Targeting nuclear import shuttles, importins/karyopherins alpha by a peptide mimicking the NFκB1/p50 nuclear localization sequence.
Targeting nuclear import shuttles, importins/karyopherins alpha by a peptide mimicking the NFκB1/p50 nuclear localization sequence.
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DOI：
10.1161/jaha.113.000386
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发表时间：
2013-09-16
影响因子：
5.4
通讯作者：
Hawiger J
中科院分区：
文献类型：
--
作者：
Zienkiewicz J;Armitage A;Hawiger J
我们最近报道，在家族性高胆固醇血症小鼠模型中，一种双功能核转运调节剂（NTM）cSN 50.1肽可降低动脉粥样硬化、血浆胆固醇、甘油三酯和葡萄糖沿着肝脏脂肪和炎症标志物。我们确定cSN50.1通过与importin β相互作用调节固醇调节元件结合蛋白的核转运来改善脂质稳态。先前的研究证实，cSN50.1和相关的NTMs也通过其核定位序列（NLS）与importins/karyopherins α的结合来调节促炎性转录因子的核转运。然而，NTMs对importins/karyopherins α的选择性和特异性尚未确定。我们分析了NTM亲水性模块N50肽（来自NFκB1/p50的NLS）与内源性人importin/karyopherins α的相互作用，以确定NTM调节importin α介导的核转运的机制。我们发现N50肽与多个importins/karyopherins α形成稳定的复合物。然而，仅与importin α5（Imp α5）的相互作用显示出特异性、高亲和力结合。N50-Imp α5相互作用的2：1化学计量（KD 1 =73 nmol/L，KD 2 =140 nmol/L）表明主要和次要NLS结合口袋均被占据。利用计算机模拟三维（3-D）对接模型和比较结构分析，我们确定了Imp α5主要NLS结合口袋的结构组分，其可能稳定N50结合。Imp α5还显示出快速刺激诱导的转换，这可能影响其在炎症反应期间用于核转运的可用性。这些结果提供了N50肽选择性靶向Imp α5的直接证据，鼓励进一步完善NLS衍生肽作为调节炎症性疾病的新工具。
We recently reported that a bifunctional nuclear transport modifier (NTM), cSN50.1 peptide, reduced atherosclerosis, plasma cholesterol, triglycerides, and glucose along with liver fat and inflammatory markers, in a murine model of familial hypercholesterolemia. We determined that cSN50.1 improved lipid homeostasis by modulating nuclear transport of sterol regulatory element‐binding proteins through interaction with importin β. Previous studies established that cSN50.1 and related NTMs also modulate nuclear transport of proinflammatory transcription factors mediated by binding of their nuclear localization sequences (NLSs) to importins/karyopherins α. However, selectivity and specificity of NTMs for importins/karyopherins α were undetermined. We analyzed interaction of the NTM hydrophilic module, N50 peptide, derived from the NLS of NFκB1/p50, with endogenous human importins/karyopherins α to determine the mechanism of NTM modulation of importin α‐mediated nuclear transport. We show that N50 peptide forms stable complexes with multiple importins/karyopherins α. However, only interaction with importin α5 (Imp α5) displayed specific, high‐affinity binding. The 2:1 stoichiometry of the N50‐Imp α5 interaction (KD1=73 nmol/L, KD2=140 nmol/L) indicated occupancy of both major and minor NLS binding pockets. Utilizing in silico 3‐dimensional (3‐D) docking models and comparative structural analysis, we identified a structural component of the Imp α5 major NLS binding pocket that may stabilize N50 binding. Imp α5 also displayed rapid stimulus‐induced turnover, which could influence its availability for nuclear transport during the inflammatory response. These results provide direct evidence that N50 peptide selectively targets Imp α5, encouraging further refinement of NLS‐derived peptides as new tools to modulate inflammatory disorders.