PUBLICATION
Quantification of gamma-secretase modulation differentiates inhibitor compound selectivity between two substrates Notch and amyloid precursor protein
- Authors
- Yang, T., Arslanova, D., Gu, Y., Augelli-Szafran, C., and Xia, W.
- ID
- ZDB-PUB-081110-1
- Date
- 2008
- Source
- Molecular brain 1(1): 15 (Journal)
- Registered Authors
- Xia, Weiming
- Keywords
- none
- MeSH Terms
-
- Amyloid Precursor Protein Secretases/antagonists & inhibitors*
- Amyloid Precursor Protein Secretases/metabolism
- Amyloid beta-Protein Precursor/metabolism*
- Animals
- Basic Helix-Loop-Helix Transcription Factors/genetics
- Basic Helix-Loop-Helix Transcription Factors/metabolism
- Dipeptides/pharmacology
- Embryo, Nonmammalian/drug effects
- Enzyme-Linked Immunosorbent Assay
- Gene Expression Regulation/drug effects
- Mutation/genetics
- Phenotype
- Protease Inhibitors/pharmacology*
- Protein Structure, Tertiary
- Receptors, Notch/antagonists & inhibitors
- Receptors, Notch/metabolism*
- Recombinant Proteins/metabolism
- Signal Transduction/drug effects
- Substrate Specificity/drug effects
- Zebrafish/embryology
- Zebrafish/genetics
- Zebrafish Proteins/genetics
- Zebrafish Proteins/metabolism
- PubMed
- 18983676 Full text @ Mol. Brain
Citation
Yang, T., Arslanova, D., Gu, Y., Augelli-Szafran, C., and Xia, W. (2008) Quantification of gamma-secretase modulation differentiates inhibitor compound selectivity between two substrates Notch and amyloid precursor protein. Molecular brain. 1(1):15.
Abstract
BACKGROUND: Deposition of amyloid beta protein (Abeta) is a major pathological hallmark of Alzheimer's disease (AD). Abeta is generated from gamma-secretase cleavage of amyloid precursor protein (APP). In addition to APP, gamma-secretase also cleaves other type I integral membrane proteins, including the Notch receptor, a key molecule involved in embryonic development. RESULTS: To explore selective gamma-secretase inhibitors, a combination of five methods was used to systematically determine these inhibitors' profiles on the gamma-secretase cleavage of APP and Notch. When two potent gamma-secretase inhibitors, compound E (cpd E) and DAPT, were used in a conventional in vitro gamma-secretase activity assay, cpd E completely blocked Abeta generation from the cleavage of substrate APP C100, but only had a minor effect on Notch cleavage and NICD generation. Next, cpd E and DAPT were applied to HEK293 cells expressing a truncated Notch substrate Notch deltaE. Both cpd E and DAPT were more potent in blocking Abeta generation than NICD generation. Third, a reporter construct was created that carried the NICD targeting promoter with three Su(H) binding sequences followed by the luciferase gene. We found that the inhibition of NICD generation by cpd E and DAPT was consistent with the reduced expression of luciferase gene driven by this Notch targeting promoter. Fourth, levels of "Notch-Abeta-like" (Nbeta*) peptide derived from two previously reported chimeric APP with its transmembrane domain or the juxtamembrane portion replaced by the Notch sequence were quantified. Measurement of Nbeta* peptides by ELISA confirmed that EC50's of cpd E were much higher for Nbeta* than Abeta. Finally, the expression levels of Notch target gene her6 in cpd E or DAPT-treated zebrafish were correlated with the degree of tail curvature due to defective somitogenesis, a well characterized Notch phenotype in zebrafish. CONCLUSION: our ELISA-based quantification of Abeta and Nbeta* in combination with the test in zebrafish provides a novel approach for efficient cell-based screening and in vivo validation of APP selective gamma-secretase inhibitors.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping