Method of identifying compounds affecting hedgehog cholesterol transfer

• US 6,057,091 A
• Filed: 10/07/1997
• Issued: 05/02/2000
• Est. Priority Date: 12/02/1994
• Status: Expired due to Term

• Current Assignee: Johns Hopkins University
• Original Assignee: Johns Hopkins University
• Inventors: Beachy, Philip A., Porter, Jeffrey A.
• Primary Examiner(s): Spector, Lorraine
• Assistant Examiner(s): Kaufman, Claire M.
• Application Number:
  US08/946,329
• Time in Patent Office: 938 Days
• Field of Search: 435/7.1, 435/7.2, 435/4, 514/2, 514/1, 424/600, 530/350, 530/300, 436/71
• US Class Current: 435/4
• CPC Class Codes: C07K 2319/21 containing a His-tag
• CPC Class Codes: C07K 2319/23 containing a GST-tag
• CPC Class Codes: C07K 2319/50 containing protease site
• CPC Class Codes: G01N 2500/10 involving cells
• CPC Class Codes: G01N 33/92 involving lipids, e.g. cholesterol , lipoproteins, or their receptors steroid hormones G01N33/743

Abstract

The present invention provides two novel polypeptides, referred to as the "N" and "C" fragments of hedgehog, or N-terminal and C-terminal fragments, respectively, which are derived after specific cleavage at a G.sup.↓ CF site recognized by the autoproteolytic domain in the native protein. Also included are sterol-modified hedgehog polypeptides and functional fragments thereof. Methods of identifying compositions which affect hedgehog activity based on inhibition of cholesterol modification of hedgehog protein are described.

Claims

1. A method for identifying a compound that affects cholesterol transfer from the COOH-terminal half of a hedgehog polypeptide to the COOH-end of the NH2-terminal half of the hedgehog polypeptide in a cell, said method comprising contacting a cell comprising a hedgehog polypeptide, or a fragment thereof which is functional for cholesterol transfer, with a test compound, and comparing the amount of cholesterol transferred in the presence and absence of the compound, wherein a difference is indicative of a compound that affects cholesterol transfer.

2. The method of claim 1, wherein the compound is selected from the group consisting of:

peptides, peptidomimetics, polypeptides, and chemical compounds.

3. The method of claim 1, wherein the compound inhibits cholesterol transfer.

4. The method of claim 1, wherein the compound stimulates cholesterol transfer.