report IDO1 and IDO2 mRNA up-regulation by exogenous stimulation with CD40L and IFN-γ. IDO is also upregulated by imiquimod, a TLR 7/8 agonist. In addition, IDO protein is detected after treatment with these exogenous factors or with supernatant from activated CD4(+) T cells. We, however, report weak or absent enzymatic activity from these IDO-expressing cells, as assessed by tryptophan consumption. We conclude that IDO may not be a counter-regulatory mechanism utilized by B lymphocytes to down-regulate immune responses, although its expression is inducible.


Assuntos
Linfócitos B/enzimologia , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Linfócitos T/metabolismo , Receptores Toll-Like/metabolismo , Linfócitos B/imunologia , Western Blotting , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/imunologia , Ativação Linfocitária/imunologia , Microscopia de Fluorescência , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/imunologia , Receptores Toll-Like/imunologia
6.
Biochem Cell Biol ; 88(2): 315-23, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20453932

RESUMO

The Core protein of hepatitis C virus is involved in several interactions other than the encapsidation of viral RNA. We recently proposed that this is related to the fact that the N-terminal half of this protein (C82) is an intrinsically unstructured protein (IUP) domain. IUP domains can adopt a secondary structure when they are interacting with another molecule, such as a nucleic acid or a protein. It is also possible to mimic these conditions by modifying the environment of the protein. We investigated the propensity of this protein to fold as a function of salt concentration, detergent, pH, and 2,2,2-trifluoro-ethanol (TFE); only the addition of TFE resulted in a structural change. The effect of TFE addition was studied by circular dichroism, structural, and dynamic data obtained by NMR. The data indicate that C82 can adopt an alpha-helical structure; this conformation is likely relevant to one of the functional roles of the HCV Core protein.


Assuntos
Trifluoretanol/farmacologia , Proteínas do Core Viral/química , Detergentes/farmacologia , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Conformação Proteica/efeitos dos fármacos , Proteínas do Core Viral/isolamento & purificação
7.
Biochem Biophys Res Commun ; 378(1): 27-31, 2009 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-18992225

RESUMO

Hepatitis C virus core protein plays an important role in the assembly and packaging of the viral genome. We have studied the structure of the N-terminal half of the core protein (C82) which was shown to be sufficient for the formation of nucleocapsid-like particle (NLP) in vitro and in yeast. Structural bioinformatics analysis of C82 suggests that it is mostly unstructured. Circular dichroism and structural NMR data indicate that C82 lacks secondary structure. Moreover, NMR relaxation data shows that C82 is highly disordered. These results indicate that the N-terminal half of the HCV core protein belongs to the growing family of intrinsically unstructured proteins (IUP). This explains the tendency of the hepatitis C virus core protein to interact with several host proteins, a well-documented characteristic of IUPs.


Assuntos
Hepacivirus/metabolismo , Proteínas do Core Viral/química , Sequência de Aminoácidos , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de Proteína , Análise de Sequência de Proteína , Proteínas do Core Viral/ultraestrutura
8.
Anal Biochem ; 366(1): 37-45, 2007 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-17493576

RESUMO

The assembly of hepatitis C virus (HCV) is not well understood. We investigated HCV nucleocapsid assembly in vitro and the role of electrostatic/hydrophobic interactions in this process. We developed a simple and rapid in vitro assay in which the progress of assembly is monitored by measuring an increase in turbidity, thereby allowing the kinetics of assembly to be determined. Assembly is performed using a truncated HCV core (C1-82), containing the minimal assembly domain, purified from Escherichia coli. The increase in turbidity is linked to the formation of nucleocapsid-like particles (NLPs) in solution, and nucleic acids are essential to initiate nucleocapsid assembly under the experimental conditions used. The sensitivity of NLP formation to salt strongly suggests that electrostatic forces govern in vitro assembly. Mutational analysis of C1-82 demonstrated that it is the global positive charge of C1-82 rather than any specific basic residue that is important for the assembly process. Our in vitro assembly assay provides an easy and efficient means of screening for assembly inhibitors, and we have identified several inhibitory peptides that could represent a starting point for drug design.


Assuntos
Hepacivirus/fisiologia , Proteínas do Core Viral/fisiologia , Sequência de Aminoácidos , Sequência de Bases , Primers do DNA/genética , DNA Viral/genética , Avaliação Pré-Clínica de Medicamentos/métodos , Hepacivirus/efeitos dos fármacos , Hepacivirus/genética , Microscopia Eletrônica , Dados de Sequência Molecular , Mutação , Nucleocapsídeo/efeitos dos fármacos , Nucleocapsídeo/fisiologia , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Sais/farmacologia , Eletricidade Estática , Proteínas do Core Viral/antagonistas & inibidores , Proteínas do Core Viral/genética , Montagem de Vírus/efeitos dos fármacos
9.
Biochem Cell Biol ; 85(1): 78-87, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17464347

RESUMO

The primary function of the hepatitis C virus (HCV) core protein is genome encapsidation. Core protein is also subject to post-translational modifications that can impact on the assembly process. In this report, we have studied the effect of cAMP-dependent protein kinase A (PKA) phosphorylation on its assembly and stability in a yeast Pichia pastoris expression system. We have recently shown that co-expression of the human signal peptide peptidase and core protein (amino acids 1-191) in yeast leads to the formation of nucleocapsid-like particles (NLPs) that are morphologically similar to the wild-type HCV capsid. In this system, we expressed mutants S53A and S116A and mutants S53D and S116D to abolish or mimic PKA phosphorylation, respectively. None of these mutations affected HCV assembly, but S116D led to the degradation of core protein. We also showed that nonenveloped NLPs were labelled in vitro by PKA, suggesting that the phosphorylation sites are available at the surface of the NLPs. The co-expression of human PKA with core and human signal peptide peptidase in yeast did not produce phosphorylated NLPs and led to a decreased accumulation of nonenveloped particles. Mutation S116A restored the core protein content. These results suggest that PKA phosphorylation can modulate HCV core levels in infected cells.


Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Hepacivirus/metabolismo , Nucleocapsídeo/metabolismo , Proteínas do Core Viral/metabolismo , Sequência de Aminoácidos , Humanos , Dados de Sequência Molecular , Mutação , Fosforilação , Proteínas do Core Viral/genética
10.
FEBS J ; 273(1): 14-25, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16367744

RESUMO

Papaya mosaic potexvirus (PapMV) coat protein (CP) was expressed (CPdeltaN5) in Escherichia coli and showed to self assemble into nucleocapsid like particles (NLPs). Twenty per cent of the purified protein was found as NLPs of 50 nm in length and 80% was found as a multimer of 450 kDa (20 subunits) arranged in a disk. Two mutants in the RNA binding domain of the PapMV CP, K97A and E128A showed interesting properties. The proteins of both mutants could be easily purified and CD spectra of these proteins showed secondary and tertiary structures similar to the WT protein. The mutant K97A was unable to self assemble and bind RNA. On the contrary, the mutant E128A showed an improved affinity for RNA and self assembled more efficiently in NLPs. E128A NLPs were longer (150 nm) than the recombinant CPdeltaN5 and 100% percent of the protein was found as NLPs in bacteria. E128A NLPs were more resistant to digestion by trypsin than the CPdeltaN5 but were more sensitive to denaturation by heat. We discuss the possible role of K97 and E128 in the assembly of PapMV.


Assuntos
Proteínas do Capsídeo/genética , Vírus do Mosaico/genética , Mutação , Potexvirus/genética , RNA Viral/metabolismo , Montagem de Vírus/genética , Sequência de Aminoácidos , Sítios de Ligação , Cromatografia em Gel , Dicroísmo Circular , Escherichia coli/genética , Escherichia coli/metabolismo , Imuno-Histoquímica , Dados de Sequência Molecular , Vírus do Mosaico/metabolismo , Vírus do Mosaico/fisiologia , Nucleocapsídeo/química , Nucleocapsídeo/genética , Nucleocapsídeo/metabolismo , Potexvirus/fisiologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Alinhamento de Sequência , Temperatura , Tripsina/metabolismo , Montagem de Vírus/fisiologia
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