کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1175434 961801 2009 13 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Measurement of cellular β-site of APP cleaving enzyme 1 activity and its modulation in neuronal assay systems
کلمات کلیدی
موضوعات مرتبط
مهندسی و علوم پایه شیمی شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
Measurement of cellular β-site of APP cleaving enzyme 1 activity and its modulation in neuronal assay systems
چکیده انگلیسی

Amyloid-β peptide (Aβ), a putatively causative agent of Alzheimer’s disease (AD), is proteolytically derived from β-amyloid precursor protein (APP). Here we describe cellular assays to detect the activity of the key protease β-site of APP cleaving enzyme 1 (BACE1) based on an artificial reporter construct containing the BACE1 cleavage site of APP. These methods allow identification of inhibitors and indirect modulators of BACE1. In primary neuronal cultures transfected with human APP constructs (huAPP), Aβ production was modified by BACE1 inhibitors similarly to the production of endogenous murine Aβ in wild-type cells and to that of different transgenic neurons. To further improve the assay, we substituted the extracellular domain of APP by secreted alkaline phosphatase (SEAP). SEAP was easily quantified in the cell culture supernatants after cleavage of SEAP–APP by BACE1 or α-secretases. To render the assay specific for BACE1, the α-secretase cleavage site of SEAP–APP was eliminated either by site-directed mutagenesis or by substituting the transmembrane part of APP by the membrane domain of the erythropoietin receptor (EpoR). The pharmacology of these constructs was characterized in detail in HEK293 cells (human embryonic kidney cell line), and the SEAP–APP–EpoR construct was also introduced into primary murine neurons and there allowed specific measurement of BACE1 activity.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Analytical Biochemistry - Volume 387, Issue 2, 15 April 2009, Pages 208–220
نویسندگان
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