NESmapper: Accurate Prediction of Leucine-Rich Nuclear Export Signals Using Activity-Based Profiles

The nuclear export of proteins is regulated largely through the exportin/CRM1 pathway, which involves the specific recognition of leucine-rich nuclear export signals (NESs) in the cargo proteins, and modulates nuclear–cytoplasmic protein shuttling by antagonizing the nuclear import activity mediated by importins and the nuclear import signal (NLS). Although the prediction of NESs can help to define proteins that undergo regulated nuclear export, current methods of predicting NESs, including computational tools and consensus-sequence-based searches, have limited accuracy, especially in terms of their specificity. We found that each residue within an NES largely contributes independently and additively to the entire nuclear export activity. We created activity-based profiles of all classes of NESs with a comprehensive mutational analysis in mammalian cells. The profiles highlight a number of specific activity-affecting residues not only at the conserved hydrophobic positions but also in the linker and flanking regions. We then developed a computational tool, NESmapper, to predict NESs by using profiles that had been further optimized by training and combining the amino acid properties of the NES-flanking regions. This tool successfully reduced the considerable number of false positives, and the overall prediction accuracy was higher than that of other methods, including NESsential and Wregex. This profile-based prediction strategy is a reliable way to identify functional protein motifs. NESmapper is available at http://sourceforge.net/projects/nesmapper.

蛋白质的核输出主要通过输出蛋白/CRM1（exportin/CRM1）通路调控，该通路可特异性识别货物蛋白内的富含亮氨酸的核输出信号（NESs），并通过拮抗输入蛋白（importins）介导的核输入活性与核输入信号（NLS），实现对核质间蛋白质穿梭的调控。尽管核输出信号（NESs）的预测有助于鉴定受调控的核输出蛋白质，但当前的NES预测方法（包括计算工具与基于保守序列的检索手段）准确率有限，尤其在特异性方面表现不足。我们发现，NES内的每个氨基酸残基均以独立且累加的方式对整体核输出活性产生贡献。我们通过在哺乳动物细胞中开展全面突变分析，构建了所有类别NES的活性特征谱。该特征谱不仅揭示了保守疏水位点处多个影响活性的氨基酸残基，还在连接区与侧翼区域中发现了此类残基。随后我们开发了一款计算工具NESmapper，通过利用经训练优化并结合NES侧翼区域氨基酸属性的特征谱来预测NES。该工具有效减少了大量假阳性结果，整体预测准确率高于包括NESsential与Wregex在内的其他同类方法。这种基于特征谱的预测策略是鉴定功能性蛋白质基序的可靠手段。NESmapper工具可通过以下网址获取：http://sourceforge.net/projects/nesmapper。