p53-Regulation in Liver Regeneration

p53-肝脏再生的调节

• 批准号: 7634451
• 负责人: Michelle Ann Barton
• 金额: $ 32.73万
• 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/7634451
• 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; TP53 gene; Tissues; Tumor Suppressor Proteins; Tumor-Derived; Work; chromatin immunoprecipitation; genome wide association study; 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）：响应手术切除（部分肝切除术，PHX）或通过化学物质或疾病破坏肝组织，刺激了完全分化的肝脏的重新器官发生。诱导的信号转导途径网络触发了肝细胞（其次是非核细胞）重新进入细胞周期，增殖和补偿性生长，这些末端终止于精确调节的终点。我们假设p53肿瘤抑制蛋白的关键功能在促进细胞周期停滞和凋亡时必须被响应部分肝切除术而阻塞或衰减。我们的目标是定义这种监管过程的机制。 有关p53调控和功能的大量信息主要源于对肿瘤衍生细胞，长期，连续培养和/或表达功能失调，突变或外源性p53的细胞的研究。尽管有证据表明p53在发育，衰老和衰老过程中p53在正常细胞中起作用，但我们知识上存在有关内源性p53调节机制的主要差距。肿瘤抑制p53以低水平表示，由多个途径紧密控制。这为内源性p53的生化纯化和机械分析提供了巨大的挑战，尤其是在正常细胞中。 我们已经开发了一种新的鼠标模型来解决这些差距。由敲门方法创建的小鼠模型表达内源性p53与c末端表位标记（TAP-P53）的框架融合。 TAP或串联亲和纯化是在相对生理条件下净化低丰度，大蛋白质复合物的一种经过验证的手段。 TAP-P53纯化和随后通过质谱分析的肽分析将用于定义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）（CHIP）和全基因组对p53-targets（CHIP-CHIP）进行p53-染色质相互作用的研究。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，并确定在肝脏再生过程中如何改变这些功能。 拟议的研究将解决p53在正常的分化细胞中的功能，该区域在研究肿瘤抑制活性的研究中通常被忽略了。这项工作将进一步理解组织再生的分子理解，其长期目标是了解如何在时间控制p53-NetWork的监视状态以促进细胞更新和组织再生。 项目叙述：这些研究的重点是理解完全分化的组织如何规避肿瘤抑制p53的调节和监视，以响应部分肝切除术而重生。我们认为，通常会停止生长和增殖的p53在再生过程中暂时阻止功能，然后在再生结束后恢复到正常的容量。 p53在正常细胞和再生过程中的功能基本未知。