Assays for Modulators of Parkin Activity Using Novel Substrates
AssigneeElan Pharmaceuticals, Inc.
InventorsJennifer A. Johnston, Alfred Goldberg
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Abstract
The invention provides in vitro and cell-based assays for parkin activity, in which parkin-mediated ubiquitination of the S5a subunit of the 26S proteasome is measured, or ubiquitination of troponin 1. The assays may be used to screen for agents that modulate parkin protein ligase activity.
Claims28
An in vitro method for measuring parkin E3 ligase activity comprising
- (a) incubating parkin protein and S5a protein together under conditions in which the S5a protein is ubiquitinated by parkin, and
- (b) measuring the rate or extent of ubiquitination of the S5a protein.
An assay for modulators of parkin activity, said assay comprising
- (a) incubating parkin protein and S5a protein together under conditions in which the S5a protein can be ubiquitinated;
- (b) incubating parkin protein and S5a protein in the presence of a test agent together under the conditions of (a);
- (c) comparing the rate or extent of S5a ubiquitination in the presence and absence of the test agent, where a relative increase in S5a ubiquitination in the presence of the test agent indicates that the test agent is a positive modulator of parkin activity and a relative decrease in S5a ubiquitination in the presence of the test agent indicates that the test agent inhibits parkin activity.
A cell-based method for measuring parkin activity comprising
- (a) providing a mammalian cell expressing parkin and expressing S5a;
- (b) measuring the rate or extent of S5a ubiquitination.
A cell-based assay for modulators of parkin activity, said assay comprising
- (a) providing a mammalian cell expressing parkin and expressing S5a
- (b) exposing the cell to a test agent;
- (c) comparing the rate or extent of S5a ubiquitination in the presence and absence of the test agent, where a relative increase in S5a ubiquitination in the presence of the test agent indicates that the test agent is a positive modulator of parkin activity and a relative decrease in S5a ubiquitination in the presence of the test agent indicates that the test agent inhibits parkin activity.
An in vitro method for measuring parkin E3 ligase activity comprising
- (a) incubating parkin protein and troponin protein together under conditions in which the troponin protein is ubiquitinated by parkin, and
- (b) measuring the rate or extent of ubiquitination of the troponin protein.
An assay for modulators of parkin activity, said assay comprising
- (a) incubating parkin protein and troponin protein together under conditions in which the Troponin protein can be ubiquitinated;
- (b) incubating parkin protein and troponin protein in the presence of a test agent together under the conditions of (a);
- (c) comparing the rate or extent of troponin ubiquitination in the presence and absence of the test agent, where a relative increase in troponin ubiquitination in the presence of the test agent indicates that the test agent is a positive modulator of parkin activity and a relative decrease in troponin ubiquitination in the presence of the test agent indicates that the test agent inhibits parkin activity.
A cell-based method for measuring parkin activity comprising
- (a) providing a mammalian cell expressing parkin and expressing troponin;
- (b) measuring the rate or extent of troponin ubiquitination.
A cell-based assay for modulators of parkin activity, said assay comprising
- (a) providing a mammalian cell expressing parkin and expressing troponin
- (b) exposing the cell to a test agent;
- (c) comparing the rate or extent of troponin ubiquitination in the presence and absence of the test agent, where a relative increase in troponin ubiquitination in the presence of the test agent indicates that the test agent is a positive modulator of parkin activity and a relative decrease in troponin ubiquitination in the presence of the test agent indicates that the test agent inhibits parkin activity.
An in vitro method to assess the specificity of a positive modulator of parkin activity comprising
- (a) incubating an E3 ligase protein other than parkin and a parkin substrate protein together under conditions in which the substrate is ubiquitinated;
- (b) incubating the E3 ligase protein and the parkin substrate protein together in the presence of a positive modulator of parkin activity, under the conditions of (a);
- (c) comparing the ligase activity of the E3 ligase in the presence and absence of the positive modulator, where an increase in E3 ligase activity when the positive modulator is present indicates the positive modulator is not completely specific for parkin, and the absence of an increase indicates positive modulator is completely specific for parkin.
The method of claim 22 wherein the E3 ligase protein is a RING E3 ligase.
Dependencies are inferred from the claim text.