Role of p300 and hHR23 proteins in p53 regulation

描述(由申请人提供)： 这项提议中的实验将试图将p300和hHR23蛋白对泛素/蛋白酶体途径对p53动态平衡调节的贡献置于生物学背景下。P300共激活子编码组蛋白乙酰化酶和泛素连接酶的功能，从而连接p53的动态平衡，编码正负调节功能，p300与p53拮抗剂和泛素连接酶MDM2相互作用，这两种蛋白在p53的多泛素化和蛋白酶体的快速转换中相互作用。在应激或DNA损伤条件下，p300通过其固有的乙酰酶活性激活P53功能。 HHR23蛋白酶体适配蛋白也深刻影响P53在人类细胞中的代谢和活性，hHR23家族蛋白通过与MDM2形成复合体，保护P53不被降解，并可能整合P53的泛素化和降解。此外，与已知的hHR23家族成员不同，hHR23家族中有一个新的、潜在的调控或显性的负性第三成员，它并不是在所有的细胞系、肿瘤和组织中都普遍表达。 本实验旨在探讨p3OO/MDM2相互作用、p300泛素连接酶活性和hHR23蛋白作为p53对电离辐射等致癌应激反应的动态生理调节中的关键成分的具体机制。P300/MDM2相互作用在确定p300作为p53激活剂或拮抗剂的活性如何平衡中的作用，将通过相互作用的图谱和对表达非相互作用突变等位基因的细胞中的p53调控的分析来检验。P300泛素连接酶活性在P53动态平衡中的具体作用将通过分析原代人类细胞和敲入小鼠的功能等位基因丢失来研究。HHR23A和hHR23B蛋白酶体适配蛋白的功能，以及hHR23/MDM2相互作用在p53调控中的作用，将通过对hHR23蛋白在培养细胞和体外重组的p53降解系统中的结构/功能分析来分析。一个新的、具有潜在调节作用的hHR23家族成员也将因其在DNA损伤调节p53稳定性中的作用而被进一步表征。

DESCRIPTION (provided by applicant): Experiments in this proposal will attempt to place into biologic context the contributions of p300 and hHR23 proteins to p53 homeostatic regulation by the ubiquitin/proteasome pathway. The p300 coactivator encodes both histone acetylase and ubiquitin ligase functions, and thus acts as a nexus for p53 homeostasis, encoding both positive and negative regulatory functions, p300 interacts with the p53 antagonist and ubiquitin ligase, MDM2, and the two proteins cooperate in the polyubiquitination and rapid proteasome turnover of p53. Under conditions of stress or DNA damage, p300 activates p53 functions through its intrinsic acetylase activity. hHR23 proteasome adaptor proteins also profoundly influence the metabolism and activity of p53 in human cells, hHR23-family proteins protect p53 from degradation, and may integrate ubiquitination and degradation of p53, by forming a complex with MDM2. Moreover, a novel, potentially regulatory or dominant negative third hHR23 family member has been identified which, unlike the known hHR23 family members, is not universally expressed in all cell lines, tumors and tissues. The proposed experimental aims address the specific mechanisms by which p3OO/MDM2 interaction, p300 ubiquitin ligase activity, and hHR23 proteins act as key components in the dynamic physiologic regulation of p53 response to oncogenic stressors, such as ionizing radiation. The role of p300/MDM2 interaction in determining how p300 activity as an activator or antagonist of p53 is balanced, will be examined by mapping of the interaction, and analysis of p53 regulation in cells expressing non-interacting mutant alleles. The specific role of p300 ubiquitin ligase activity in p53 homeostasis will be studied by analysis of loss of function alleles in primary human cells and a knock-in mouse model. The function of the hHR23A and hHR23B proteasome adaptor proteins, and the role of hHR23/MDM2 interaction in p53 regulation, will be analyzed by structure/function analysis of hHR23 proteins in cultured ceils and in a reconstituted in vitro p53 degradation system. A novel, potentially regulatory, hHR23 family member will also be further characterized for its role in DNA damage regulation of p53 stability.