D-box附近泛素化位点突变对Cdc20介导的Sp100蛋白降解的影响

Point Mutation of D-box Nearby Ubiquitin Binding Site Affect Cdc20Mediated Sp100Degradation

前期的研究发现Cdc20介导Sp100通过泛素-蛋白酶体途径降解,Sp100中的D-box在底物识别过程中具有重要作用.尽管在PML和DAXX中都存在典型的D-box序列,但是这两种蛋白都不能被Cdc20所识别.本研究中,我们构建了一系列含D-box的Sp100截断体来探讨Cdc20底物识别的必要序列.研究发现GFP-Sp100(152-182)截断体包含D-box但是不能被APC/C-Cdc20降解.GFP-Sp100(151K→G)突变潜在泛素结合位点后,Sp100降解显著受到抑制.远离D-box的潜在泛素化位点(217K,219K,225K)破坏后对Sp100降解没有影响.我们的结果表明,Sp100蛋白中D-box附近泛素化位点(151K)在Cdc20介导的蛋白降解中是必要的.

Abstract： Our previous work found that Cde20 mediated Spl00 degradation in ubiquitin-proteasome pathway, and D-box of Sp100 plays a role in the process of substrate recognition. Although there are typical D-box sequence existing in PML and Daxx, both of them cannot be recognized by Cdc20. In this study, a set of Spl00 truncation mutants with D-box were established to investigate the necessary sequences for the substrate recognition of Cdc20. We found that the truncation Sp100（152-182）that contained the D-box cannot be degraded by the APC/C-Cdc20. Further, we found a mutation Spl00（151K-G） that damaged the potential ubiquitin binding site was also cannot be degraded by the APC/C-Cdc20. But several potential ubiquitin binding sites（217K, 219K, 225K） far from D- box presented no effect on Spl00 degradation. Our results suggested that a D-box nearby ubiquitin binding site （151K） of Sp100 might be necessary for its Cdc20 mediated substrate degradation.