SPECIFICITY OF UBIQUITINATION--THE HPV E6/E6 AP PARADIGM

描述：E6-AP（E6相关蛋白）是一种细胞蛋白， 在E6依赖性细胞凋亡中与p53的泛素化相关并催化p53的泛素化。 方式 E6-AP的表征显示其代表了大的 泛素蛋白连接酶（或E3蛋白）家族，已被 假定在底物选择性中起主要作用。 申请人 提出了E6-AP相关E3蛋白的功能模型， 它们由两个广泛的结构域组成：N-末端底物识别 结构域和C-末端催化结构域（hect结构域，用于同源 至E6-AP C-末端），其泛素化结合的底物。 具体 本建议的目的是：1）通过确定是否 底物特异性可以通过N-末端取代 如果hect结构域在E3之间是功能上可互换的， 蛋白质，2）通过精确地测定E6-AP来评估该模型， 定位p53结合和hect结构域所需的决定因素 功能，和3）鉴定Rsp 5的底物，一种必需的酵母hectE 3， 从2域模型的预测开始， 遗传和生物化学方法。 这一长期目标 应用的目的是了解hect E3的作用机制 蛋白质及其作用，以及高危HPV E6的作用 蛋白质（和可能的细胞类似物），在指导特异性 泛素系统 最终目标是更全面地了解 HPV相关的致癌作用和蛋白质调控的其他过程 泛素化

DESCRIPTION: E6-AP (E6-Associated Protein) is a cellular protein that associates with and catalyzes ubiquitination of p53 in anE6-dependent manner. Characterization of E6-AP showed it to be representative of a large family of ubiquitin-protein ligases (or E3 proteins), which have been postulated to play the major role in substrate selectivity. The applicant proposes a model for function of the E6-AP-related E3 proteins which states that they consist of two broad domains: an N-terminal substrate recognition domain, and a C-terminal catalytic domain (the hect domain, for homologous to E6-AP C-terminus) which ubiquitinates bound substrates. The specific aims of this proposal are: 1) to evaluate this model by determining if substrate specificity can be redirected by substitution of N-terminal sequences and if hect domains are functionally interchangeable among E3 proteins, 2) to evaluate this model with respect to E6-AP by precisely mapping the determinants necessary for p53 association and hect domain function, and 3) to identify substrates of Rsp5, an essential yeast hectE3, beginning with predictions of the 2-domain model and taking a combined genetic and biochemical approach. The long-term objectives of this application are to understand the mechanisms of action of the hect E3 proteins and their role, as well as the role of the high-risk HPV E6 proteins (and possible cellular analogs), in directing the specificity of the ubiquitin system. The ultimate goal is a more complete understanding of HPV-associated carcinogenesis and other processes regulated by protein ubiquitination.