RESUMO
Despite recent progresses in the use of eukaryotic expression system, production of membrane proteins for structural studies still relies on microbial expression systems. In this review, we provide protocols to achieve high level expression of membrane proteins in Escherichia coli, especially using the T7 RNA polymerase based expression system. From the design of the construct, the choice of the appropriate vector-host combination, the assessment of the bacterial fitness, to the selection of bacterial mutant adapted to the production of the target membrane protein, the chapter covers all necessary methods for a rapid optimization of a specific target membrane protein. In addition, we provide a protocol for membrane protein solubilization based on our recent analysis of the Protein Data Bank.
Assuntos
Escherichia coli , Proteínas de Membrana , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Recombinantes/metabolismo , Literatura de Revisão como AssuntoRESUMO
O-GlcNAcylation is a post-translational modification of tau understood to lower the speed and yield of its aggregation, a pathological hallmark of Alzheimer's disease (AD). O-GlcNAcase (OGA) is the only enzyme that removes O-linked N-acetyl-d-glucosamine (O-GlcNAc) from target proteins. Therefore, inhibition of OGA represents a potential approach for the treatment of AD by preserving the O-GlcNAcylated tau protein. Herein, we report the multifactorial optimization of high-throughput screening hit 8 to a potent, metabolically stable, and orally bioavailable diazaspirononane OGA inhibitor (+)-56. The human OGA X-ray crystal structure has been recently solved, but bacterial hydrolases are still widely used as structural homologues. For the first time, we reveal how a nonsaccharide series of inhibitors binds bacterial OGA and discuss the suitability of two different bacterial orthologues as surrogates for human OGA. These breakthroughs enabled structure-activity relationships to be understood and provided context and boundaries for the optimization of druglike properties.
Assuntos
Compostos Aza/farmacologia , Inibidores Enzimáticos/farmacologia , Doenças Neurodegenerativas/tratamento farmacológico , beta-N-Acetil-Hexosaminidases/antagonistas & inibidores , beta-N-Acetil-Hexosaminidases/metabolismo , Animais , Compostos Aza/química , Catálise , Inibidores Enzimáticos/química , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Estrutura Molecular , Mutagênese , Relação Estrutura-AtividadeRESUMO
Copper(i) catalyzes the borylative cyclization of γ-alkenyl aldehydes through chemo- and regioselective addition of Cu-B to C[double bond, length as m-dash]C and concomitant intramolecular 1,2-addition of Cu-C on C[double bond, length as m-dash]O. The products are formed in an exclusive diastereoselective manner and computational analysis identifies the key points for the observed chemo- and diastereoselectivity.
RESUMO
The insertion of the diazo derivative Me3SiCHN2 into pinB-SR σ bonds (R = Ph, Tol, Bn) allows a direct synthesis of multisubstituted H-C(SR)(Bpin)(SiMe3) compounds. Consecutive base-assisted transformations of HC(S)(B) (Si) systems lead to deborylative alkylations, Sommelet-Haüser rearrangements, and deprotoalkylations. Intramolecular cyclizations can be selectively performed either via desilylative or deborylative manifolds by fine-tuning the base employed.
