RESUMEN
The ß-site amyloid precursor protein-cleaving enzyme BACE1 is a prime drug target for Alzheimer disease. However, the function and the physiological substrates of BACE1 remain largely unknown. In this work, we took a quantitative proteomic approach to analyze the secretome of primary neurons after acute BACE1 inhibition, and we identified several novel substrate candidates for BACE1. Many of these molecules are involved in neuronal network formation in the developing nervous system. We selected the adhesion molecules L1 and CHL1, which are crucial for axonal guidance and maintenance of neural circuits, for further validation as BACE1 substrates. Using both genetic BACE1 knock-out and acute pharmacological BACE1 inhibition in mice and cell cultures, we show that L1 and CHL1 are cleaved by BACE1 under physiological conditions. The BACE1 cleavage sites at the membrane-proximal regions of L1 (between Tyr(1086) and Glu(1087)) and CHL1 (between Gln(1061) and Asp(1062)) were determined by mass spectrometry. This work provides molecular insights into the function and the pathways in which BACE1 is involved, and it will help to predict or interpret possible side effects of BACE1 inhibitor drugs in current clinical trials.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Moléculas de Adhesión Celular/metabolismo , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Neuronas/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Animales , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Encéfalo/efectos de los fármacos , Encéfalo/enzimología , Encéfalo/metabolismo , Células COS , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/genética , Células Cultivadas , Chlorocebus aethiops , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Molécula L1 de Adhesión de Célula Nerviosa/química , Molécula L1 de Adhesión de Célula Nerviosa/genética , Neuronas/enzimología , Fragmentos de Péptidos/química , Cultivo Primario de Células , Inhibidores de Proteasas/farmacología , Proteolisis , Proteoma/metabolismo , Sinapsis/efectos de los fármacos , Sinapsis/enzimología , Sinapsis/metabolismoRESUMEN
REALISIS is a software system for reagent selection, library design, and profiling, developed to fit the workflow of bench chemists and medicinal chemists. Designed to be portable, the software offers a comprehensive graphical user interface and rapid, integrated functionalities required for reagent retrieval and filtering, product enumeration, and library profiling. REALISIS is component-based, consisting of four main modules: reagent searching; reagent filtering; library enumeration; and library profiling. Each module allows the chemist to access specific functionalities and diverse filtering and profiling mechanisms. By implementing the entire process of reagent selection, library design, and profiling and by integrating all the necessary functionalities for this process, REALISIS cuts the time required to design combinatorial and noncombinatorial libraries from several days to a few hours.