Mechanisms regulating protein stability of Gli

Gli 蛋白稳定性调节机制

• 批准号: 7055776
• 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 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7055776
• 关键词: 3T3 cells; chemical kinetics; chemical stability; chimeric proteins; gene deletion mutation; mass spectrometry; phosphorylation; predoctoral investigator; protein degradation; protein sequence; sonic hedgehog gene /protein; transcription factor; 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地通过调节Gli转录因子蛋白质的稳定性而发生。 初步数据表明，Gli的两个区域介导了破坏。 C-末端序列包含一个泛素-蛋白酶体破坏信号或降解决定子，它被衔接蛋白，一种泛素连接酶，识别。 然而，推定的N-末端降解决定子和与其结合的复合物的身份仍然未知。 本提案的目标是确定N-末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限定降解决定子并且足以赋予嵌合蛋白质蛋白质不稳定性的最小序列。 我们将评估已知的Shh信号调节剂如何影响N和C末端降解决定子; 2）通过蛋白质下拉测定和质谱技术鉴定Gli 1 N末端相关蛋白。 我们将在Gli 1蛋白稳定性和泛素化分析中使用功能分析的丧失和获得来表征Gli 1 N-末端相关蛋白。