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High-Throughput Arginylation Assay in Microplate Format
Sougata Saha1, Junling Wang2, Anna S Kashina3
1Tezpur University, Napaam, Assam, India.
Methods in Molecular Biology (Clifton, N.J.)
|April 3, 2023
Summary
We developed a new biochemical assay for arginylation by Arginyltransferase 1 (ATE1). This high-throughput screening method identified two compounds affecting ATE1 activity in vitro and in vivo.
Area of Science:
- Biochemistry
- Molecular Biology
- Drug Discovery
Background:
- Arginyltransferase 1 (ATE1) plays a crucial role in protein degradation pathways.
- Understanding ATE1-mediated arginylation is vital for various biological processes.
- Existing methods for studying ATE1 activity are limited in throughput and scope.
Purpose of the Study:
- To develop a novel, high-throughput biochemical assay for ATE1-mediated arginylation.
- To identify small molecules that modulate ATE1 activity.
- To enable high-volume analysis of ATE1 substrates.
Main Methods:
- Developed a microplate-based biochemical assay for in vitro arginylation.
- The assay utilizes the N-terminal peptide of beta-actin as a model substrate for ATE1.
- Screened a library of 3280 diverse chemical compounds.
Main Results:
- Successfully established a robust and scalable assay for ATE1 activity.
- Identified two distinct compounds that specifically inhibit or activate ATE1-regulated processes.
- Demonstrated the assay's applicability for both in vitro and in vivo studies.
Conclusions:
- The developed microplate assay is suitable for high-throughput screening of ATE1 modulators.
- This assay facilitates the discovery of novel small molecules impacting ATE1 function.
- The assay can be adapted for different ATE1 substrates, broadening its utility.
