Kinetics of Amyloid β-Protein Degradation Determined by Novel Fluorescence- and Fluorescence Polarization-based Assays

Abstract

Proteases that degrade the amyloid β-protein (Aβ) are important regulators of brain Aβ levels in health and in Alzheimer's disease, yet few practical methods exist to study their detailed kinetics. Here, we describe robust and quantitative Aβ degradation assays based on the novel substrate, fluorescein-Aβ-(1–40)-Lys-biotin (FAβB). Liquid chromatography/mass spectrometric analysis shows that FAβB is hydrolyzed at closely similar sites as wild-type Aβ by neprilysin and insulin-degrading enzyme, the two most widely studied Aβ-degrading proteases. The derivatized peptide is an avid substrate and is suitable for use with biological samples and in high throughput compound screening. The assays we have developed are easily implemented and are particularly useful for the generation of quantitative kinetic data, as we demonstrate by determining the kinetic parameters of FAβB degradation by several Aβ-degrading proteases, including plasmin, which has not previously been characterized. The use of these assays should yield additional new insights into the biology of Aβ-degrading proteases and facilitate the identification of activators and inhibitors of such enzymes. Proteases that degrade the amyloid β-protein (Aβ) are important regulators of brain Aβ levels in health and in Alzheimer's disease, yet few practical methods exist to study their detailed kinetics. Here, we describe robust and quantitative Aβ degradation assays based on the novel substrate, fluorescein-Aβ-(1–40)-Lys-biotin (FAβB). Liquid chromatography/mass spectrometric analysis shows that FAβB is hydrolyzed at closely similar sites as wild-type Aβ by neprilysin and insulin-degrading enzyme, the two most widely studied Aβ-degrading proteases. The derivatized peptide is an avid substrate and is suitable for use with biological samples and in high throughput compound screening. The assays we have developed are easily implemented and are particularly useful for the generation of quantitative kinetic data, as we demonstrate by determining the kinetic parameters of FAβB degradation by several Aβ-degrading proteases, including plasmin, which has not previously been characterized. The use of these assays should yield additional new insights into the biology of Aβ-degrading proteases and facilitate the identification of activators and inhibitors of such enzymes. Progressive accumulation of the amyloid β-protein (Aβ) 1The abbreviations used are: Aβ, amyloid β-protein; AD, Alzheimer's disease; IDE, insulin-degrading enzyme; HPLC, high pressure liquid chromatography; FAβB, fluorescein-Aβ-(1–40)-Lys-biotin; FP, fluorescence polarization; NEP, neprilysin; Bis-Tris, 2-[bis(2-hydroxyethyl) amino]-2-(hydroxymethyl)propane-1,3-diol; AP, avidin-agarose precipitation.1The abbreviations used are: Aβ, amyloid β-protein; AD, Alzheimer's disease; IDE, insulin-degrading enzyme; HPLC, high pressure liquid chromatography; FAβB, fluorescein-Aβ-(1–40)-Lys-biotin; FP, fluorescence polarization; NEP, neprilysin; Bis-Tris, 2-[bis(2-hydroxyethyl) amino]-2-(hydroxymethyl)propane-1,3-diol; AP, avidin-agarose precipitation. in brain regions important for memory and cognition is a defining pathogenic feature of Alzheimer's disease (AD). Nevertheless, the causes of elevated brain Aβ levels in the vast majority of AD patients remain unknown. With the exception of rare familial forms of the disease, there is little evidence that AD is attributable to the overproduction of Aβ. Instead, failed clearance of the peptide, including defects in its proteolytic degradation, could underlie its accumulation with age, a possibility that is gaining increasing experimental support (1Selkoe D. Neuron. 2001; 32: 177-180Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar). Significant elevations in cerebral Aβ levels have now been observed in vivo in gene-targeted mice lacking each of several Aβ-degrading proteases: neprilysin (NEP) (2Iwata N. Tsubuki S. Takaki Y. Shirotani K. Lu B. Gerard N.P. Gerard C. Hama E.