RESUMO
The enzymatic activity of the SARS coronavirus main proteinase dimer was characterized by a sensitive, quantitative assay. The new, fluorogenic substrate, (Ala-Arg-Leu-Gln-NH)(2)-Rhodamine, contained a severe acute respiratory syndrome coronavirus (SARS CoV) main proteinase consensus cleavage sequence and Rhodamine 110, one of the most detectable compounds known, as the reporter group. The gene for the enzyme was cloned in the absence of purification tags, expressed in Escherichia coli and the enzyme purified. Enzyme activity from the SARS CoV main proteinase dimer could readily be detected at low pM concentrations. The enzyme exhibited a high K(m), and is unusually sensitive to ionic strength and reducing agents.
Assuntos
Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/enzimologia , Clonagem Molecular , Proteases 3C de Coronavírus , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/isolamento & purificação , Dimerização , Expressão Gênica , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Concentração Osmolar , Peptídeos/química , Peptídeos/metabolismo , Estrutura Quaternária de Proteína , Sensibilidade e Especificidade , Especificidade por Substrato , TemperaturaRESUMO
Cross-species studies enable rapid translational discovery and produce the broadest impact when both mechanism and phenotype are consistent across organisms. We developed a knock-in mouse that biologically recapitulates a common human mutation in the gene for fatty acid amide hydrolase (FAAH) (C385A; rs324420), the primary catabolic enzyme for the endocannabinoid anandamide. This common polymorphism impacts the expression and activity of FAAH, thereby increasing anandamide levels. Here, we show that the genetic knock-in mouse and human variant allele carriers exhibit parallel alterations in biochemisty, neurocircuitry and behaviour. Specifically, there is reduced FAAH expression associated with the variant allele that selectively enhances fronto-amygdala connectivity and fear extinction learning, and decreases anxiety-like behaviours. These results suggest a gain of function in fear regulation and may indicate for whom and for what anxiety symptoms FAAH inhibitors or exposure-based therapies will be most efficacious, bridging an important translational gap between the mouse and human.