The role of p53 redox-modification in cell fate signaling

p53 氧化还原修饰在细胞命运信号传导中的作用

• 批准号: 8569469
• 负责人: Jason M. Held
• 金额: $ 20.48万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8569469
• 关键词: Apoptosis; Apoptotic; BCL2 gene; Biochemical; Biochemical Process; Biochemistry; Caspase; Cell Aging; Cell Death; Cell Fate Control; Cell Nucleus; Cell Proliferation; Cells; Chemicals; Codon Nucleotides; Complex; Cysteine; DNA; DNA Binding; DNA Binding Domain; DNA Damage; Data; Detection; Diamide; Disulfides; Dominant-Negative Mutation; Fibroblasts; Genetic Transcription; H1299; Homeostasis; Hydrogen Peroxide; In Vitro; Investigation; Knock-in Mouse; Laboratories; Learning; Link; Longitudinal Studies; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Mediator of activation protein; Membrane; Metabolism; Mitochondria; Modification; Molecular; Mutagenesis; Mutation; Necrosis; Neoplasm Metastasis; Nuclear; Oxidation-Reduction; Oxidative Stress; Permeability; Physiological; Physiology; Play; Post-Translational Protein Processing; Process; Protein Biochemistry; Protein Family; Protein p53; Proteins; Proteomics; RNA chemical synthesis; Reaction; Recombinant Proteins; Recombinants; Regulation; Role; Signal Pathway; Signal Transduction; Site; Site-Directed Mutagenesis; Specificity; Staging; Stress; Structure; System; Techniques; Technology; Testing; Time; Transactivation; Translations; Tumor Suppressor Proteins; Zinc Fingers; angiogenesis; arm; base; cancer cell; carcinogenesis; cell growth regulation; in vivo; interdisciplinary approach; knock-down; link protein; major outer membrane protein; multidisciplinary; mutant; novel; oxidation; programs; public health relevance; response; senescence; small hairpin RNA; trafficking; tumor growth; tumorigenesis

DESCRIPTION (provided by applicant): The tumor suppressor p53 is a central hub in multiple cancer-related signaling networks that control cell proliferation and cell fate decisions. However, the more we learn about p53 the more complex and dynamic its role becomes. Recent studies have revealed that p53 is involved in an increasingly diverse range of cellular fates, including necrosis and senescence in addition to apoptosis. The remarkable biochemical complexity of p53 is essential to its diverse physiological roles, yet our understanding of these mechanisms is far from complete. For example, as shown in studies by dozens of laboratories, non-nuclear p53 functions are nearly as important as DNA transactivation with regard to cell fate signaling. In fact, p53 can induce apoptosis in cells through transcription-independent mechanisms even without a functional DNA binding domain, in the absence of RNA synthesis, protein translation, or even without a nucleus. However, the mechanistic details of this process remains much less characterized than classical p53 DNA transactivation response and warrants further investigation. Oxidation-reduction (redox) reactions are essential biochemical processes in many pro- and anti-apoptotic cellular programs that direct cancer cell metabolism, tumor growth, angiogenesis and metastasis. The specificity of these reactions is primarily defined by the high oxidizability of reactive sulfhydryls in select proteins. p53 is a zinc finger protein with ten cysteines, all within its DNA binding domain, and redox-regulated is widely speculated to play a role in its regulation. However, due to technological limitations, nearly all of the studies assessing p53 redox regulation and function have investigated isolated, recombinant protein. There is very little evidence that p53 is redox-regulated in cells on one of its cysteines under physiologically-relevant conditions or stresses. Using a novel mass spectrometry-based redox analysis technology, OxMRM, as well as non-reducing SDS-PAGE, we've discovered that p53 is redox-regulated in a cellular context in response to DNA damage and oxidative stress. These results, along with our biochemical and functional studies, suggest that redox- regulation of p53 is a critical step in cell fate signaling including apoptosis, senescence, and necrosis. This proposal will further characterize the redox-regulation of p53, including 1) identification of the disulfide-linked p53 partner, 2) quantify and define the redox-status of p53 at the mitochondria, and 3) determine the stage of transcription-independent p53 signaling in which redox-regulation is essential. In the long term, altered redox homeostasis is a hallmark of carcinogenesis and may impact p53 function. For example, deregulation of p53 redox-regulation is a potential mechanism by which DNA damaged cells could evade apoptosis and drive tumorigenesis.

描述（由申请人提供）：肿瘤抑制p53是控制细胞增殖和细胞命运决策的多个与癌症相关信号网络中的中心枢纽。然而， 我们了解p53的越多，它的作用就越复杂和动态。最近的研究表明，p53涉及越来越多的细胞命运，包括坏死和衰老。 p53的显着生化复杂性对于其多样化的生理作用至关重要，但是我们对这些机制的理解远非完整。例如，如数十个实验室的研究所示，对于细胞命运信号，非核p53功能几乎与DNA反式激活一样重要。实际上，在没有RNA合成，蛋白质翻译甚至没有核的情况下，p53即使没有功能性DNA结合结构域也可以通过独立的DNA结合结构域诱导细胞凋亡。但是，此过程的机械细节与经典p53 DNA反式激活响应的特征差不多，并需要进一步研究。 氧化还原（氧化还原）反应是许多促癌细胞代谢，肿瘤生长，血管生成和转移的许多促和抗凋亡细胞程序中必不可少的生化过程。这些反应的特异性主要是由选择蛋白中反应性硫丙烯酰的高氧化能力来定义。 p53是一种具有十个半胱氨酸的锌指蛋白，全部在其DNA结合结构域内，氧化还原调节被广泛推测以在其调节中起作用。但是，由于技术局限性，几乎所有评估p53氧化还原调控和功能的研究都研究了分离的重组蛋白。很少有证据表明，在生理上相关的条件或应力下，p53在其半胱氨酸之一的细胞中对氧化还原进行了调节。 使用基于质谱的新型氧化还原分析技术OXMRM以及非还原的SDS-PAGE，我们发现p53在细胞环境中对DNA损伤和氧化应激进行了氧化还原调节。这些结果以及我们的生化和功能研究表明，p53的氧化还原调节是细胞命运信号传导的关键步骤，包括凋亡，衰老和坏死。该提案将进一步表征p53的氧化还原调节，包括1）识别二硫键链接的p53合作伙伴，2）量化并定义了线粒体上p53的氧化还原态，以及3）确定转录与独立的p53信号的阶段，其中氧化还原是必不可少的。从长远来看，改变的氧化还原稳态是致癌作用的标志，可能会影响p53功能。例如，p53氧化还原调节的放松管制是一种潜在的机制，通过该机制，DNA受损的细胞可以逃避凋亡并驱动肿瘤发生。