Parkin Substrate and Assay
AssigneeElan Pharmaceuticals, Inc.
InventorsJennifer A. Johnston, Colleen Tsui Cutcliffe
Life of this patent
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Abstract
The invention provides in vitro, ex vivo, and in vivo assays for Parkin activity, in which Parkin-mediated ubiquitination of a Sept4 protein is measured. The assays may be used to screen for agents that modulate Parkin protein ligase activity.
Claims24
An method for detecting Parkin activity comprising combining a Parkin protein and a Septin-4 protein under conditions in which the Sept4 protein is ubiquitinated by the Parkin protein ligase activity, and measuring the presence, rate or extent of ubiquitination of the Sept4 protein.
An assay for modulators of Parkin activity, said assay comprising
- (1) incubating Parkin protein and Septin-4 protein together in vitro under conditions in which the Septin-4 protein can be ubiquitinated;
- (2) incubating Parkin protein and Septin-4 protein in the presence of a test agent together under the conditions of (1);
- (3) comparing the rate or extent of Septin-4 ubiquitination in the presence of the test agent with the rate or extent of Sept4 ubiquitination in the absence of the test agent, where a relative increase in Septin-4 ubiquitination in the presence of the test agent indicates that the test agent enhances Parkin ubiquitination activity and a relative decrease in Sept4 ubiquitination in the presence of the test agent indicates that the test agent inhibits Parkin ubiquitination activity.
A cell-based method for measuring Parkin activity comprising
- (a) providing a mammalian cell expressing Parkin and expressing Sept4;
- (b) measuring the rate or extent of Sept4 ubiquitination in the cell.
A ex vivo cell-based assay for modulators of Parkin activity, said assay comprising
- (a) providing a mammalian cell expressing Parkin and expressing Sept4;
- (b) exposing the cell to a test agent;
- (c) comparing the rate or extent of Sept4 ubiquitination in the presence of the test agent with the rate or extent of Sept4 ubiquitination in a control cell not exposed to the test agent, where a relative increase in Sept4 ubiquitination in the presence of the test agent indicates that the test agent enhances Parkin ubiquitination activity and a relative decrease in Sept4 ubiquitination in the presence of the test agent indicates that the test agent inhibits Parkin ubiquitination activity.
A method for identifying modulators of Parkin activity, said method comprising
- (a) providing a non-human animal co-expressing Parkin and Sept4 in neuronal cells;
- (b) administering a test agent to the animal;
- (c) comparing the rate or extent of Sept4 ubiquitination in said neuronal cells of the animal in to which the test agent was administered with the rate or extent of Sept4 ubiquitination in a control animal not administered the agent, wherein a relative increase in Sept4 ubiquitination in the presence of the therapeutic agent indicates that the therapeutic agent enhances Parkin ubiquitination activity and a relative decrease in Sept4 ubiquitination in the presence of the therapeutic agent indicates that the therapeutic agent inhibits Parkin ubiquitination activity.
Dependencies are inferred from the claim text.