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High-Throughput Arginylation Assay in Microplate Format

  • Sougata Saha
  • Junling Wang
  • Anna S. KashinaEmail author
Protocol
  • 1.6k Downloads
Part of the Methods in Molecular Biology book series (MIMB, volume 1337)

Abstract

Here we describe the biochemical assay for ATE1-mediated arginylation in microplate format, which can be applied to high-throughput screens for identification of small-molecule inhibitors and activators of ATE1, high-volume analysis of ATE1 substrates, and other similar applications. Originally, we have applied this screen to a library of 3280 compounds and identified two 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.

Key words

ATE1 inhibitors Arginylation Tannic acid Cell motility Angiogenesis 

References

  1. 1.
    Saha S, Wang J, Buckley B, Wang Q, Lilly B, Chernov M, Kashina A (2012) Small molecule inhibitors of arginyltransferase regulate arginylation-dependent protein degradation, cell motility, and angiogenesis. Biochem Pharmacol 83(7):866–873PubMedCentralCrossRefPubMedGoogle Scholar
  2. 2.
    Wang J, Han X, Saha S, Xu T, Rai R, Zhang F, Wolf YI, Wolfson A, Yates JR III, Kashina A (2011) Arginyltransferase is an ATP-independent self-regulating enzyme that forms distinct functional complexes in vivo. Chem Biol 18(1):121–130PubMedCentralCrossRefPubMedGoogle Scholar
  3. 3.
    Wang J, Han X, Wong CC, Cheng H, Aslanian A, Xu T, Leavis P, Roder H, Hedstrom L, Yates JR III, Kashina A (2014) Arginyltransferase ATE1 catalyzes midchain arginylation of proteins at side chain carboxylates in vivo. Chem Biol 21(3):331–337PubMedCentralCrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of Molecular Biology and BiotechnologyTezpur UniversitySonitpurIndia
  2. 2.Department of Animal BiologyUniversity of PennsylvaniaPhiladelphiaUSA
  3. 3.Department of Animal Biology, School of Veterinary MedicineUniversity of PennsylvaniaPhiladelphiaUSA

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