Mechanisms regulating protein stability of Gli

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转录因子蛋白的稳定性来实现的。初步数据显示，GLI的两个区域参与了破坏。C-端序列含有泛素-蛋白酶体破坏信号或降解蛋白，可被泛素连接酶的接头蛋白TrCP识别。然而，推测的N-末端降解子及其结合的复合体的身份仍不清楚。这项提议的目的是确定N-末端序列和与其结合的反式作用因子，以介导Gli蛋白的降解。我们的目标是：1)通过缺失突变分析和降解分析，确定定义变性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和C末端降解物；2)通过蛋白质下拉分析和质谱学技术鉴定Gli1 N末端相关蛋白质。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分析来表征Gli1 N末端相关蛋白。

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.