RESUMO
Understanding the core replication complex of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential to the development of novel coronavirus-specific antiviral therapeutics. Among the proteins required for faithful replication of the SARS-CoV-2 genome are nonstructural protein 14 (NSP14), a bifunctional enzyme with an N-terminal 3'-to-5' exoribonuclease (ExoN) and a C-terminal N7-methyltransferase, and its accessory protein, NSP10. The difficulty in producing pure and high quantities of the NSP10/14 complex has hampered the biochemical and structural study of these important proteins. We developed a straightforward protocol for the expression and purification of both NSP10 and NSP14 from Escherichia coli and for the in vitro assembly and purification of a stoichiometric NSP10/14 complex with high yields. Using these methods, we observe that NSP10 provides a 260-fold increase in kcat/Km in the exoribonucleolytic activity of NSP14 and enhances protein stability. We also probed the effect of two small molecules on NSP10/14 activity, remdesivir monophosphate and the methyltransferase inhibitor S-adenosylhomocysteine. Our analysis highlights two important factors for drug development: first, unlike other exonucleases, the monophosphate nucleoside analog intermediate of remdesivir does not inhibit NSP14 activity; and second, S-adenosylhomocysteine modestly activates NSP14 exonuclease activity. In total, our analysis provides insights for future structure-function studies of SARS-CoV-2 replication fidelity for the treatment of coronavirus disease 2019.
Assuntos
Antivirais/farmacologia , Exorribonucleases/metabolismo , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia , Proteínas não Estruturais Virais/metabolismo , Ativação Enzimática , Replicação Viral/efeitos dos fármacosRESUMO
Maintenance and replication of the mitochondrial genome (mtDNA) is essential to mitochondrial function and eukaryotic energy production through the electron transport chain. mtDNA is replicated by a core set of proteins: Pol γ, Twinkle, and the single-stranded DNA binding protein. Fewer pathways exist for repair of mtDNA than nuclear DNA, and unrepaired damage to mtDNA may accumulate and lead to dysfunctional mitochondria. The mitochondrial genome is susceptible to damage by both endogenous and exogenous sources. Missense mutations to the nuclear genes encoding the core mtDNA replisome (POLG, POLG2, TWNK, and SSBP1) cause changes to the biochemical functions of their protein products. These protein variants can damage mtDNA and perturb oxidative phosphorylation. Ultimately, these mutations cause a diverse set of diseases that can affect virtually every system in the body. Here, we briefly review the mechanisms of mtDNA damage and the clinical consequences of disease variants of the core mtDNA replisome.
Assuntos
DNA Helicases/genética , DNA Polimerase gama/genética , Proteínas de Ligação a DNA/genética , DNA Polimerase Dirigida por DNA/genética , Doenças Mitocondriais/genética , Proteínas Mitocondriais/genética , Mutação , DNA Helicases/metabolismo , DNA Polimerase gama/metabolismo , Replicação do DNA , DNA Mitocondrial/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Genoma Mitocondrial , Humanos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismoRESUMO
Faithful replication of the mitochondrial genome is carried out by a set of key nuclear-encoded proteins. DNA polymerase γ is a core component of the mtDNA replisome and the only replicative DNA polymerase localized to mitochondria. The asynchronous mechanism of mtDNA replication predicts that the replication machinery encounters dsDNA and unique physical barriers such as structured genes, G-quadruplexes, and other obstacles. In vitro experiments here provide evidence that the polymerase γ heterotrimer is well-adapted to efficiently synthesize DNA, despite the presence of many naturally occurring roadblocks. However, we identified a specific G-quadruplex-forming sequence at the heavy-strand promoter (HSP1) that has the potential to cause significant stalling of mtDNA replication. Furthermore, this structured region of DNA corresponds to the break site for a large (3,895 bp) deletion observed in mitochondrial disease patients. The presence of this deletion in humans correlates with UV exposure, and we have found that efficiency of polymerase γ DNA synthesis is reduced after this quadruplex is exposed to UV in vitro.
Assuntos
DNA Polimerase gama/metabolismo , DNA Mitocondrial/metabolismo , Quadruplex G , Biocatálise , Replicação do DNA/efeitos da radiação , Humanos , Mitocôndrias/genética , Doenças Mitocondriais/genética , Doenças Mitocondriais/patologia , Regiões Promotoras Genéticas , Especificidade por Substrato , Raios UltravioletaRESUMO
Epigenetic regulation is a key factor in cellular homeostasis. Post-translational modifications (PTMs) are a central focus of this regulation as they function as signaling markers within the cell. Lysine acetylation is a dynamic, reversible PTM that has garnered recent attention due to alterations in various types of cancer. Acetylation levels are regulated by two opposing enzyme families: lysine acetyltransferases (KATs) and histone deacetylases (HDACs). HDACs are key players in epigenetic regulation and have a role in the silencing of tumor suppressor genes. The dynamic equilibrium of acetylation makes HDACs attractive targets for drug therapy. However, substrate selectivity and biological function of HDAC isozymes is poorly understood. This review outlines the current understanding of the roles and specific epigenetic interactions of the metal-dependent HDACs in addition to their roles in cancer.
Assuntos
Histona Desacetilases/metabolismo , Metais/metabolismo , Neoplasias/metabolismo , Animais , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , HumanosRESUMO
Histone deacetylase inhibitors (HDACi) target abnormal epigenetic states associated with a variety of pathologies, including cancer. Here, the development of a prodrug of the canonical broad-spectrum HDACi suberoylanilide hydroxamic acid (SAHA) is described. Although hydroxamic acids are utilized universally in the development of metalloenzyme inhibitors, they are considered to be poor pharmacophores with reduced activity in vivo. We developed a prodrug of SAHA by appending a promoiety, sensitive to thiols, to the hydroxamic acid warhead (termed SAHA-TAP). After incubation of SAHA-TAP with an HDAC, the thiol of a conserved HDAC cysteine residue becomes covalently tagged with the promoiety, initiating a cascade reaction that leads to the release of SAHA. Mass spectrometry and enzyme kinetics experiments validate that the cysteine residue is covalently appended with the TAP promoiety. SAHA-TAP demonstrates cytotoxicity activity against various cancer cell lines. This strategy represents an original prodrug design with a dual mode of action for HDAC inhibition.
Assuntos
Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Pró-Fármacos/farmacologia , Proteínas Repressoras/antagonistas & inibidores , Animais , Antineoplásicos/química , Western Blotting , Células Cultivadas , Cromatografia Líquida , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Inibidores de Histona Desacetilases/química , Histona Desacetilases , Humanos , Ácidos Hidroxâmicos/química , Leucemia de Células T/tratamento farmacológico , Leucemia de Células T/enzimologia , Leucemia de Células T/patologia , Camundongos , Modelos Moleculares , Estrutura Molecular , Pró-Fármacos/química , Relação Estrutura-Atividade , Espectrometria de Massas em Tandem , VorinostatRESUMO
Despite being extensively characterized structurally and biochemically, the functional role of histone deacetylase 8 (HDAC8) has remained largely obscure due in part to a lack of known cellular substrates. Herein, we describe an unbiased approach using chemical tools in conjunction with sophisticated proteomics methods to identify novel non-histone nuclear substrates of HDAC8, including the tumor suppressor ARID1A. These newly discovered substrates of HDAC8 are involved in diverse biological processes including mitosis, transcription, chromatin remodeling, and RNA splicing and may help guide therapeutic strategies that target the function of HDAC8.
Assuntos
Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Proteínas Nucleares/metabolismo , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Acetilação , Proteínas de Ligação a DNA , Humanos , Proteômica , Proteínas Repressoras/antagonistas & inibidores , Especificidade por SubstratoRESUMO
Histone deacetylases catalyze the hydrolysis of an acetyl group from post-translationally modified acetyl-lysine residues in a wide variety of essential cellular proteins, including histones. Because these lysine modifications can alter the activity and properties of affected proteins, aberrant acetylation/deacetylation may contribute to disease states. Many fundamental questions regarding the substrate specificity and regulation of these enzymes have yet to be answered. Here, we optimize an enzyme-coupled assay to measure low micromolar concentrations of acetate, coupling acetate production to the formation of NADH (nicotinamide adenine dinucleotide, reduced form) that is measured by changes in either absorbance or fluorescence. Using this assay, we measured the steady-state kinetics of peptides representing the H4 histone tail and demonstrate that a C-terminally conjugated methylcoumarin enhances the catalytic efficiency of deacetylation catalyzed by cobalt(II)-bound histone deacetylase 8 [Co(II)-HDAC8] compared with peptide substrates containing a C-terminal carboxylate, amide, and tryptophan by 50-, 2.8-, and 2.3-fold, respectively. This assay can be adapted for a high-throughput screening format to identify HDAC substrates and inhibitors.
Assuntos
Acetatos/metabolismo , Ensaios Enzimáticos/métodos , Histona Desacetilases/metabolismo , Sequência de Aminoácidos , Biocatálise , Inibidores de Histona Desacetilases/farmacologia , NAD/metabolismo , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Especificidade por SubstratoRESUMO
The penultimate reaction in the oxidative degradation of nicotinate (vitamin B(3)) to fumarate in several species of aerobic bacteria is the hydrolytic deamination of maleamate to maleate, catalyzed by maleamate amidohydrolase (NicF). Although it has been considered a model system for bacterial degradation of N-heterocyclic compounds, only recently have gene clusters that encode the enzymes of this catabolic pathway been identified to allow detailed investigations concerning the structural basis of their mechanisms. Here, the Bb1774 gene from Bordetella bronchiseptica RB50, putatively annotated as nicF, has been cloned, and the recombinant enzyme, overexpressed and purified from Escherichia coli, is shown to catalyze efficiently the hydrolysis of maleamate to maleate and ammonium ion. Steady-state kinetic analysis of the reaction by isothermal titration calorimetry (ITC) established k(cat) and K(M) values (pH 7.5 and 25 °C) of 11.7 ± 0.2 s(-1) and 128 ± 6 µM, respectively. The observed K(D) of the NicF·maleate (E·P) complex, also measured by ITC, is approximated to be 3.8 ± 0.4 mM. The crystal structure of NicF, determined at 2.4 Å using molecular replacement, shows that the enzyme belongs to the cysteine hydrolase superfamily. The structure provides insight concerning the roles of potential catalytically important residues, most notably a conserved catalytic triad (Asp29, Lys117, and Cys150) observed in the proximity of a conserved non-proline cis-peptide bond within a small cavity that is likely the active site. On the basis of this structural information, the hydrolysis of maleamate is proposed to proceed by a nucleophilic addition-elimination sequence involving the thiolate side chain of Cys150.