Method of screening for gamma-secretase activity
View Patent ↗The present invention relates to an improved process for determining γ-secretase activity and for detecting γ-secretase, a subunit protein of γ-secretase, or a γ-secretase-like proteinase. In particular, the present invention relates to processes for the identification of a γ-secretase or of a cDNA which codes for a γ-secretase, a subunit protein of γ-secretase, or a γ-secretase-like proteinase. Another embodiment of the present invention relates to processes for the identification of substances which can inhibit the activity of γ-secretase, or a γ-secretase-like proteinase.
1. A method for detecting the activity of γ-secretase, the method comprising:
A. providing a transgene encoding a fusion protein, said transgene comprising:
a) a first nucleotide sequence encoding the amino acid sequence LDA EFRHDSGYEV HHQKLVFFAE DVGSNKGAII GLMVGGVVIA TVIVITLVML KK (SEQ ID NO. 6);
b) a second nucleotide sequence, at the 5′ end of the first nucleotide sequence, encoding a signal peptide; and
c) a promoter, and a nucleotide sequence at the 3′ end of the first nucleotide sequence coding for a protein consisting of a DNA-binding domain and a transcription-activating domain to be expressed as a fusion protein with SEQ ID NO. 6;
B. expressing the fusion protein in an isolated cell or transgenic C. elegans that contains γ-secretase said isolated cell or C. elegans having a reporter system activatable by release of a polypeptide that comprises VIVITLVML (SEQ ID NO. 3) said released polypeptide that comprises SEQ ID NO. 3 not comprising SEQ ID NO. 6; and
C. detecting said polypeptide that comprises VIVITLVML (SEQ ID NO. 3) by observing a report of said reporter system.
2. The method of claim 1 , further comprising determining, from the amount of the polypeptide of SEQ ID NO. 3, the amount of γ-secretase activity.
3. The method of claim 1 , wherein the fusion protein does not comprise, with the exception of SEQ ID NO. 6, one or more sequences consisting of a caspase cleavage site.
4. The method of claim 1 , wherein the cell is a eukaryotic cell.
5. The method of claim 1 , wherein the cell is cotransfected with a reporter plasmid comprising a reporter gene under the control of a regulatable promoter.
6. The method of claim 5 , wherein the reporter plasmid comprises a reporter gene for EGFP (Enhanced Green Fluorescent Protein), Ura 3, His 3 or Lac Z and the regulatable promoter comprises a GAL4 binding site and a minimal promoter of HIV.
7. The method of claim 1 , wherein the transgene is present in a vector.
8. The method of claim 7 , wherein the vector is pcDNA 3.1+.
9. An assay for detecting inhibition of γ-secretase, said assay comprising:
a. providing an isolated transgenic cell or a transgenic C. elegans , said transgenic cell or a transgenic C. elegans comprising:
i. a first nucleotide sequence encoding amino acid sequence LDA EFRHDSGYEV HHQKLVFFAE DVGSNKGAII GLMVGGVVIA TVIVITLVML KK (SEQ ID NO. 6)
ii. a second nucleotide sequence, at the 5′ end of the first nucleotide sequence, encoding a signal peptide; and
iii. a promoter, and a nucleotide sequence at the 3′ end of the first nucleotide sequence coding for a protein consisting of a DNA-binding domain and a transcription-activating domain to be expressed as a fusion protein with SEQ ID NO. 6, said fusion protein to be used for determining as in c. below
b. incubating the transgenic C. elegans or isolated transgenic cell with a substance; and
c. determining whether a protein comprising the amino acid sequence of SEQ ID NO. 3, but not comprising SEQ ID NO: 6 is formed
wherein said isolated transgenic cell or transgenic C. elegans is cotransfected with a reporter plasmid comprising a reporter gene under the control of a regulatable promoter comprising a binding site for the DNA-binding domain of said protein consisting of a DNA binding domain and a transcription-activating domain, and
wherein c. comprises detecting the polypeptide comprising SEQ ID NO: 3 by observing a report of said reporter under the control of said regulatable promoter.
10. The method of claim 1 wherein the protein having the DNA-binding domain and the transcription-activating domain is the protein of SEQ ID NO: 7 having the GAL4-binding domain and the transcription-activating domain of VP16 or is a protein having a LexA-binding domain and the transcription-activating domain of VP16.
11. The assay of claim 9 wherein the protein having the DNA-binding domain and the transcription-activating domain is the protein of SEQ ID NO: 7 having the GAL4-binding domain and the transcription-activating domain of VP16 or is a protein having a LexA-binding domain and the transcription-activating domain of VP16.