Dimerisation of the UBA Domain of p62 Inhibits Ubiquitin Binding and Regulates NF-κB Signalling
Dimerisation of the UBA Domain of p62 Inhibits Ubiquitin Binding and Regulates NF-κB Signalling
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DOI：
10.1016/j.jmb.2009.11.032
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发表时间：
2010-02-12
影响因子：
5.6
通讯作者：
Searle, Mark S.
中科院分区：
文献类型：
--
作者：
Long, Jed;Garner, Thomas P.;Searle, Mark S.
泛素（Ub）结合的p62支架蛋白（由SQSTM1基因编码）调节多种信号通路，导致核因子κ B （nf - κ B）家族转录因子的激活，是巨噬的重要调节因子。编码p62基因的突变常见于骨佩吉特病患者，并且主要聚集在c端泛素相关（UBA）结构域，损害其结合Ub的能力，导致nf - κ B信号传导失调。然而，ub结合在分子水平上是如何调控的尚不清楚。核磁共振弛豫色散实验，结合浓度依赖性核磁共振、CD、等温滴定量热法和荧光动力学测量，表明p62 UBA结构域在298 K下形成高度稳定的二聚体（K-dim类似于4-12 μ M）。核磁共振分析表明，二聚体界面部分遮挡了ub结合表面，特别是在螺旋3的c端，使UBA二聚化和ub结合过程相互排斥。有些不寻常的是，单体UBA似乎是生物活性形式，而二聚体似乎是无活性形式。环1中的工程点突变（E409K和G410K）被证明破坏了二聚体界面的稳定性，导致更高比例的结合单体，并且在NF-kappa B荧光素酶报告基因检测中，与wt-p62相比，与NF-kappa B活性降低有关。2009爱思唯尔有限公司版权所有。
The ubiquitin (Ub)-binding p62 scaffold protein (encoded by the SQSTM1 gene) regulates a diverse range of signalling pathways leading to activation of the nuclear factor kappa B (NF-kappa B) family of transcription factors and is an important regulator of macroautophagy. Mutations within the gene encoding p62 are commonly found in patients with Paget's disease of bone and largely cluster within the C-terminal ubiquitin-associated (UBA) domain, impairing its ability to bind Ub, resulting in dysregulated NF-kappa B signalling. However, precisely how Ub-binding is regulated at the molecular level is unclear. NMR relaxation dispersion experiments, coupled with concentration-dependent NMR, CD, isothermal titration calorimetry and fluorescence kinetic measurements, reveal that the p62 UBA domain forms a highly stable dimer (K-dim similar to 4-12 mu M at 298 K). NMR analysis shows that the dimer interface partially occludes the Ub-binding surface, particularly at the C-terminus of helix 3, making UBA dimerisation and Ub-binding mutually exclusive processes. Somewhat unusually, the monomeric UBA appears to be the biologically active form and the dimer appears to be the inactive one. Engineered point mutations in loop 1 (E409K and G410K) are shown to destabilise the dimer interface, lead to a higher proportion of the bound monomer and, in NF-kappa B luciferase reporter assays, are associated with reduced NF-kappa B activity compared with wt-p62. (C) 2009 Elsevier Ltd. All rights reserved.