Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597.
J Mol Biol. 2011 Oct 21;413(2):430-7. doi: 10.1016/j.jmb.2011.08.030. Epub 2011 Aug 29.
Cycle inhibiting factor (Cif) is produced by pathogenic intracellular bacteria and injected into the host cells via a type III secretion system. Cif is known to interfere with the eukaryotic cell cycle by inhibiting the function of cullin RING E3 ubiquitin ligases (CRLs). Cullin proteins form the scaffold protein of CRLs and are modified with the ubiquitin-like protein Nedd8, which exerts important conformational control required for CRL activity. Cif has recently been shown to catalyze the deamidation of Gln40 in Nedd8 to Glu. Here, we addressed how Nedd8 deamidation inhibits CRL activity. Our results indicate that Burkholderia pseudomallei Cif (also known as CHBP) inhibits the deconjugation of Nedd8 in vivo by inhibiting binding of the deneddylating COP9 signalosome (CSN) complex. We provide evidence that the reduced binding of CSN and the inhibition of CRL activity by Cif are due to interference with Nedd8-induced conformational control, which is dependent on the interaction between the Nedd8 hydrophobic patch and the cullin winged-helix B subdomain. Of note, mutation of Gln40 to Glu in ubiquitin, an additional target of Cif, inhibits the interaction between the hydrophobic surface of ubiquitin and the ubiquitin-binding protein p62/SQSTM1, showing conceptually that Cif activity can impair ubiquitin/ubiquitin-like protein non-covalent interactions. Our results also suggest that Cif may exert additional cellular effects by interfering with the association between ubiquitin and ubiquitin-binding proteins.
周期抑制因子（Cif）由致病性胞内细菌产生，并通过 III 型分泌系统注入宿主细胞。已知 Cif 通过抑制细胞周期蛋白 RING E3 泛素连接酶（CRLs）的功能来干扰真核细胞周期。Cullin 蛋白构成 CRLs 的支架蛋白，并被泛素样蛋白 Nedd8 修饰，Nedd8 对 CRL 活性所需的重要构象控制发挥作用。最近的研究表明，Cif 催化 Nedd8 中 Gln40 的脱酰胺作用，形成 Glu。在这里，我们研究了 Nedd8 脱酰胺作用如何抑制 CRL 活性。我们的结果表明，伯克霍尔德氏菌假单胞菌 Cif（也称为 CHBP）通过抑制去泛素化 COP9 信号小体（CSN）复合物的结合，抑制体内 Nedd8 的去缀合。我们提供的证据表明，CSN 的结合减少和 Cif 对 CRL 活性的抑制是由于干扰 Nedd8 诱导的构象控制，这取决于 Nedd8 疏水区与 Cullin 翅膀螺旋 B 亚基的相互作用。值得注意的是，在泛素（Cif 的另一个靶标）中，将 Gln40 突变为 Glu 会抑制泛素与泛素结合蛋白 p62/SQSTM1 之间的相互作用，这表明 Cif 活性可以损害泛素/泛素样蛋白的非共价相互作用。我们的结果还表明，Cif 通过干扰泛素与泛素结合蛋白之间的关联，可能会对细胞产生额外的影响。
