Investigating the mechanisms for mutant p53 stability in cancer cells

项目摘要 P53是一种在肿瘤抑制中起关键作用的转录因子，它经常在 人类癌症。P53 DNA结合域的错义突变可导致功能增强 表型，导致细胞增殖增加和肿瘤形成。而其他抑癌基因的突变 像RB1和NF1这样的蛋白质是缺失突变，导致蛋白质表达减少，点突变 在P53中增加了P53的稳定性，延长了半衰期。为了改进和开发新的治疗方法 对于含有突变型p53(Mtp53)的肿瘤，其增加肿瘤稳定性和积聚的机制 需要在很大程度上扩大。具体地说，无论mtp53需要关键的相互作用蛋白还是 对其增加蓄积和功能增益效应的翻译后修饰仍有待于 已澄清。在识别mtp53的新的相互作用或修饰方面，大多数工作都依赖于靶向特定的 调节P53活性的信号通路。然而，mtp53受到广泛的外部信号的调控， 因此，全面和公正地研究其监管对于理解其机制至关重要。 因此，这项提议旨在利用基于定量质谱学的方法来识别 不同mtp53突变和癌症类型之间的相互作用和修饰。这项拟议的研究将 揭示一层新的信息，有助于确定mtp53稳定性的机制，并将导致 癌症治疗新靶点的发现。这一建议阐明了mtp53的作用机制。 两个目标：(1)在多个携带GOF mtp53的癌细胞系中鉴定和验证mtp53的相互作用 利用免疫沉淀与质谱仪联用，确定候选的稳定剂是否 使用CRISPR gRNA筛查方法对癌症生长至关重要。(2)识别组合 通过开发定量质谱学对mtp53稳定性至关重要的翻译后修饰 线粒体P53基因的检测方法。总体而言，这些目标将阐明人类癌症中mtp53稳定的机制， 这将为癌症提供有前景的治疗靶点。

Project Summary p53 is a transcription factor that plays a crucial role in tumor suppression, and it is frequently mutated in human cancers. Missense mutations in the DNA binding domain of p53 can result in a gain-of-function phenotype, leading to increased cell proliferation and tumor formation. While mutations in other tumor suppressor proteins such as RB1 and NF1 are deletion mutations leading to decreased protein expression, point mutations in p53 lead to increased p53 stability and prolonged half-life. In order to improve and develop novel treatments for cancers containing mutant p53 (mtp53), its mechanisms for increased stability and accumulation in tumors needs to be largely expanded. Specifically, whether mtp53 requires critical interacting proteins or posttranslational modifications for its increased accumulation and gain-of-function effect remains to be elucidated. Most work in identifying novel interactors or modifications on mtp53 has relied on targeting specific signaling pathways that modulate p53 activity. However, mtp53 is regulated by a wide array of external signals, so a comprehensive and unbiased approach in studying its regulation is crucial in understanding its mechanisms. Therefore, this proposal aims to utilize a quantitative mass spectrometry-based approach in identifying interactors and modifications across different mtp53 mutations and cancer types. This proposed research will reveal a new layer of information that will aid in determining mechanisms of mtp53 stability, and it will lead to the discovery of novel targets for cancer therapeutics. This proposal elucidates the mechanisms of mtp53 through two Aims: (1) Identifying and validating mtp53 interactors across multiple cancer cell lines harboring GOF mtp53 using immunoprecipitation coupled to mass spectrometry, and determining whether the candidate “stabilizers” are essential for cancer growth using CRISPR gRNA screening approaches. (2) Identifying combinatorial posttranslational modifications essential for mtp53 stability by developing a quantitative mass spectrometry method for mtp53. Overall, these aims will shed light on the mechanisms of mtp53 stability in human cancers, which will offer promising therapeutic targets in cancer.