Krist David T, Statsyuk Alexander V
Chemistry of Life Processes Institute, Department of Chemistry, Northwestern University, Silverman Hall, 2145 Sheridan Road, Evanston, Illinois 60208, United States.
Biochemistry. 2015 Jul 28;54(29):4411-4. doi: 10.1021/acs.biochem.5b00625. Epub 2015 Jul 20.
Inactivation of the E6AP E3 ubiquitin ligase (UBE3A gene) causes Angelman syndrome, while aberrant degradation of p53 by E6AP is implicated in cervical cancers. Herein, we describe the development of photo-cross-linkers to discover catalytic residues of E6AP. Using these cross-linkers, we identified covalent modifications of the E6AP catalytic cysteine and two lysines: Lys(847) and Lys(799). Lys(847) is required for the formation of Lys(48)-linked polyubiquitin chains, while the K799A E6AP mutant was more active at producing Lys(48)-linked polyubiquitin chains. Thus, opposing roles of Lys(799) and Lys(847) pave the path forward to pharmacological inhibitors or activators of E6AP for therapeutic purposes.
E66E6AP E3泛素连接酶(UBE3A基因)的失活会导致天使综合征,而E6AP对p53的异常降解与宫颈癌有关。在此,我们描述了用于发现E6AP催化残基的光交联剂的开发。使用这些交联剂,我们鉴定了E6AP催化半胱氨酸和两个赖氨酸(Lys(847)和Lys(799))的共价修饰。Lys(847)是形成K48连接的多聚泛素链所必需的,而K799A E6AP突变体在产生K48连接的多聚泛素链方面更具活性。因此,Lys(799)和Lys(847)的相反作用为开发用于治疗目的的E6AP药理抑制剂或激活剂铺平了道路。