Characterization of a Non-UBA Domain Missense Mutation of Sequestosome 1 (SQSTM1) in Paget's Disease of Bone
Characterization of a Non-UBA Domain Missense Mutation of Sequestosome 1 (SQSTM1) in Paget's Disease of Bone
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DOI：
10.1359/jbmr.081204
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发表时间：
2009-04-01
影响因子：
6.2
通讯作者：
Layfield, Robert
中科院分区：
文献类型：
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作者：
Najat, Dereen;Garner, Thomas;Layfield, Robert
影响隔离体1泛素相关结构域(SQSTM1/p62)的突变常见于Paget病，并削弱SQSTM1的S结合泛素的能力，导致核转录因子-kappaB信号失控。相比之下，非UBA结构域突变更罕见，人们对它们如何表现其影响知之甚少。我们首次在分子、细胞和功能水平上对SQSTM1的一个非UBA结构域错义突变(A381V)进行了表征。对所有意大利PDB患者的SQSTM1基因外显子7进行直接测序，在1182位检测到杂合子C到T颠换，导致密码子381处所有丙氨酸到Valine替换。下拉实验显示了SQSTM1的非UBA区域。A381在介导泛素结合亲和力中起重要作用，A381V突变对泛素结合有微弱的负面影响。利用核磁共振波谱和圆二色谱对含有A381的较长UBA结构进行结构和结合分析，表明该蛋白质的这一区域基本上是非结构的，并证实了它对提高泛素结合亲和力的贡献。U20S细胞共转染实验表明，A381V突变体SQSTM1与泛素共定位，细胞表型与野生型难以区分。最后，在HEK293细胞中检测野生型和突变型SQSTM1对NF-kappa B信号转导的影响。A381V突变体SQSTM1对NF-kappa B信号的激活程度高于野生型，与UBA结构域突变相似，表明非UBA结构域突变和UBA结构域突变可能通过参与NF-kappa B信号失控的共同机制发挥作用。
Mutations affecting the ubiquitin-associated (UBA) domain of sequestosome 1 (SQSTM1/p62) are commonly found in Paget's disease of bone (PDB) and impair SQSTM1's ability to bind ubiquitin, resulting in dysregulated NF-kappa B signaling. In contrast, non-UBA domain mutations are rarer, and little is known about how they manifest their effects. We present the first characterization at the molecular, cellular, and functional level of a non-UBA domain missense mutation (A381V) of SQSTM1. Direct sequencing of exon 7 of the SQSTM1 gene in all Italian PDB patient detected a heterozygous C to T transversion at position 1182, resulting in all alanine to valine substitution at codon 381. Pull-down assays showed the non-UBA region of SQSTM1. that contains A381 is important in mediating ubiquitin-binding affinity and that the A381V mutation exerts weak negative effects on ubiquitin binding. Structural and binding analyses of longer UBA constructs containing A381, using NMR spectroscopy and circular dichroism, showed this region of the protein to be largely unstructured and confirmed its contribution to increased ubiquitin-binding, affinity. Co-transfections of U20S cells showed that the A381V mutant SQSTM1 co-localized with ubiquitin with a cellular phenotype indistinguishable from wildtype. Finally, effects of the wildtype and mutant SQSTM1 on NF-kappa B signaling were assessed in HEK293 cells co-transfected with an NF-kappa B luciferase reporter construct. A381V mutant SQSTM1 produced a level of activation of NF-kappa B signaling greater than wildtype and similar to that of UBA domain mutants, indicating, that non-UBA and UBA domain mutations may exert their effects through a common mechanism involving dysregulated NF-kappa B signaling.