Assays for modulators of parkin activity using S5a as a parkin substrate
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.
1. An assay to determine whether a test agent is a modulator of parkin activity, said assay comprising
(a) incubating parkin protein and S5a protein together under conditions in which the S5a protein can be ubiquitinated, wherein said incubating is in the absence of the test agent, and determining the rate or extent of S5a ubiquitination;
(b) incubating parkin protein and S5a protein together in the presence of the test agent under the conditions of (a), and determining the rate or extent of S5a ubiquitination;
(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 is a negative modulator (inhibitor) of parkin activity.
2. The assay of claim 1 wherein the parkin and S5a are human.
3. An assay according to claim 1 , wherein step (a) comprises incubating a mammalian cell expressing parkin and expressing S5a under conditions in which the S5a protein can be ubiquitinated, wherein said incubating is in the absence of the test agent, and determining the rate or extent of S5a ubiquitination in the cell: and
wherein step (b) comprises incubating a mammalian cell expressing parkin and expressing S5a in the presence of the test agent under the conditions of (a), and determining the rate or extent of S5a ubiquitination in the cell.
4. The assay of claim 3 wherein at least one of the parkin and S5a is heterologous to the cell.
5. A method to assess the specificity of a positive modulator of parkin activity comprising identifying a test agent as a positive modulator of parkin activity according to the method of claim 1 , and then
(d) incubating in vitro an E3 ligase protein other than parkin and S5a together in the absence of the positive modulator under conditions in which the S5a is ubiquitinated;
(e) incubating the E3 ligase protein and the S5a together in the presence of the positive modulator of parkin activity, under the conditions of (d);
(f) 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.
6. The assay of claim 5 wherein an increase in substrate ubiquitination in the presence of the positive modulator indicates the positive modulator is not completely specific for parkin, but the positive modulator is determined to be partially specific,
wherein partial specificity is defined as an EC 10 for the non-parkin E3 that is not more than 100 micromolar and is at least 4-fold higher than the EC 10 for parkin.
7. The method of claim 5 wherein the E3 ligase protein is a RING E3 ligase.
8. The method of claim 5 wherein the E3 ligase protein is selected from the group consisting of Mdm2, Nedd4, Murf1, and E6AP.