p53-Regulation in Liver Regeneration

p53-肝脏再生的调节

• 批准号: 8094399
• 负责人: Michelle Ann Barton
• 金额: $ 32.07万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-10 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8094399
• 关键词: Address; Affinity Chromatography; Aging; Apoptosis; Area; Attenuated; Binding; Biochemical; C-terminal; Cell Cycle; Cell Cycle Arrest; Cell physiology; Cells; Chemicals; Chromatin; Complement; Development; Disease; Epitopes; Excision; Family member; Financial compensation; Goals; Growth; Hepatocyte; Investigation; Knock-in Mouse; Knowledge; Liver; Liver Regeneration; Masks; Mass Spectrum Analysis; Mediating; Methodology; Modification; Molecular; Natural regeneration; Normal Cell; Organogenesis; Partial Hepatectomy; Pathway interactions; Peptides; Physiological; Post-Translational Protein Processing; Process; Protein p53; Proteome; Regulation; Research; Signal Transduction Pathway; Tissues; Tumor Suppressor Proteins; Tumor-Derived; Work; chromatin immunoprecipitation; genome-wide analysis; innovation; mouse model; mutant; protein complex; response; senescence; stem; tissue regeneration

DESCRIPTION (provided by applicant): Renewed organogenesis of the fully differentiated liver is stimulated in response to surgical removal (partial hepatectomy, PHx) or destruction of liver tissue by chemicals or disease. An induced network of signal transduction pathways triggers re-entry of hepatocytes (followed by non-parenchymal cells) into cell cycle, proliferation and compensatory growth, which terminates at a precisely regulated endpoint. We hypothesize that critical functions of the p53 tumor suppressor protein, in promoting cell cycle arrest and apoptosis, must be blocked or attenuated in response to partial hepatectomy. Our goal is to define the mechanisms of this regulatory process. The wealth of information, regarding regulation and functions of p53, stems primarily from studies of tumor-derived cells, cells under long term, continuous culture, and/or cells expressing dysfunctional, mutant or exogenous p53. Although evidence continues to mount that p53 acts in normal cells during development, aging and senescence, major gaps in our knowledge exist regarding mechanisms of endogenous p53-regulation. Tumor suppressor p53 is expressed at low levels, which are tightly controlled by multiple pathways. This offers a considerable challenge to biochemical purification and mechanistic analyses of endogenous p53, especially in normal cells. We have developed a new mouse model to address these gaps in our knowledge. The mouse model, created by knock-in methodology, expresses endogenous p53 fused in-frame with a C-terminal epitope-tag (TAP-p53). TAP or Tandem Affinity Purification is a proven means of purifying low-abundance, large protein complexes under relatively physiological conditions. TAP-p53 purification and subsequent peptide analyses by mass spectrometry will be used to define p53-protein interactions (the p53 "proteome") and to determine post-translational modifications of p53. Epitope-tagging of p53 further facilitates studies of p53-chromatin interactions by chromatin immunoprecipitation (ChIP) and genome-wide analysis of p53-targets (ChIP-chip). We will use these and other approaches to establish the ground state of p53 function and regulation in normal hepatic cells and to determine how these functions may be altered during liver regeneration. The proposed research will address how p53 functions in normal, differentiated cells, an area generally overlooked in investigations of tumor suppressor activities. This work will further molecular understanding of tissue regeneration with a long-term goal of understanding how the surveillance status of the p53-network may be temporally controlled to facilitate cellular renewal and tissue regeneration. PROJECT NARRATIVE: These studies are focused on understanding how a fully differentiated tissue circumvents regulation and surveillance by tumor suppressor p53 to regenerate itself in response to partial hepatectomy. We believe that p53, which normally stops growth and proliferation, is temporarily blocked from functioning during regeneration and then restored to normal capacities when regeneration is finished. How p53 functions in normal cells and during regeneration is essentially unknown.

描述（由申请人提供）：完全分化的肝脏的更新器官发生是由于手术切除（部分肝切除术，PHx）或化学物质或疾病对肝组织的破坏而受到刺激。信号转导途径的诱导网络触发肝细胞（随后是非实质细胞）重新进入细胞周期、增殖和代偿性生长，并在精确调节的终点处终止。我们假设 p53 肿瘤抑制蛋白在促进细胞周期停滞和细胞凋亡方面的关键功能必须因部分肝切除术而被阻断或减弱。我们的目标是定义这一监管过程的机制。 关于 p53 的调节和功能的大量信息主要源于对肿瘤来源的细胞、长期连续培养的细胞和/或表达功能失调、突变或外源性 p53 的细胞的研究。尽管越来越多的证据表明 p53 在发育、衰老和衰老过程中在正常细胞中发挥作用，但我们对内源性 p53 调节机制的了解仍存在重大差距。肿瘤抑制因子 p53 低水平表达，并受到多种途径的严格控制。这给内源性p53的生化纯化和机制分析带来了相当大的挑战，尤其是在正常细胞中。 我们开发了一种新的小鼠模型来解决我们的这些知识空白。通过敲入方法创建的小鼠模型表达与 C 端表位标签框内融合的内源性 p53 (TAP-p53)。 TAP 或串联亲和纯化是一种在相对生理条件下纯化低丰度大蛋白复合物的行之有效的方法。 TAP-p53 纯化和随后的质谱肽分析将用于定义 p53-蛋白质相互作用（p53“蛋白质组”）并确定 p53 的翻译后修饰。 p53 的表位标记进一步促进了通过染色质免疫沉淀 (ChIP) 和 p53 靶标的全基因组分析（ChIP 芯片）来研究 p53-染色质相互作用。我们将使用这些和其他方法来建立正常肝细胞中 p53 功能和调节的基态，并确定这些功能在肝再生过程中如何改变。 拟议的研究将解决 p53 在正常分化细胞中如何发挥作用，这是肿瘤抑制活性研究中通常被忽视的一个领域。这项工作将进一步加深对组织再生的分子理解，长期目标是了解如何暂时控制 p53 网络的监视状态以促进细胞更新和组织再生。 项目叙述：这些研究的重点是了解完全分化的组织如何规避肿瘤抑制因子 p53 的调节和监视，从而响应部分肝切除术而自我再生。我们认为，通常会停止生长和增殖的p53在再生过程中暂时无法发挥作用，然后在再生完成时恢复到正常功能。 p53 在正常细胞和再生过程中如何发挥作用基本上是未知的。