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

Abstract

Proteases that degrade the amyloid ␤-protein (A␤) are important regulators of brain A␤ levels in health and in Alzheimer's disease, yet few practical methods exist to study their detailed kinetics. Here, we describe robust and quantitative A␤ degradation assays based on the novel substrate, fluorescein-A␤-(1-40)-Lys-biotin (FA␤B). Liquid chromatography/mass spectrometric analysis shows that FA␤B is hydrolyzed at closely similar sites as wild-type A␤ by neprilysin and insulindegrading enzyme, the two most widely studied A␤-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 FA␤B degradation by several A␤-degrading proteases, including plasmin, which has not previously been characterized. The use of these assays should yield additional new insights into the biology of A␤-degrading proteases and facilitate the identification of activators and inhibitors of such enzymes.Progressive accumulation of the amyloid ␤-protein (A␤) 1 in brain regions important for memory and cognition is a defining pathogenic feature of Alzheimer's disease (AD). Nevertheless, the causes of elevated brain A␤ 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 A␤. 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 (1). Significant elevations in cerebral A␤ levels have now been observed in vivo in gene-targeted mice lacking each of several A␤-degrading proteases: neprilysin (NEP) (2), endothelin-converting enzyme-1 and -2 (3), and insulin-degrading enzyme (IDE) (4, 5). Moreover, several genetic studies have reported linkage and/or allelic association between late onset AD and polymorphisms near or within the IDE gene on chromosome 10q (6 -8).Progress in elucidating the mechanisms underlying the production of A␤ from its protein precursor, APP, by the ␤-and ␥-secretases has depended critically on the development of sensitive, reliable, and accessible assays for quantifying A␤ levels in biological samples. More recently, assays for directly measuring the activity of the secretases have been described (9 -11), enabling significant progress in the biochemical characterization of these proteases and the identification and characterization of small-molecule inhibitors. In contrast, relatively few techniques and assays for studying A␤ degradation have been reported, and the most commonly used general methods (e.g. measurement of radiolabeled peptides by trichloroacetic acid precipitation or HPLC) are cumbersome and ill suited for accurate quantification of kinetic constants or for hig...

Decreased Catalytic Activity of the Insulin-degrading Enzyme in Chromosome 10-Linked Alzheimer Disease Families

“…The post-nuclear homogenate was centrifuged at 20,000 ϫ g for 20 min to separate cytosolic (supernatant) and membrane (pellet) fractions, and protein concentration was determined by bicinchoninic acid assay (Pierce). Fluorometric quantification of A␤ degradation was performed using FA␤B (fluorescein-A␤-(1-40)-Lys(LC-biotin)) as described by Leissring et al (28). Briefly, 5 mg of protein was incubated with 0.5 mM FA␤B for 90 min at 20°C in a degradation buffer (50 mM HEPES, 100 mM NaCl, 10 mM MgCl 2 , 0.05% bovine serum albumin, pH 7.4).…”

Metal ions affect insulin-degrading enzyme activity

“…The precipitate was then filtered, dried under vacuum, redissolved in water and lyophilized. The crude peptide was purified by preparative RP-HPLC, using the following instrumental conditions: from 0 to 15 min of isocratic elution in 10% of B, then linear gradient from 10% to 25% B in 15 …”

“…It is also interesting to outline that if we compare these parameters with those obtained for the amyloid peptides Aβ [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16] and Aβ [16][17][18][19][20][21][22][23][24][25][26][27][28] several similarities and some differences emerge [54]. In particular, the overall proteolytic activity toward the main cleavage sites (i.e., Phe 24 [16][17][18][19][20][21][22][23][24][25][26][27][28] ) is somewhat higher in the case of the B20-30 peptide due to a slightly more efficient cleavage rate-limiting step (see Table 2), whereas the substrate affinity of B20-30 appears intermediate between that of Aβ [1][2][3]…”

“…Since the discovery of insulin and Aβ cleavage by IDE [11], much effort has been put in trying to understand some key questions regarding cleavage sites [12,13], kinetics of substrate interaction [14,15] and the mechanism of IDE modulation [16,17].…”

The Effects of Choto-san on the mRNA Expression of Alzheimer's Disease Related Factors in the Permanent Ischemic Rat Brain

“…IDE is also well known as an Ab degrading enzyme. 14,15) The expression was enhanced by 2VO and Chotosan inhibited this enhancement to the basal level. Since, as discussed above, the up-regulation of Ab degrading enzyme may be a reflection of the protective or compensatory roles for neuronal damage, Choto-san may produce a state in which the induction of IDE expression is not necessary.…”