1‐Hydroxy‐xanthine derivatives inhibit the human Caf1 nuclease and Caf1‐containing nuclease complexes via Mg2+‐dependent binding
1‐Hydroxy‐xanthine derivatives inhibit the human Caf1 nuclease and Caf1‐containing nuclease complexes via Mg2+‐dependent binding
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DOI：
10.1002/2211-5463.12605
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发表时间：
2019-03
期刊：
影响因子：
2.6
通讯作者：
B. Airhihen;Lorenzo Pavanello;Gopal P Jadhav;P. Fischer;G. Winkler
中科院分区：
文献类型：
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作者：
B. Airhihen;Lorenzo Pavanello;Gopal P Jadhav;P. Fischer;G. Winkler
在真核细胞中，胞质mRNA的特征在于3′ poly（A）尾。Ccr 4-Not核酸酶复合物缩短和去除poly（A）尾（去腺苷酸化）导致翻译效率降低和RNA降解。使用重组人Caf 1（hocT 7）酶作为筛选工具，我们最近描述了一系列取代的1-羟基-3，7-二氢-1H-嘌呤-2，6-二酮（1-羟基-黄嘌呤）的发现和合成，作为Ccr 4-Not复合物的Caf 1催化亚基的抑制剂。在这里，我们使用了基于化学发光的AMP检测试验，以表明活性1-羟基黄嘌呤抑制分离的Caf 1酶和人Caf 1-含有复合物，也含有第二个核酸酶亚基Ccr 4（hocT 6L）在相似的程度，表明Caf 1核酸酶亚基的活性位点在与其他Ccr 4-Not亚基结合时不会发生实质性构象变化。使用差示扫描荧光法，我们还表明，活性1-羟基-黄嘌呤的结合需要Mg 2+离子的存在下，这是目前在活性位点的咖啡因。
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.