DiGly analysis of autoubiquitinated UBR4 (residues 4730-5183)

Human recombinant UBR4 protein, consisting of residues 4730-5183, undergoes in vitro autoubiquitination when incubated with the E2 conjugating enzyme UBE2A and the other reaction components. The autoubiquitinated protein was analysed by mass spectrometry to identify modification sites and the ubiquitin linkage types present. This was achieved by searching for diGly as a variable modification on Ser, Thr, Tyr, Cys and Lys residues. A single autoubiquitination site at K4814 was detected. However, mutation of this residue impaired autoubiquitination, indicating the presence of additional sites. The ubiquitin linkages identified were Lys33, Lys48, Lys63, Lys11 and Lys6, suggesting that UBR4 assembles these ubiquitin linkages on its substrates.

由氨基酸残基4730-5183构成的重组人源UBR4蛋白，在与泛素偶联酶E2（E2 conjugating enzyme）UBE2A及其他反应组分共同孵育时，可发生体外自身泛素化反应。随后通过质谱法对该自身泛素化蛋白进行分析，以鉴定其修饰位点与存在的泛素连接类型。该分析通过将丝氨酸（Ser）、苏氨酸（Thr）、酪氨酸（Tyr）、半胱氨酸（Cys）与赖氨酸（Lys）残基上的双甘氨酸（diGly）作为可变修饰进行检索得以实现。研究检测到位于K4814位的一处自身泛素化修饰位点。然而，该位点的突变会削弱自身泛素化水平，提示还存在其他额外的修饰位点。鉴定得到的泛素连接类型包括Lys33、Lys48、Lys63、Lys11与Lys6，这表明UBR4可在其底物上组装此类泛素链。