RESUMEN
A variety of covalent modifications of RNA have been identified and demonstrated to affect RNA processing, stability, and translation. Methylation of adenosine at the N6 position (m6A) in messenger RNA (mRNA) is currently the most well-studied RNA modification and is catalyzed by the RNA methyltransferase complex METTL3/METTL14. Once generated, m6A can modulate mRNA splicing, export, localization, degradation, and translation. Although potent and selective inhibitors exist for several members of the Type I S-adenosylmethionine (SAM)-dependent methyltransferase family, no inhibitors have been reported for METTL3/METTL14 to date. To facilitate drug discovery efforts, a sensitive and robust mass spectrometry-based assay for METTL3/METTL14 using self-assembled monolayer desorption/ionization (SAMDI) technology has been developed. The assay uses an 11-nucleotide single-stranded RNA compared to a previously reported 27-nucleotide substrate. IC50 values of mechanism-based inhibitors S-adenosylhomocysteine (SAH) and sinefungin (SFG) are comparable between the SAMDI and radiometric assays that use the same substrate. This work demonstrates that SAMDI technology is amenable to RNA substrates and can be used for high-throughput screening and compound characterization for RNA-modifying enzymes.
Asunto(s)
Espectrometría de Masas/métodos , Metiltransferasas/genética , Procesamiento Postranscripcional del ARN/efectos de los fármacos , Adenosina/análogos & derivados , Adenosina/genética , Adenosina/farmacología , Descubrimiento de Drogas/tendencias , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Humanos , Metilación/efectos de los fármacos , Complejos Multiproteicos/antagonistas & inhibidores , Complejos Multiproteicos/genética , Procesamiento Postranscripcional del ARN/genética , Estabilidad del ARN/efectos de los fármacos , Estabilidad del ARN/genética , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , S-Adenosilhomocisteína/farmacologíaRESUMEN
SIRT1, the founding member of the mammalian family of seven NAD(+)-dependent sirtuins, is composed of 747 amino acids forming a catalytic domain and extended N- and C-terminal regions. We report the design and characterization of an engineered human SIRT1 construct (mini-hSIRT1) containing the minimal structural elements required for lysine deacetylation and catalytic activation by small molecule sirtuin-activating compounds (STACs). Using this construct, we solved the crystal structure of a mini-hSIRT1-STAC complex, which revealed the STAC-binding site within the N-terminal domain of hSIRT1. Together with hydrogen-deuterium exchange mass spectrometry (HDX-MS) and site-directed mutagenesis using full-length hSIRT1, these data establish a specific STAC-binding site and identify key intermolecular interactions with hSIRT1. The determination of the interface governing the binding of STACs with human SIRT1 facilitates greater understanding of STAC activation of this enzyme, which holds significant promise as a therapeutic target for multiple human diseases.
Asunto(s)
Lisina/metabolismo , Sirtuina 1/química , Secuencia de Aminoácidos , Sitios de Unión/genética , Dominio Catalítico/genética , Cristalización , Cristalografía por Rayos X , Medición de Intercambio de Deuterio , Escherichia coli , Vectores Genéticos , Humanos , Espectrometría de Masas , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Sirtuina 1/genética , Sirtuina 1/metabolismo , TransfecciónRESUMEN
A molecule that treats multiple age-related diseases would have a major impact on global health and economics. The SIRT1 deacetylase has drawn attention in this regard as a target for drug design. Yet controversy exists around the mechanism of sirtuin-activating compounds (STACs). We found that specific hydrophobic motifs found in SIRT1 substrates such as PGC-1α and FOXO3a facilitate SIRT1 activation by STACs. A single amino acid in SIRT1, Glu(230), located in a structured N-terminal domain, was critical for activation by all previously reported STAC scaffolds and a new class of chemically distinct activators. In primary cells reconstituted with activation-defective SIRT1, the metabolic effects of STACs were blocked. Thus, SIRT1 can be directly activated through an allosteric mechanism common to chemically diverse STACs.
Asunto(s)
Sirtuina 1/química , Sirtuina 1/metabolismo , Estilbenos/farmacología , Regulación Alostérica , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Células Cultivadas , Activación Enzimática , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/química , Factores de Transcripción Forkhead/genética , Ácido Glutámico/química , Ácido Glutámico/genética , Compuestos Heterocíclicos de 4 o más Anillos/química , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Ratones , Datos de Secuencia Molecular , Mioblastos/efectos de los fármacos , Mioblastos/enzimología , Estructura Terciaria de Proteína , Resveratrol , Sirtuina 1/genética , Estilbenos/química , Especificidad por SustratoRESUMEN
A strategy for preparing high relaxivity, metabolically stable peptide-based MR contrast agents is described.