ABSTRACT
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.
Subject(s)
Amyloid Precursor Protein Secretases/metabolism , Aspartic Acid Endopeptidases/metabolism , Cell Adhesion Molecules/metabolism , Neural Cell Adhesion Molecule L1/metabolism , Neurons/metabolism , Amino Acid Motifs , Amino Acid Sequence , Amino Acid Substitution , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Animals , Aspartic Acid Endopeptidases/antagonists & inhibitors , Brain/drug effects , Brain/enzymology , Brain/metabolism , COS Cells , Cell Adhesion Molecules/chemistry , Cell Adhesion Molecules/genetics , Cells, Cultured , Chlorocebus aethiops , Mice , Mice, Inbred C57BL , Mice, Knockout , Molecular Sequence Data , Mutagenesis, Site-Directed , Neural Cell Adhesion Molecule L1/chemistry , Neural Cell Adhesion Molecule L1/genetics , Neurons/enzymology , Peptide Fragments/chemistry , Primary Cell Culture , Protease Inhibitors/pharmacology , Proteolysis , Proteome/metabolism , Synapses/drug effects , Synapses/enzymology , Synapses/metabolismABSTRACT
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.
