Proteome-wide Identification of Ubiquitylation Sites by Conjugation of Engineered Lysine-less Ubiquitin

Ubiquitin conjugation (ubiquitylation) plays important roles not only in protein degradation but also in many other cellular functions. However, the sites of proteins that are targeted for such modification have remained poorly characterized at the proteomic level. We have now developed a method for the efficient identification of ubiquitylation sites in target proteins with the use of an engineered form of ubiquitin (K0-Ub), in which all seven lysine residues are replaced with arginine. K0-Ub is covalently attached to lysine residues of target proteins via an isopeptide bond, but further formation of a polyubiquitin chain does not occur on K0-Ub. We identified a total of 1392 ubiquitylation sites of 794 proteins from HEK293T cells. Profiling of ubiquitylation sites indicated that the sequences surrounding lysine residues targeted for ubiquitin conjugation do not share a common motif or structural feature. Furthermore, we identified a critical ubiquitylation site of the cyclin-dependent kinase inhibitor p27Kip1. Mutation of this site thus inhibited ubiquitylation of and stabilized p27Kip1, suggesting that this lysine residue is the target site of p27Kip1 for ubiquitin conjugation in vivo. In conclusion, our method based on K0-Ub is a powerful tool for proteome-wide identification of ubiquitylation sites of target proteins.

泛素结合（ubiquitylation，泛素化）不仅在蛋白质降解过程中发挥关键作用，还参与诸多其他细胞生理功能。然而，在蛋白质组学层面，受该修饰靶向的蛋白质位点仍未得到充分表征。我们现已开发出一种可高效鉴定靶蛋白泛素化位点的方法，该方法借助经工程改造的泛素变体（K0-Ub）——其全部7个赖氨酸残基均被精氨酸取代。K0-Ub可通过异肽键（isopeptide bond）共价结合至靶蛋白的赖氨酸残基，但无法在其之上进一步形成多聚泛素链。我们从HEK293T细胞中共鉴定出794种蛋白的1392个泛素化位点。对泛素化位点的序列分析显示，靶向泛素结合的赖氨酸残基周围的序列并无共同基序或结构特征。此外，我们还鉴定出细胞周期蛋白依赖性激酶抑制剂（cyclin-dependent kinase inhibitor）p27Kip1的关键泛素化位点；该位点的突变可抑制p27Kip1的泛素化修饰并使其稳定性增强，表明该赖氨酸残基即为体内p27Kip1泛素结合的靶向位点。综上，我们基于K0-Ub开发的方法是实现靶蛋白全蛋白质组范围泛素化位点鉴定的有力工具。