Dynamics, Regulation and Function of p53 in Single Cells

单细胞中 p53 的动态、调控和功能

• 批准号: 10796079
• 负责人: Galit Lahav
• 金额: $ 21.17万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2025-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10796079
• 关键词: 3-Dimensional; Address; Affect; Architecture; Cell Death; Cell Fate Control; Cells; Cellular Stress; Clinical; DNA Damage; Data; Development; Dose; Environment; Gene Expression; Genes; Goals; Human; Image; Immune; Immune system; Immunotherapy; Individual; Knowledge; Link; Malignant Neoplasms; Mediating; Normal Cell; Outcome; Pathway interactions; Pattern; Population; Post-Translational Protein Processing; Predisposition; Process; Proteins; RNA; Radiation; Regulation; Schedule; Signal Transduction; Stimulus; System; TP53 gene; Therapeutic; Time; Translating; Tumor Suppressor Proteins; Work; cancer cell; cancer therapy; cell killing; clinically relevant; combinatorial; imaging system; in vivo; inhibitor; insight; mutant; neoplastic cell; new technology; novel; overexpression; programs; protective pathway; response; single-cell RNA sequencing; targeted treatment; transcription factor; tumor

Project Summary The dynamics of signaling systems are critical for controlling gene expression programs and cellular outcomes. The tumor suppressor protein p53 is a transcription factor orchestrating the response to cellular stresses, and we previously found that its dynamics (changes in its protein levels over time) following DNA damage depend on the stimulus and play a role in determining whether a cell will survive or die. However, many questions remain about how different cellular contexts influence p53 dynamics and ultimate cellular outcomes, how p53 chooses between conflicting cellular outcomes, and how p53 dynamics can best be leveraged for therapeutic purposes. The goals of our work are to obtain a comprehensive quantitative understanding of how p53 dynamics regulate cellular outcomes in single cells and to apply our findings to address clinical needs. The cellular environment can influence p53 dynamics, therefore we will first investigate how p53 dynamics are regulated by factors such as 3D cellular architecture in cultured tumor spheroids and in in vivo tumors. We will then investigate the dynamics and cellular outcomes of cancer-associated p53 mutants in cultured and in vivo settings. The effects of p53 dynamical patterns on gene expression will be determined in single cells by using novel technology that supports integrating live imaging data of p53 dynamics with single-cell RNA sequencing. We will also investigate how p53 dynamics influence gene expression at the RNA and protein levels, as well as the dynamics of p53 post-translational modifications in bulk populations. These studies will reveal the impact that p53 dynamical patterns have on the RNA and protein of its target genes, and how the combinations of these dynamical patterns guide cellular outcomes. We will also use our live-imaging systems to determine how clinically-relevant therapeutic approaches can be optimized to induce the desired p53 dynamics and cellular outcomes in cancer. We will determine how the doses and timings of radiation fractions affect p53 dynamics and function, and optimize the schedule of fractions for inducing tumor cell death via p53-mediated mechanisms. Many cancers overexpress the p53 inhibitors Mdm2 or Mdmx and are susceptible to their inhibition. Through quantifying and modulating p53 dynamics we will determine how to fine-tune their inhibition to sensitize Mdm2 or Mdmx overexpressing cells to DNA damage while sparing healthy cells. Tumor cells can be cleared by the immune system, and this process is influenced by the tumors' gene expression programs. Therefore, we will investigate how p53 dynamics influence interactions between tumor cells and immune cells, and work towards optimizing combinations of p53-targeting therapeutics with immunotherapies to maximize tumor cell killing by the immune system. In total, these studies will provide new mechanistic insights into the links between p53 dynamics and function in controlling cell fates, and will inform novel combinatorial therapeutic approaches to cancer treatments.

项目摘要 信号系统的动力学对于控制基因表达程序和细胞结果至关重要。 肿瘤抑制蛋白p53是一种转录因子，策划了对细胞应激的反应，并且 我们先前发现DNA损伤后其动力学（随着时间的推移随时间变化）取决于 在刺激上并在确定细胞是否生存还是死亡中发挥作用。但是，很多问题 仍然存在不同的细胞环境如何影响p53动力学和最终的细胞结果，p53如何 在相互冲突的蜂窝结果之间进行选择，以及如何最好地利用p53动力学来治疗 目的。 我们工作的目标是获得对p53动态如何调节的全面定量理解 单个细胞中的细胞结局并应用我们的发现以满足临床需求。细胞环境 可以影响p53动力学，因此我们将首先研究p53动态如何受到此类因素的调节 作为培养的肿瘤球体和体内肿瘤中的3D细胞结构。然后，我们将调查 培养和体内环境中与癌症相关的p53突变体的动力学和细胞结局。效果 通过使用新技术来确定基因表达上的p53动力学模式 支持通过单细胞RNA测序集成p53动力学的实时成像数据。我们也会 研究p53动态如何影响RNA和蛋白质水平的基因表达，以及 p53大量种群翻译后修饰的动力学。这些研究将揭示出这样的影响 p53动力学模式在其靶基因的RNA和蛋白质上具有 动态模式指导细胞结局。我们还将使用我们的实影系统来确定如何 可以优化与临床相关的治疗方法，以诱导所需的p53动力学和细胞 癌症的结果。我们将确定辐射分数的剂量和时机如何影响p53动力学 和功能，并优化通过p53介导的肿瘤细胞死亡的分数时间表 机制。许多癌症过表达p53抑制剂MDM2或MDMX，并且易受其影响 抑制。通过量化和调节p53动力学，我们将确定如何微调其抑制作用 在保留健康细胞的同时，使MDM2或MDMX过表达细胞对DNA损伤敏感。肿瘤细胞可以 通过免疫系统清除，该过程受肿瘤的基因表达程序的影响。 因此，我们将研究p53动态如何影响肿瘤细胞与免疫细胞之间的相互作用， 并致力于优化p53靶向治疗剂和免疫疗法的组合以最大化 免疫系统杀死肿瘤细胞。总的来说，这些研究将为您提供新的机械见解 p53动力学与控制细胞命运方面的功能之间的链接，并将为新型组合提供信息 癌症治疗的治疗方法。

项目成果

Direct interaction between the transmembrane helices stabilize cytochrome P450 2B4 and cytochrome b5 redox complex.

跨膜螺旋之间的直接相互作用稳定细胞色素 P450 2B4 和细胞色素 b5 氧化还原复合物。

• DOI: 10.1016/j.bpc.2023.107092
• 发表时间: 2023
• 期刊: Biophysical chemistry
• 影响因子: 3.8
• 作者: Sahoo,BikashR;Ramamoorthy,Ayyalusamy
• 通讯作者: Ramamoorthy,Ayyalusamy

Principles, mechanisms and functions of entrainment in biological oscillators.

• DOI: 10.1098/rsfs.2021.0088
• 发表时间: 2022-06-06
• 期刊: Interface focus
• 影响因子: 4.4

Detergent-free isolation of CYP450-reductase's FMN-binding domain in E. coli lipid-nanodiscs using a charge-free polymer.

• DOI: 10.1039/d1cc07193a
• 发表时间: 2022-04-14
• 期刊: Chemical communications (Cambridge, England)

Factors influencing the detergent-free membrane protein isolation using synthetic nanodisc-forming polymers.

影响使用合成纳米盘形成聚合物进行无去污剂膜蛋白分离的因素。

• DOI: 10.1101/2023.05.12.540572
• 发表时间: 2023
• 期刊: bioRxiv : the preprint server for biology
• 作者: Krishnarjuna,Bankala;Sharma,Gaurav;Ravula,Thirupathi;Ramamoorthy,Ayyalusamy
• 通讯作者: Ramamoorthy,Ayyalusamy

Reading oscillatory instructions: How cells achieve time-dependent responses to oscillating transcription factors.

• DOI: 10.1016/j.ceb.2022.102099
• 发表时间: 2022-08
• 期刊: CURRENT OPINION IN CELL BIOLOGY
• 影响因子: 7.5
• 作者: Venkatachalam, Veena;Jambhekar, Ashwini;Lahav, Galit
• 通讯作者: Lahav, Galit