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- W3022838900 abstract "The central pathological cause of Alzheimer disease (AD) is an accumulation of beta–amyloid (Aβ) into toxic fibrillar deposits in brain. These deposits disrupt neural and synaptic function and ultimately lead to neuronal degeneration and dementia. Previously, we discovered an activity that inhibits Aβ aggregation from Streptomyces sp. KK565 growth media. To identify and characterize the inhibiter of Aβ aggregation and to elucidate the mechanism of inhibition. The active component was purified from the aqueous fraction of methanol extracted culture media and identified as a protein. With the N–terminal amino acid sequence of the purified protein, degenerate primers were designed and the gene was cloned and sequenced. The recombinant Streptomyces sp. KK565 aminopeptidase (rSKAP) was expressed in E. coli as a secreted form with 6–Histidines on C–terminus and purified by Ni–NTA affinity chromatography. The enzyme activity was measured with Leu–pNA as a subtrate. The cleavage of Aβ peptides by rSKAP was analyzed by MALDI–TOF. SH–SY5Y cell viability was measured using alamarBlue (Biosource Inc.) reagent. The amino acid sequence revealed high degree of similarity to aminopeptidases from Streptomyces fradia and Streptomyces griseus that belong to the M28 family of metallo–peptidases. The molecular weight was approximately 30 kDa by SDS–PAGE. The enzyme was activated by Ca2+ and strongly inhibited by EDTA, EGTA and 1,10–phenathroline, an inhibitor of aminopeptidases. The purified rSKAP cleaved soluble Aβ peptides and consequently inhibited Aβ aggregation. MALDI–TOF analysis showed sequential digestion of amino acids 1–7 of both Aβ40 and Aβ42 by rSKAP. In addition, preincubation of the rSKAP with Aβ reduced Aβ–induced cytotoxicity in neuronal cells. The activity of SKAP toward Aβ peptide in vitro suggests the possible role of M28 family peptidases in brain to reduce the Aβ levels, hence preventing the pathogenesis of AD." @default.
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- W3022838900 date "2006-07-01" @default.
- W3022838900 modified "2023-09-27" @default.
- W3022838900 title "P4-068: An aminopeptidase from Strepomyces sp . KK565 (SKAP) inhibits β-amyloid peptide aggregation by cleaving Aβ N-terminal amino acids" @default.
- W3022838900 doi "https://doi.org/10.1016/j.jalz.2006.05.1806" @default.
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