Mechanisms regulating protein stability of the Gli transcription factor

Gli 转录因子蛋白稳定性的调节机制

• 批准号: 7450815
• 负责人: Ivette S. Estay
• 金额: $ 5.18万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7450815
• 关键词: Affect; Basal cell carcinoma; Binding; Biological Assay; C-terminal; Cells; Chimeric Proteins; Complex; Data; Development; Dominant-Negative Mutation; Epithelium; Erinaceidae; Fellowship; Goals; Hair follicle structure; Human; In Vitro; Individual; Kinetics; Luciferases; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Modeling; Mutation Analysis; N-terminal; Names; Protein Overexpression; Proteins; Proteolysis; RNA Interference; Regulation; Role; SKP Cullin F-Box Protein Ligases; Serine; Signal Pathway; Signal Transduction; Skin; Technology; Trans-Activators; Ubiquitin; Ubiquitination; adapter protein; base; gain of function; gene induction; in vivo; keratinocyte; multicatalytic endopeptidase complex; mutant; prevent; protein degradation; tool; transcription factor; ubiquitin ligase; ubiquitin-protein ligase

DESCRIPTION (provided by applicant): Inappropriate Sonic hedgehog (Shh) target gene induction has been implicated in many human cancers, including skin Basal cell carcinomas (BCCs). Previous studies have shown that regulation of Shh signal reception in skin keratinocytes occurs in part by regulating Gli transcription factor protein stability. Preliminary data has demonstrated two regions of Gli that mediate destruction. C-terminal sequences contain a ubiquitin-proteasome destruction signal or degron that is recognized by the adapter protein, ¿TrCP, a ubiquitin ligase. However, the identity of the putative N-terminal degron and the complex that binds to it remains unknown. The goal of this proposal is to identify the N-terminal sequences and trans-acting factors that bind to it to mediate Gli protein degradation. We aim to: 1) Determine the minimal sequences that define the degron and are sufficient to confer protein instability to chimeric proteins via deletion mutant analysis and degradation assays. We will assess how known regulators of Shh signaling affect both the N and C terminal degrons; 2) Identify Gli1 N-terminal associated proteins via protein pull-down assays and mass spectrometry technology. We will characterize Gli1 N-terminal associated proteins in Gli1 protein stability and ubiquitination assays using loss and gain of function analyses.

描述（由申请人提供）：不适当的 Sonic Hedgehog (Shh) 靶基因诱导与许多人类癌症有关，包括皮肤基底细胞癌 (BCC)。 先前的研究表明，皮肤角质形成细胞中Shh信号接收的调节部分是通过调节Gli转录因子蛋白稳定性来实现的。 初步数据表明 Gli 的两个区域可以介导破坏。 C 端序列包含泛素蛋白酶体破坏信号或降解决定子，可被接头蛋白 TrCP（一种泛素连接酶）识别。 然而，假定的 N 端降解决定子以及与其结合的复合物的身份仍然未知。 该提案的目标是鉴定 N 端序列和与其结合以介导 Gli 蛋白降解的反式作用因子。 我们的目标是：1）通过缺失突变体分析和降解测定确定定义降解决定子并足以赋予嵌合蛋白蛋白质稳定性的最小序列。 我们将评估已知的 Shh 信号调节因子如何影响 N 和 C 末端降解决定子； 2) 通过蛋白质下拉分析和质谱技术鉴定 Gli1 N 末端相关蛋白。 我们将使用功能丢失和获得分析来表征 Gli1 蛋白稳定性和泛素化测定中的 Gli1 N 末端相关蛋白。