Restore the Tumor-Suppressive Activities of p53 Mutants

恢复 p53 突变体的肿瘤抑制活性

• 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
• 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

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.

项目总结/摘要 该项目的重点是p53突变体的肽酶D（PEPD）的再激活。p53肿瘤抑制因子是 癌症中最常见的突变蛋白质大多数p53突变是错义突变，导致单个突变。 氨基酸在每个突变体中发生变化，并聚集在其DNA结合结构域内。p53突变无效 其肿瘤抑制功能和/或赋予致癌功能。PEPD，也称为氨脯氨酸二肽酶，是一种 二肽酶对胶原代谢很重要。然而，我们最近发现PEPD与野生型和非野生型都结合， 型p53（p53 WT）和各种突变体通过其富含脯氨酸的结构域（PRD），并破坏结合， PEPD敲低（KD）不仅激活p53 WT，而且再激活其突变体。这是一个新的功能， PEPD，其不需要其酶活性。我们的长期目标是促进对 p53 WT及其突变体的调控和功能。本提案的目的是界定 通过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 细胞中的每种p53突变体，尽管PEPD不是其致癌活性所必需的，但破坏PEPD 与p53突变体的结合诱导突变体的翻译后修饰（PTM）， 再折叠和再活化。我们将在三个具体目标中检验该假设：1）确定p53的结合 突变体的PEPD和他们的重新激活PEPD KD; 2）以确定分子机制， PEPD KD重新激活p53突变体，重点是PTM，特别是K373乙酰化在驱动中的作用 突变体的重折叠和再激活; 3）确定再激活的突变体的肿瘤抑制活性。 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（R175 H，G245 C和R249 S）和接触突变体（R248 Q， R273 H和R280 K）。将使用细胞系和小鼠肿瘤模型，包括患者来源的异种移植物。 PEPD KD将使用siRNA和多西环素调节系统实现。拟议的研究是 意义重大，因为它可能会带来对生物学和生物学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。预期的结果是， 项目包括：1）显示PEPD与细胞核和胞质溶胶中几乎一半的p53突变体结合; 2)这表明通过PEPD KD破坏PEPD-p53突变体复合物释放了突变体的PTM， 驱动突变体的重折叠和再活化，无论其是构象突变体还是接触突变体，以及 p300/CBP对K373的乙酰化是这一过程的关键; 3）显示了K373的肿瘤抑制活性， p53突变体与p53野生型相似。因此，我们的研究将揭示 p53突变体的一个关键的内在再激活机制，这可能对p53 研究，并可能为开发新的癌症治疗策略开辟新天地。