Shimizu Harumi, Saliba David, Wallace Maura, Finlan Lee, Langridge-Smith Patrick R R, Hupp Ted R
Cancer Research UK Cell Signalling Unit, p53 Signal Transduction Group, University of Edinburgh, South Crewe Road, Edinburgh EH4 2XR, Scotland, UK.
Biochem J. 2006 Jul 15;397(2):355-67. doi: 10.1042/BJ20051521.
p53 ubiquitination catalysed by MDM2 (murine double minute clone 2 oncoprotein) provides a biochemical assay to dissect stages in E3-ubiquitin-ligase-catalysed ubiquitination of a conformationally flexible protein. A mutant form of p53 (p53(F270A)) containing a mutation in the second MDM2-docking site in the DNA-binding domain of p53 (F270A) is susceptible to modification of long-lived and high-molecular-mass covalent adducts in vivo. Mutant F270A is hyperubiquitinated in cells as defined by immunoprecipitation and immunoblotting with an anti-ubiquitin antibody. Transfection of His-tagged ubiquitin along with p53(R175H) or p53(F270A) also results in selective hyperubiquitination in cells under conditions where wild-type p53 is refractory to covalent modification. The extent of mutant p53(R175H) or p53(F270A) unfolding in cells as defined by exposure of the DO-12 epitope correlates with the extent of hyperubiquitination, suggesting a link between substrate conformation and E3 ligase function. The p53(F270A:6KR) chimaeric mutant (where 6KR refers to the simultaneous mutation of lysine residues at positions 370, 372, 373, 381, 382 and 386 to arginine) maintains the high-molecular-mass covalent adducts and is modified in an MDM2-dependent manner. Using an in vitro ubiquitination system, mutant p53(F270A) and the p53(F270A:6KR) chimaeric mutant is also subject to hyperubiquitination outwith the C-terminal domain, indicating direct recognition of the mutant p53 conformation by (a) factor(s) in the cell-free ubiquitination system. These data identify an in vitro and in vivo assay with which to dissect how oligomeric protein conformational alterations are linked to substrate ubiquitination in cells. This has implications for understanding the recognition of misfolded proteins during aging and in human diseases such as cancer.
由MDM2（小鼠双微体克隆2癌蛋白）催化的p53泛素化提供了一种生化分析方法，用于剖析E3泛素连接酶催化的构象灵活蛋白泛素化过程中的各个阶段。p53（p53(F270A)）的一种突变形式，其在p53（F270A）的DNA结合域中的第二个MDM2对接位点发生突变，在体内易受长寿命和高分子量共价加合物的修饰。如通过用抗泛素抗体进行免疫沉淀和免疫印迹所定义的，突变体F270A在细胞中被高度泛素化。在野生型p53对共价修饰不敏感的条件下，转染His标签的泛素以及p53(R175H)或p53(F270A)也会导致细胞中的选择性高度泛素化。如由DO-12表位的暴露所定义的，细胞中突变型p53(R175H)或p53(F270A)的解折叠程度与高度泛素化程度相关，这表明底物构象与E3连接酶功能之间存在联系。p53(F270A:6KR)嵌合突变体（其中6KR指第370、372、373、381、382和386位赖氨酸残基同时突变为精氨酸）维持高分子量共价加合物，并以MDM2依赖的方式被修饰。使用体外泛素化系统，突变型p53(F270A)和p53(F270A:6KR)嵌合突变体在C末端结构域外也会被高度泛素化，这表明在无细胞泛素化系统中，（一种或多种）因子可直接识别突变型p53构象。这些数据确定了一种体外和体内分析方法，可用于剖析细胞中寡聚蛋白构象改变与底物泛素化之间的联系。这对于理解衰老过程中以及癌症等人类疾病中错误折叠蛋白的识别具有重要意义。
