Design of Peptide Substrate for Sensitively and Specifically Detecting Two Aβ-Degrading Enzymes: Neprilysin and Angiotensin-Converting Enzyme.
Upregulation of neprilysin (NEP) to reduce Aβ accumulation in the brain is a promising strategy for the prevention of Alzheimer's disease (AD). This report describes the design and synthesis of a quenched fluorogenic peptide substrate qf-Aβ(12-16)AAC (with the sequence VHHQKAAC), which has a fluorophore, Alexa-350, linked to the side-chain of its C-terminal cysteine and a quencher, Dabcyl, linked to its N-terminus. This peptide emitted strong fluorescence upon cleavage. Our results showed that qf-Aβ(12-16)AAC is more sensitive to NEP than the previously reported peptide substrates, so that concentrations of NEP as low as 0.03 nM could be detected at peptide concentration of 2 μM. Moreover, qf-Aβ(12-16)AAC had superior enzymatic specificity for both NEP and angiotensin-converting enzyme (ACE), but was inert with other Aβ-degrading enzymes. This peptide, used in conjunction with a previously reported peptide substrate qf-Aβ(1-7)C [which is sensitive to NEP and insulin-degrading enzyme (IDE)], could be used for high-throughput screening of compounds that only upregulate NEP. The experimental results of cell-based activity assays using both qf-Aβ(1-7)C and qf-Aβ(12-16)AAC as the substrates confirm that somatostatin treatment most likely upregulates IDE, but not NEP, in neuroblastoma cells.
Institute of Biochemical Sciences, National Taiwan University, Taipei, 10617, Taiwan.|Institute of Biological Chemistry, Academia Sinica, Taipei, 11529, Taiwan.
Po-Ting
PT
Chen
Department of Chemical Engineering, National Taiwan University, Taipei, 10617, Taiwan.
Chao-Long
CL
Chen
Department of Chemical Engineering, National Taiwan University, Taipei, 10617, Taiwan.
Lilian Tsai-Wei
LT
Lin
Department of Chemical Engineering, National Taiwan University, Taipei, 10617, Taiwan.
Chun-Hsien
CH
Lo
Department of Neurology, Shuang-Ho Hospital, Taipei Medical University, Taipei, 110, Taiwan.
Chaur-Jong
CJ
Hu
Institute of Biochemical Sciences, National Taiwan University, Taipei, 10617, Taiwan.|Institute of Biological Chemistry, Academia Sinica, Taipei, 11529, Taiwan.
Rita P-Y
RP
Chen
Department of Chemical Engineering, National Taiwan University, Taipei, 10617, Taiwan.
Steven S-S
SS
Wang
D000085:Acetates / Q000737:chemistry / Q000378:metabolism; D000544:Alzheimer Disease / Q000201:enzymology* / Q000378:metabolism; D000595:Amino Acid Sequence; D016229:Amyloid beta-Peptides / Q000737:chemistry / Q000378:metabolism*; D045744:Cell Line, Tumor; D002867:Chromones / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007339:Insulysin / Q000378:metabolism; D015260:Neprilysin / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D007703:Peptidyl-Dipeptidase A / Q000378:metabolism*; D059748:Proteolysis; D013050:Spectrometry, Fluorescence / Q000379:methods; D013379:Substrate Specificity
D000085:Acetates; C400304:Alexa 350; D016229:Amyloid beta-Peptides; D002867:Chromones; D010446:Peptide Fragments; D007703:Peptidyl-Dipeptidase A; D015260:Neprilysin; D007339:Insulysin