Restore the Tumor-Suppressive Activities of p53 Mutants

恢复 p53 突变体的肿瘤抑制活性

基本信息

批准号：
10716397
负责人：
YUESHENG ZHANG
金额：
$ 37.52万
依托单位：
VIRGINIA COMMONWEALTH UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-07-01 至 2028-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10716397
关键词：
Acceleration Acetylation Affinity Amino Acids Automobile Driving Binding Biology C-terminal Cancer Burden Cancer Patient Cell Line Cell Nucleus Cells Collagen Complex Cytosol DNA Binding Domain Data Dipeptidases Dominant-Negative Mutation Doxycycline EP300 gene Endowment Genes Goals In Vitro Inhibition of Cancer Cell Growth Malignant Neoplasms Metabolism Missense Mutation Mission Modeling Molecular Molecular Conformation Mus Mutate Mutation Oncogenic Outcome Post-Translational Protein Processing Process Proline-Rich Domain Proteins Public Health Reactive Oxygen Species Regulation Research Role Signal Transduction Small Interfering RNA Stress System TP53 gene Testing Therapeutic Time Tumor Suppressor Proteins Xaa-Pro dipeptidase cancer cell cancer therapy gain of function in vivo innovation knock-down mutant novel patient derived xenograft model therapeutic target treatment strategy tumor

项目摘要

Project Summary/Abstract This project is focused on reactivation of p53 mutants by peptidase D (PEPD). p53 tumor suppressor is the most frequently mutated protein in cancer. Most p53 mutations are missense mutations, causing a single amino acid change in each mutant, and are clustered within its DNA binding domain. p53 mutations nullify its tumor suppressor functions and/or endow oncogenic functions. PEPD, also known as prolidase, is a dipeptidase important for collagen metabolism. However, we recently found that PEPD binds to both wild- type p53 (p53WT) and various mutants via their proline-rich domain (PRD) and that disrupting the binding by PEPD knockdown (KD) not only activates p53WT but also reactivates its mutants. This is a novel function of PEPD, which does not require its enzymatic activity. Our long-term goal is to advance the understanding of regulation and function of p53WT and its mutants. The objective of the present proposal is to delineate the reactivation of oncogenic hotspot p53 mutants by PEPD KD and to assess the tumor-suppressing activities of the reactivated p53 mutants. The central hypothesis of the proposal is that PEPD binds to nearly half of each p53 mutant in cells and that, while PEPD is not required for their oncogenic activities, disrupting PEPD binding to p53 mutants induces post-translational modifications (PTMs) of the mutants that cause their refolding and reactivation. We will test the hypothesis in three specific aims: 1) to determine binding of p53 mutants to PEPD and their reactivation by PEPD KD; 2) to determine the molecular mechanism by which PEPD KD reactivates p53 mutants, focusing on the roles of PTMs, K373 acetylation in particular, in driving refolding and reactivation of the mutants; 3) to determine the tumor-inhibitory activities of the reactivated p53 mutants. We will pursue these aims by focusing on some of the most common oncogenic p53 mutants in cancer, including conformation mutants (R175H, G245C, and R249S) and contact mutants (R248Q, R273H, and R280K). Cell lines and mouse tumor models, including patient-derived xenografts, will be used. PEPD KD will be achieved using siRNA and a doxycycline-regulated system. The proposed research is significant, because it may bring about a paradigm shift in understanding of the biology and regulation of p53 mutants, which in turn may offer innovative cancer treatment strategies. The expected outcome of this project includes: 1) showing that PEPD binds to nearly half of each p53 mutant in the nucleus and cytosol; 2) showing that disrupting the PEPD-p53 mutant complex by PEPD KD frees the mutant for PTMs which drive refolding and reactivation of the mutant, whether it is a conformation mutant or a contact mutant, and K373 acetylation by p300/CBP is key to this process; and 3) showing that the tumor-suppressive activities of the reactivated p53 mutants are similar to that of activated p53WT. As such, our research will bring to light a critical intrinsic reactivation mechanism of p53 mutants, which may have far-reaching implications in p53 research and may break new ground for developing novel cancer therapeutic strategies.

项目概要/摘要该项目的重点是通过肽酶 D (PEPD) 重新激活 p53 突变体。 p53抑癌基因是癌症中最常见的突变蛋白。大多数 p53 突变是错义突变，导致单一每个突变体中的氨基酸发生变化，并聚集在其 DNA 结合域内。 p53突变无效其肿瘤抑制功能和/或赋予致癌功能。 PEPD，也称为脯氨酸酶，是一种二肽酶对胶原蛋白代谢很重要。然而，我们最近发现 PEPD 与两种野生 p53 型 (p53WT) 和各种突变体通过其富含脯氨酸的结构域 (PRD) 并通过破坏结合 PEPD 敲低 (KD) 不仅可以激活 p53WT，还可以重新激活其突变体。这是一个新颖的功能 PEPD，不需要其酶活性。我们的长期目标是增进对 p53WT 及其突变体的调控和功能。本提案的目的是界定 PEPD KD 重新激活致癌热点 p53 突变体并评估肿瘤抑制活性重新激活的p53突变体。该提案的核心假设是 PEPD 与近一半的细胞中的每个 p53 突变体，虽然 PEPD 不是其致癌活性所必需的，但会破坏 PEPD 与 p53 突变体的结合会诱导突变体的翻译后修饰 (PTM)，从而导致其重折叠和重新激活。我们将在三个具体目标上检验该假设：1) 确定 p53 的结合 PEPD 突变体及其通过 PEPD KD 的重新激活； 2）确定其分子机制 PEPD KD 重新激活 p53 突变体，重点关注 PTM 的作用，特别是 K373 乙酰化在驱动中的作用突变体的重折叠和重新激活； 3) 确定重新激活的肿瘤抑制活性 p53 突变体。我们将通过关注一些最常见的致癌 p53 突变体来实现这些目标在癌症中，包括构象突变体（R175H、G245C 和 R249S）和接触突变体（R248Q、 R273H 和 R280K）。将使用细胞系和小鼠肿瘤模型，包括源自患者的异种移植物。 PEPD KD 将使用 siRNA 和多西环素调节系统来实现。拟议的研究是意义重大，因为它可能会带来对生物学和调控的理解的范式转变 p53 突变体，这反过来可能提供创新的癌症治疗策略。本次活动的预期结果项目包括：1) 表明 PEPD 与细胞核和细胞质中每个 p53 突变体的近一半结合； 2) 表明通过 PEPD KD 破坏 PEPD-p53 突变体复合物会释放突变体的 PTM，这驱动突变体的重折叠和重新激活，无论是构象突变体还是接触突变体，以及 p300/CBP 乙酰化 K373 是该过程的关键； 3）显示肿瘤抑制活性重新激活的 p53 突变体与激活的 p53WT 相似。因此，我们的研究将揭示 p53突变体的关键内在再激活机制，可能对p53产生深远影响研究并可能为开发新型癌症治疗策略开辟新天地。