Deconjugation of Nedd8 from Cul1 is directly regulated by Skp1-F-box and substrate, and the COP9 signalosome inhibits deneddylated SCF by a noncatalytic mechanism.
Deconjugation of Nedd8 from Cul1 is directly regulated by Skp1-F-box and substrate, and the COP9 signalosome inhibits deneddylated SCF by a noncatalytic mechanism.
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DOI：
10.1074/jbc.m112.352484
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发表时间：
2012-08-24
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影响因子：
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通讯作者：
Deshaies RJ
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文献类型：
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作者：
Emberley ED;Mosadeghi R;Deshaies RJ
背景：目前还缺乏对CSN对cullin脱羧动力学的详细描述。结果：所选因子和SCF亚基均能不同程度地抑制去皮化。CSN通过非催化机制干扰scf介导的泛素化。结论：CSN对Cul1的去角质作用受F-box蛋白、底物等因素的调控。意义：我们在这里报道的工作可以促进定向治疗的发展。COP9信号体（CSN）介导SCF cullin亚基和其他cullin- ring泛素连接酶（CRLs）的泛素样蛋白Nedd8的解偶联。这一过程对于维持细胞中crl的正常活性至关重要。在这里，我们报告了csn介导的SCF中Nedd8的解偶联的详细动力学表征。CSN是一种高效酶，kcat为~ 1 s−1，而类木化Cul1-Rbx1的kcat为~ 200 nm， kcat/Km接近预期的扩散控制极限。与F-box-Skp1复合物的组装显著抑制了去甲酰基化，尽管幅度差异很大，Fbw7-Skp1抑制了约5倍，而Skp2-Cks1-Skp1仅抑制了约15%。通过添加底物，SCFFbw7和SCFSkp2-Cks1的去毛化进一步被抑制约2.5倍。Fbw7-Skp1及其底物cyclin E的联合抑制作用大于10倍。出乎意料的是，我们的研究结果还揭示了去偶联Cul1对产物的显著抑制作用，这是由于Cul1与CSN紧密结合的能力。反过来，CSN抑制去醛化Cul1的泛素连接酶活性。我们提出了一种模型，其中组装的CRL配合物与底物接合通常难以去基化。在消耗底物和随后的去基化后，CSN可以稳定地与CRL结合，并将其保持在低活性状态。
Background: A detailed description of the kinetics of deneddylation of cullin by CSN has been lacking. Results: Selected factors and SCF subunits are able to inhibit deneddylation to varying degrees. CSN interferes with SCF-mediated ubiquitination through a noncatalytic mechanism. Conclusion: Deneddylation of Cul1 by CSN is regulated by F-box protein, substrate, and other factors. Significance: Our work reported here could facilitate the development of directed therapies. COP9 signalosome (CSN) mediates deconjugation of the ubiquitin-like protein Nedd8 from the cullin subunits of SCF and other cullin-RING ubiquitin ligases (CRLs). This process is essential to maintain the proper activity of CRLs in cells. Here, we report a detailed kinetic characterization of CSN-mediated deconjugation of Nedd8 from SCF. CSN is an efficient enzyme, with a kcat of ∼1 s−1 and Kmfor neddylated Cul1-Rbx1 of ∼200 nm, yielding a kcat/Km near the anticipated diffusion-controlled limit. Assembly with an F-box-Skp1 complex markedly inhibited deneddylation, although the magnitude varied considerably, with Fbw7-Skp1 inhibiting by ∼5-fold but Skp2-Cks1-Skp1 by only ∼15%. Deneddylation of both SCFFbw7 and SCFSkp2-Cks1 was further inhibited ∼2.5-fold by the addition of substrate. Combined, the inhibition by Fbw7-Skp1 plus its substrate cyclin E was greater than 10-fold. Unexpectedly, our results also uncover significant product inhibition by deconjugated Cul1, which results from the ability of Cul1 to bind tightly to CSN. Reciprocally, CSN inhibits the ubiquitin ligase activity of deneddylated Cul1. We propose a model in which assembled CRL complexes engaged with substrate are normally refractory to deneddylation. Upon consumption of substrate and subsequent deneddylation, CSN can remain stably bound to the CRL and hold it in low state of reduced activity.