Mechanisms regulating protein stability of the Gli transcription factor

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（BCCs）。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转录因子蛋白的稳定性来实现的。初步数据显示，Gli有两个区域负责调节破坏。c端序列包含泛素-蛋白酶体破坏信号或降解子，该信号或降解子可被适配蛋白TrCP（一种泛素连接酶）识别。然而，推测的n端degron的身份和与它结合的复合物仍然未知。本研究的目的是鉴定n端序列及其结合介导Gli蛋白降解的反式作用因子。我们的目标是：1)通过缺失突变分析和降解试验，确定定义降解子并足以赋予嵌合蛋白蛋白质不稳定性的最小序列。我们将评估已知的Shh信号调节因子如何影响N端和C端退化；2)通过蛋白下拉分析和质谱技术鉴定Gli1 n端相关蛋白。我们将在Gli1蛋白稳定性和泛素化分析中使用功能损失和增益来表征Gli1 n端相关蛋白。

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.