RESUMO
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.
Assuntos
Espectrometria de Massas/métodos , Metiltransferases/genética , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , Adenosina/análogos & derivados , Adenosina/genética , Adenosina/farmacologia , Descoberta de Drogas/tendências , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , Metilação/efeitos dos fármacos , Complexos Multiproteicos/antagonistas & inibidores , Complexos Multiproteicos/genética , Processamento Pós-Transcricional do RNA/genética , Estabilidade de RNA/efeitos dos fármacos , Estabilidade de RNA/genética , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , S-Adenosil-Homocisteína/farmacologiaRESUMO
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.
Assuntos
Lisina/metabolismo , Sirtuína 1/química , Sequência de Aminoácidos , Sítios de Ligação/genética , Domínio Catalítico/genética , Cristalização , Cristalografia por Raios X , Medição da Troca de Deutério , Escherichia coli , Vetores Genéticos , Humanos , Espectrometria de Massas , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Sirtuína 1/genética , Sirtuína 1/metabolismo , TransfecçãoRESUMO
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.
Assuntos
Sirtuína 1/química , Sirtuína 1/metabolismo , Estilbenos/farmacologia , Regulação Alostérica , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Células Cultivadas , Ativação Enzimática , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/química , Fatores de Transcrição Forkhead/genética , Ácido Glutâmico/química , Ácido Glutâmico/genética , Compostos Heterocíclicos de 4 ou mais Anéis/química , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Dados de Sequência Molecular , Mioblastos/efeitos dos fármacos , Mioblastos/enzimologia , Estrutura Terciária de Proteína , Resveratrol , Sirtuína 1/genética , Estilbenos/química , Especificidade por SubstratoRESUMO
A strategy for preparing high relaxivity, metabolically stable peptide-based MR contrast agents is described.