Regulating Gli Function in Hair Follicle Progenitors

项目摘要 基底细胞癌(BCC)，由刺猬的不适当激活引起 (HH)途径，代表了一种常见的皮肤癌，在手术治疗时，也可以表现为 快速侵袭和转移。虽然平滑抑制剂(SMOI)显示出有效性，但大多数 高级BCC获得抵抗力。5ARO54780显示BCC的表达增加 极性激酶非典型蛋白激酶C(PRKCi)增强Gli活性并赋予 抗性，尽管机制尚不清楚。PRKCi与组蛋白合作 脱乙酰酶1(HDAC1)促进谷氨酸脱乙酰化和染色质结合。一位邻居 蛋白质组学的方法导致了令人惊讶的鉴定，叶相关的亚型 多肽2(LAP2)与Gli锌指结构域结合。核膜捆绑LAP2b和 相关的乙酰赖氨酸阅读器促进谷氨酸在被称为暂停核的位置积累 保护GLi免受退化或核出口的复合体。相比之下，核质LAP2a 结合HDAC1和Gli，使其稳定地积聚在染色质上。反过来，PRKCi 防止LAP2a-HDAC1-GLI降级，同时促进从LAP2b到LAP2a的切换。现在 在它的第10个年头，5ARO54780将测试PRKCI依赖Gli的首要假设 去乙酰化直接与不同的LAP2亚型相关联，这些亚型放大了 抵抗力BCC。我们将：1)阐明Gli-LAP2b停顿核的结构和功能 当我们确定Gli活性所需的LAP2b功能结构域时，请确定 Gli-LAP2b乙酰赖氨酸阅读器的组成，并剖析LAP2是如何调节Gli的 2)阐明GLI-LAP2a激活的结构和功能 当我们确定LAP2a是否需要GLI进行协同定位时，请定义非碱基 LAP2-Gli1的接触面，并阐明PRKCi如何调节LAP2a-HDAC1-Gli的稳定性； 3)建立抗性途径的有效性和上位性，因为我们确定了 BCC耐药途径及LAP2-LLD在人和小鼠BCC中的作用 外植体。该项目的完成将加深我们对转录因子的机械性见解 核内的贩运和监管，有助于优化合理的BCC治疗，并确定新的 治疗靶点。

Project Summary Basal cell carcinoma (BCC), caused by the inappropriate activation of the hedgehog (Hh) pathway, represents a common skin cancer that, while treated surgically, can also exhibit rapid invasion and metastasis. While Smoothened inhibitors (Smoi) show efficacy, the majority of advanced BCCs acquire resistance. 5ARO54780 showed that increased expression in BCCs of the polarity kinase atypical protein kinase C (PRKCi) amplifies Gli activity and confers resistance, although the mechanism remains unknown. PRKCi cooperates with histone deacetylase 1 (HDAC1) to promote Gli deacetylation and chromatin association. A vicinal proteomics approach led to the surprising identification that isoforms of the Lamina-Associated Polypeptide 2 (LAP2) bind to the Gli zinc finger domain. Nuclear envelope tethered LAP2b and an associated acetyl-lysine reader promote Gli accumulation at a site termed the paused nuclear complex that protects Gli from degradation or nuclear export. By contrast, nucleoplasmic LAP2a binds both HDAC1 and Gli allowing it to stably accumulate on chromatin. In turn, PRKCi prevents LAP2a-HDAC1-GLI degradation while promoting switching from LAP2b to LAP2a. Now in its 10th year, 5ARO54780 will test the overarching hypothesis that PRKCi-dependent Gli deacetylation directs association with distinct LAP2 isoforms that amplify pathway activity in resistant BCCs. We will: 1) Elucidate the structure and function of the Gli-LAP2b paused nuclear complex as we identify the LAP2b functional domains required for Gli activity, identify components of the Gli-LAP2b acetyl-lysine reader, and dissect how LAP2 regulates Gli localization and mobility; 2) Elucidate the structure and function of the GLI-LAP2a activation complex as we determine whether LAP2a requires Gli for colocalization, define the non-base contact surface of LAP2-Gli1, and elucidate how PRKCi regulates LAP2a-HDAC1-Gli stability; 3) Establish resistance pathway efficacy and epistasis as we determine the prevalence of the BCC resistance pathways and determine efficacy of the LAP2-LLD in human and mouse BCC explants. Completion of the project will deepen our mechanistic insights into transcription factor trafficking and regulation in the nucleus, help optimize rational BCC therapy, and identify new therapeutic targets.