Methods and compositions for gamma-secretase assay
Presented herein are polypeptide substrates based on Notch polypeptides, assay methods based on the use of these substrates, and screening methods directed toward identifying inhibitors of γ-secretase activity. The assay methods and the screening methods are adapted for use in high throughput multi-well plate assay apparatuses. In many embodiments the substrate polypeptides are labeled for ease of detection, and/or may bind specific ligands that themselves are labeled. Generally the labels promote high specificity as well as high sensitivity of detection. These features render the assay and screening methods that employ the labeled substrates especially suited for use in high throughput assay formats.
1. A method of assaying the activity of γ-secretase comprising:
providing a composition suspected of containing γ-secretase activity;
contacting the composition with a polypeptide substrate for γ-secretase comprising a Notch substrate, wherein the Notch substrate comprises a fragment of a Notch protein, wherein the fragment comprises an amino acid sequence consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 10, or SEQ ID NO: 13, wherein the fragment comprises an S3 cleavage site and an amino acid sequence VLLSRKRRR (SEQ ID NO:2) adjacent to the S3 cleavage site and wherein the fragment comprises an amino acid sequence bound to a biotin label located on the C-terminal side of the S3 site, and wherein γ-secretase cleaves the Notch substrate at the S3 cleavage site to provide a detectably labeled product comprising an amino acid sequence having the biotin at the C-terminal side and the sequence VLLSRKRRR (SEQ ID NO: 2) at the N-terminal side that is specifically recognized by a second antibody;
contacting the detectably labeled product with
a first ligand comprising a first tag wherein the first ligand specifically binds the biotin label of the detectably labeled product, and
the second antibody comprising a second tag wherein the second antibody specifically binds the VLLSRKRRR (SEQ ID NO: 2) epitope at the N-terminal side of the detectably labeled product, wherein the second antibody is raised against the polypeptide sequence VLLSRKRRR (SEQ ID NO:2) and the second antibody binds the γ-secretase cleaved detectably labeled product but not the uncleaved notch substrate; and
determining the presence, the amount, or the combination thereof of the detectably labeled product bound to the first ligand and to the second antibody.
2. The method of claim 1 wherein the contacting occurs in a container.
3. The method of claim 2 wherein the method is a high throughput assay method conducted in a plurality of containers.
4. The method of claim 3 wherein each container is a well in a multi-well assay plate, thereby providing a high throughput method of assaying.
5. The method of claim 1 wherein the first ligand comprises avidin and the first tag comprises a detectable fluorescence acceptor.
6. The method of claim 5 wherein the second antibody specifically binds the Notch substrate after cleavage by the γ-secretase and does not specifically bind the Notch substrate before cleavage by the γ-secretase, the second antibody being bound to a third antibody bearing a fluorescence donor that excites the fluorescence acceptor tag bound to the first ligand.
7. A method of assaying the activity of γ-secretase in a cell comprising:
providing cells suspected of harboring γ-secretase activity;
contacting the cells with media comprising a polypeptide substrate for γ-secretase wherein the substrate comprises a Notch substrate, wherein cleavage of the Notch substrate by the γ-secretase provides a detectably labeled product, wherein the Notch substrate comprises a fragment of a Notch protein, wherein the fragment comprises an amino acid sequence consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:13, wherein the fragment comprises an S3 cleavage site and an amino acid sequence VLLSRKRRR (SEQ ID NO:2) adjacent to the S3 cleavage site and wherein the fragment comprises an amino acid sequence bound to a biotin label located on the C-terminal side of the S3 cleavage site; wherein the γ-secretase cleaves the Notch substrate at the S3 cleavage site to provide the detectably labeled product comprising an amino acid sequence having the biotin at the C-terminal side and the sequence VLLSRKRRR (SEQ ID NO: 2) at the N-terminal side that is specifically recognized by a second antibody raised using the polypeptide VLLSRKRR (SEQ ID NO: 2) and the second antibody binds the γ-secretase cleaved detectably labeled product but not the uncleaved notch substrate; and
determining the presence of the detectably labeled product using a first ligand that specifically binds the biotin and the second antibody.
8. The method of claim 7 wherein the determining comprises:
separating the cells from the media after a suitable incubation period with the polypeptide substrate for γ-secretase to provide a supernatant; and determining the presence of the detectably labeled product in the supernatant.
9. The method of claim 8 wherein the determining the presence of the detectably labeled product comprises:
contacting the detectably labeled product with
the first ligand comprising a first tag wherein the first ligand specifically binds the biotin label, and
the second antibody comprising a second tag; and
determining the presence, the amount, or the combination thereof of the labeled product bound to the first ligand and to the second antibody.
10. The method of claim 7 wherein the media further comprises a detergent.
11. The method of claim 9 , wherein the first tag comprises a detectable probe.
12. The method of claim 11 wherein the detectable probe comprises ruthenium.
13. The method of claim 7 wherein the contacting occurs in a container.
14. The method of claim 13 wherein the method is a high throughput assay method conducted in a plurality of containers.
15. The method of claim 14 wherein each container is a well in a multi-well assay plate, thereby providing a high throughput method of assaying.
16. The method of claim 9 wherein the first ligand comprises avidin and the first tag comprises a detectable fluorescence acceptor.
17. The method of claim 1 wherein the fragment of the Notch protein consists of the amino acid sequence SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:13.
18. The method of claim 7 wherein the fragment of the Notch protein consists of the amino acid sequence SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:13.