Discovery and applications of the proteolytic function of N-terminal acetylation of cellular proteins
AssigneeCalifornia Institute of Technology
InventorsCheol-Sang Hwang, Anna Shemorry, Alexander Varshavsky
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Abstract
Screening assays that allow for the identification of agents that modulate the activity of N-terminal acetylation of a polypeptide and the Doa10 branch of the N-end rule pathway are provided. Also provided are methods of using an agent that modulates the activity of N-terminal acetylation of a polypeptide and the Doa10 branch of the N-end rule pathway to increase or decrease protein degradation in a cell, and to modulate physiologic and pathologic associated with N-terminal acetylation of a polypeptide and the Doa10 branch of the N-end rule pathway.
Claims19
A method of identifying an agent that modulates Nα-terminal acetyl transferase-mediated N-end rule pathway-mediated acetylation of an amino terminal (N-terminal) Met, Ala, Val, Ser, Thr, Cys, Gly, or Pro residue of a peptide, comprising:
- a) contacting at least one sample comprising a peptide having an N-terminal Met, Ala, Val, Ser, Thr, Cys, Gly, or Pro residue of the peptide, wherein the peptide comprises an N-terminal portion of a fusion protein and wherein the fusion protein comprises a reporter polypeptide C-terminal to the peptide, with at least one test agent, under conditions suitable for the N-terminal Met, Ala, Val, Ser, Thr, Cys, Gly, or Pro residue of the peptide to act as a substrate for an N-end rule pathway reaction;
- b) using mass spectroscopy or capillary electrophoresis, analyzing the results to detect a change in the N-end rule pathway substrate activity of the N-terminal Met, Ala, Val, Ser, Thr, Cys, Gly, or Pro residue of the peptide of a) in the presence of the test agent as compared to the activity in the absence of the test agent;
- wherein a change identifies the test agent as an agent that modulates Nα-terminal acetyl transferase-mediated N-end rule pathway-mediated acetylation of an amino terminal (N-terminal) Met, Ala, Val, Ser, Thr, Cys, Gly, or Pro residue of the peptide, wherein the acetylated peptide is a substrate of Doa10 E3 Ub ligase.
Dependencies are inferred from the claim text.