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

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 12, 2019
• 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
• Contract Date: Feb 14, 2019