RESUMEN
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
Carba-NAD is a synthetic compound identical to NAD except for one substitution, where an oxygen atom adjacent to the anomeric linkage bearing nicotinamide is replaced with a methylene group. Because it is inert in nicotinamide displacement reactions, carba-NAD is an unreactive substrate analogue for NAD-consuming enzymes. SIRT3 and SIRT5 are NAD-consuming enzymes that are potential therapeutic targets for the treatment of metabolic diseases and cancers. We report an improved carba-NAD synthesis, including a pyrophosphate coupling method that proceeds in approximately 60% yield. We also disclose the X-ray crystal structures of the ternary complexes of SIRT3 and SIRT5 bound to a peptide substrate and carba-NAD. These X-ray crystal structures provide critical snapshots of the mechanism by which human sirtuins function as protein deacylation catalysts.
Asunto(s)
Carba-azúcares/química , Carba-azúcares/síntesis química , NAD/química , NAD/síntesis química , Sirtuina 3/química , Sirtuinas/química , Carba-azúcares/metabolismo , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Estructura Molecular , NAD/metabolismo , Sirtuina 3/metabolismo , Sirtuinas/metabolismo , EstereoisomerismoRESUMEN
O-Acetyl-ADP-ribose (OAADPR) is a metabolite produced from nicotinamide adenine dinucleotide (NAD) as a product of sirtuin-mediated protein deacetylation. We present here a simple, one-step, nonenzymatic synthesis of OAADPR from NAD and sodium acetate in acetic acid. We extended the reaction to other carboxylic acids, demonstrating that the reaction between NAD and nonaqueous carboxylate buffers produces mixtures of the corresponding 2'- and 3'-carboxylic esters.
Asunto(s)
Ácidos Carboxílicos/química , NAD/química , O-Acetil-ADP-Ribosa/síntesis química , O-Acetil-ADP-Ribosa/metabolismo , Sirtuina 2/metabolismo , Sirtuinas/metabolismo , Secuencia de Aminoácidos , Histona Desacetilasas , Datos de Secuencia Molecular , Estructura Molecular , NAD/metabolismo , O-Acetil-ADP-Ribosa/química , Sirtuina 2/química , Sirtuinas/químicaRESUMEN
Synthesis of potent adenosine A(2A) and A(3) receptor agonist from the modification of adenosine-5'-N-ethylcarboxamide (NECA) has been reported. Diastereoisomer possessing an (R) 3,4-dihydro-2H-pyranyl (DHP) moiety exhibited the highest affinity at the A(2A) and A(3) receptors. The key steps involve the synthesis of (R)-3,4-dihydro-2H-pyran-2-carboxaldehyde (7), which was obtained through the enzyme catalyzed kinetic resolution of (±)-2-acetoxymethyl-3,4-dihydro-2H-pyran (5).