Paget disease of bone-associated UBA domain mutations of SQSTM1 exert distinct effects on protein structure and function.
Paget disease of bone-associated UBA domain mutations of SQSTM1 exert distinct effects on protein structure and function.
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DOI：
10.1016/j.bbadis.2014.03.006
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发表时间：
2014-07
影响因子：
6.2
通讯作者：
Layfield, Robert
中科院分区：
文献类型：
--
作者：
Goode, Alice;Long, Jed E.;Shaw, Barry;Ralston, Stuart H.;Visconti, Micaela Rios;Gianfrancesco, Fernando;Esposito, Teresa;Gennari, Luigi;Merlotti, Daniela;Rendina, Domenico;Rea, Sarah L.;Sultana, Melanie;Searle, Mark S.;Layfield, Robert
SQSTM1 突变在佩吉特骨病 (PDB) 患者中很常见，其中大多数影响 SQSTM1 蛋白的 C 端泛素相关 (UBA) 结构域。我们对两个 UBA 结构域突变进行了结构和功能分析，其中一种是英国 PDB 患者中相对常见的 I424S 突变，以及与意大利南部患者的严重表型相关的 A427D 突变。在 Pull-down 实验中，两者都会损害 SQSTM1 的泛素结合功能，并在报告基因实验中，与野生型相比，导致基础 NF-κB 信号传导的激活。我们在来自英国和意大利的 1152 名 PDB 患者中发现了不同 UBA 结构域突变体体外激活 NF-κB 信号传导的能力与体内受影响位点数量之间存在关系的证据，其中 A427D-SQSTM1 在迄今为止测试的所有 PDB 突变体中产生最高水平的激活（相对于野生型）。 NMR 和等温滴定量热法研究能够证明 I424S 与 UBA 结构域的整体结构变化相关，导致 UBA 二聚体稳定性比野生型弱 10 倍，并且 UBA 单体的泛素结合亲和力降低。我们的观察提供了对 SQSTM1 介导的 NF-κB 信号在 PDB 病因学中的作用的见解，并证明 SQSTM1 UBA 结构域的环 2/螺旋 3 内的不同突变对蛋白质结构和稳定性产生不同的影响，包括对 UBA/泛素结合界面的间接影响。 I424S 和 A427D SQSTM1 突变影响泛素结合和 NF-κB 信号传导。突变体SQSTM1激活NF-κB信号传导的能力可能与PDB的疾病程度有关。 A427D-SQSTM1 在所有 PDB 突变体中产生最大的激活（相对于野生型）。 I424S 突变体使 UBA 二聚体不稳定，导致单体解折叠。 SQSTM1 的 UBA 结构域突变对蛋白质结构和稳定性产生明显影响。
SQSTM1 mutations are common in patients with Paget disease of bone (PDB), with most affecting the C-terminal ubiquitin-associated (UBA) domain of the SQSTM1 protein. We performed structural and functional analyses of two UBA domain mutations, an I424S mutation relatively common in UK PDB patients, and an A427D mutation associated with a severe phenotype in Southern Italian patients. Both impaired SQSTM1's ubiquitin-binding function in pull-down assays and resulted in activation of basal NF-κB signalling, compared to wild-type, in reporter assays. We found evidence for a relationship between the ability of different UBA domain mutants to activate NF-κB signalling in vitro and number of affected sites in vivo in 1152 PDB patients from the UK and Italy, with A427D-SQSTM1 producing the greatest level of activation (relative to wild-type) of all PDB mutants tested to date. NMR and isothermal titration calorimetry studies were able to demonstrate that I424S is associated with global structural changes in the UBA domain, resulting in 10-fold weaker UBA dimer stability than wild-type and reduced ubiquitin-binding affinity of the UBA monomer. Our observations provide insights into the role of SQSTM1-mediated NF-κB signalling in PDB aetiology, and demonstrate that different mutations in close proximity within loop 2/helix 3 of the SQSTM1 UBA domain exert distinct effects on protein structure and stability, including indirect effects at the UBA/ubiquitin-binding interface. The I424S and A427D SQSTM1 mutations affect ubiquitin-binding and NF-κB signalling. Mutant SQSTM1's ability to activate NF-κB signalling may be related to disease extent in PDB. A427D-SQSTM1 produces the greatest activation (relative to wild-type) of all PDB mutants. The I424S mutant destabilises the UBA dimer causing unfolding of the monomer. UBA domain mutations of SQSTM1 exert distinct effects on protein structure and stability.
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