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1-Hydroxy-xanthine derivatives inhibit the human Caf1 nuclease and Caf1-containing nuclease complexes via Mg2+-dependent binding

Airhihen, Blessing; Pavanello, Lorenzo; Jadhav, Gopal P.; Fischer, Peter M.; Winkler, Gerlof Sebastiaan

1-Hydroxy-xanthine derivatives inhibit the human Caf1 nuclease and Caf1-containing nuclease complexes via Mg2+-dependent binding Thumbnail


Authors

Blessing Airhihen

Lorenzo Pavanello

Gopal P. Jadhav

Peter M. Fischer



Abstract

In eukaryotic cells, cytoplasmic mRNA is characterised by a 3’ poly(A) tail. The shortening and removal of poly(A) tails (deadenylation) by the Ccr4‐Not nuclease complex leads to reduced translational efficiency and RNA degradation. Using recombinant human Caf1 (CNOT7) enzyme as a screening tool, we recently described the discovery and synthesis of a series of substituted 1‐hydroxy‐3,7‐dihydro‐1H‐purine‐2,6‐diones (1‐hydroxy‐xanthines) as inhibitors of the Caf1 catalytic subunit of the Ccr4‐Not complex. Here, we used a chemiluminescence‐based AMP detection assay to show that active 1‐hydroxy‐xanthines inhibit both isolated Caf1 enzyme and human Caf1‐containing complexes that also contain the second nuclease subunit Ccr4 (CNOT6L) to a similar extent, indicating that the active site of the Caf1 nuclease subunit does not undergo substantial conformational change when bound to other Ccr4‐Not subunits. Using differential scanning fluorimetry, we also show that binding of active 1‐hydroxy‐xanthines requires the presence of Mg2+ ions, which are present in the active site of Caf1.

Citation

Airhihen, B., Pavanello, L., Jadhav, G. P., Fischer, P. M., & Winkler, G. S. (2019). 1-Hydroxy-xanthine derivatives inhibit the human Caf1 nuclease and Caf1-containing nuclease complexes via Mg2+-dependent binding. FEBS Open Bio, 9(4), 717-727. https://doi.org/10.1002/2211-5463.12605

Journal Article Type Article
Acceptance Date Feb 7, 2019
Online Publication Date Feb 13, 2019
Publication Date Feb 13, 2019
Deposit Date Feb 14, 2019
Publicly Available Date Mar 28, 2024
Journal FEBS Open Bio
Electronic ISSN 2211-5463
Publisher Wiley
Peer Reviewed Peer Reviewed
Volume 9
Issue 4
Pages 717-727
DOI https://doi.org/10.1002/2211-5463.12605
Keywords Ribonuclease; deadenylase; Caf1/CNOT7; Pop2; Ccr4-Not; thermal stability assay; 1-hydroxy-xanthine; Caf1 nuclease;mRNA; deadenylation; chemiluminescence-based AMP detection assay
Public URL https://nottingham-repository.worktribe.com/output/1547867
Publisher URL https://febs.onlinelibrary.wiley.com/doi/abs/10.1002/2211-5463.12605

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