Proteomics Unit, CIC bioGUNE, CIBERehd, Bizkaia Technology Park, Building 801A, Derio 48160, Spain.
EMBO Rep. 2009 Nov;10(11):1250-8. doi: 10.1038/embor.2009.192. Epub 2009 Oct 2.
Post-translational modification with ubiquitin is one of the most important mechanisms in the regulation of protein stability and function. However, the high reversibility of this modification is the main obstacle for the isolation and characterization of ubiquitylated proteins. To overcome this problem, we have developed tandem-repeated ubiquitin-binding entities (TUBEs) based on ubiquitin-associated (UBA) domains. TUBEs recognize tetra-ubiquitin with a markedly higher affinity than single UBA domains, allowing poly-ubiquitylated proteins to be efficiently purified from cell extracts in native conditions. More significant is the fact that TUBEs protect poly-ubiquitin-conjugated proteins, such as p53 and IkappaBalpha, both from proteasomal degradation and de-ubiquitylating activity present in cell extracts, as well as from existing proteasome and cysteine protease inhibitors. Therefore, these new 'molecular traps' should become valuable tools for purifying endogenous poly-ubiquitylated proteins, thus contributing to a better characterization of many essential functions regulated by these post-translational modifications.
泛素化的翻译后修饰是调节蛋白质稳定性和功能的最重要机制之一。然而，这种修饰的高可逆性是分离和鉴定泛素化蛋白质的主要障碍。为了克服这个问题，我们基于泛素结合（UBA）结构域开发了串联重复的泛素结合结构域（TUBEs）。TUBEs 以明显更高的亲和力识别四聚泛素，使得多泛素化蛋白质能够在天然条件下从细胞提取物中有效纯化。更重要的是，TUBEs 保护多泛素化连接的蛋白质，如 p53 和 IkappaBalpha，免受细胞提取物中存在的蛋白酶体降解和去泛素化活性以及现有的蛋白酶体和半胱氨酸蛋白酶抑制剂的影响。因此，这些新的“分子陷阱”应该成为纯化内源性多泛素化蛋白质的有用工具，从而有助于更好地描述这些翻译后修饰调节的许多基本功能。
