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， 结合并催化E6依赖蛋白中P53的泛素化 举止。E6-AP的特性表明它代表了一种 泛素蛋白连接酶家族(或E3蛋白)，已被 推测在底物选择性中起主要作用。申请人 提出了一个与E6-AP相关的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，以及C端催化结构域(hect结构域，用于同源 至E6-AP C-末端)，泛素化结合底物。具体的 本提案的目的是：1)通过确定是否 底物专一性可以通过N-末端的取代来重定向 序列以及hect结构域是否在E3之间功能上可互换 蛋白质，2)以E6-AP为参照对该模型进行精确评估 映射P53关联和hect结构域所必需的决定因素 功能，以及3)鉴定Rsp5的底物，Rsp5是一种必需的酵母HhE3， 从对双域模型的预测开始，并结合 遗传和生化方法。这个项目的长期目标是 应用是为了了解hect E3的作用机制。 蛋白质及其作用，以及高危HPVE6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 人乳头瘤病毒相关致癌及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.