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 language | English (US) |
|---|---|
| Pages (from-to) | 717-727 |
| Number of pages | 11 |
| Journal | FEBS Open Bio |
| Volume | 9 |
| Issue number | 4 |
| DOIs | |
| State | Published - Apr 1 2019 |
| Externally published | Yes |
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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 journal › Article
}
TY - JOUR
T1 - 1-Hydroxy-xanthine derivatives inhibit the human Caf1 nuclease and Caf1-containing nuclease complexes via Mg 2+ -dependent binding
AU - Airhihen, Blessing
AU - Pavanello, Lorenzo
AU - Jadhav, Gopal
AU - Fischer, Peter M.
AU - Winkler, Gerlof Sebastiaan
PY - 2019/4/1
Y1 - 2019/4/1
N2 - 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.
AB - 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.
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U2 - 10.1002/2211-5463.12605
DO - 10.1002/2211-5463.12605
M3 - Article
VL - 9
SP - 717
EP - 727
JO - FEBS Open Bio
JF - FEBS Open Bio
SN - 2211-5463
IS - 4
ER -