1-Hydroxy-xanthine derivatives inhibit the human Caf1 nuclease and Caf1-containing nuclease complexes via Mg 2+ -dependent binding

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

Research output: Contribution to journalArticle

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 Mg 2+ ions, which are present in the active site of Caf1.

Original languageEnglish (US)
Pages (from-to)717-727
Number of pages11
JournalFEBS Open Bio
Volume9
Issue number4
DOIs
StatePublished - Apr 1 2019
Externally publishedYes

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Xanthines
Xanthine
Catalytic Domain
Derivatives
Fluorometry
Messenger RNA
Chemiluminescence
RNA Stability
Eukaryotic Cells
Enzymes
Adenosine Monophosphate
Luminescence
Tail
Assays
Screening
RNA
Ions
Scanning
Degradation

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

1-Hydroxy-xanthine derivatives inhibit the human Caf1 nuclease and Caf1-containing nuclease complexes via Mg 2+ -dependent binding . / Airhihen, Blessing; Pavanello, Lorenzo; Jadhav, Gopal; Fischer, Peter M.; Winkler, Gerlof Sebastiaan.

In: FEBS Open Bio, Vol. 9, No. 4, 01.04.2019, p. 717-727.

Research output: Contribution to journalArticle

Airhihen, Blessing ; Pavanello, Lorenzo ; Jadhav, Gopal ; Fischer, Peter M. ; Winkler, Gerlof Sebastiaan. / 1-Hydroxy-xanthine derivatives inhibit the human Caf1 nuclease and Caf1-containing nuclease complexes via Mg 2+ -dependent binding In: FEBS Open Bio. 2019 ; Vol. 9, No. 4. pp. 717-727.
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