High-Throughput Arginylation Assay in Microplate Format

Sougata Saha; Junling Wang; Anna S Kashina

doi:10.1007/978-1-0716-2942-0_16

High-Throughput Arginylation Assay in Microplate Format

Methods Mol Biol. 2023:2620:119-122. doi: 10.1007/978-1-0716-2942-0_16.

Authors

Sougata Saha¹, Junling Wang², Anna S Kashina³

Affiliations

¹ Tezpur University, Napaam, Assam, India.
² University of Pennsylvania, Philadelphia, PA, USA.
³ Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA. akashina@upenn.edu.

PMID: 37010757
DOI: 10.1007/978-1-0716-2942-0_16

Abstract

Here, we describe the biochemical assay for ATE1-mediated arginylation in microplate format, which can be applied to high-throughput screens for the identification of small molecule inhibitors and activators of ATE1, high-volume analysis of AE1 substrates, and other similar applications. Originally, we have applied this screen to a library of 3280 compounds and identified 2 compounds which specifically affect ATE1-regulated processes in vitro and in vivo. The assay is based on in vitro ATE1-mediated arginylation of beta-actin's N-terminal peptide, but it can also be applied using other ATE1 substrates.

Keywords: ATE1 inhibitors; Angiogenesis; Arginylation; Cell motility; Tannic acid.

MeSH terms

Aminoacyltransferases* / chemistry
Arginine / metabolism
High-Throughput Screening Assays
Protein Processing, Post-Translational*

Substances

Aminoacyltransferases
Arginine