Mechanism of Gli3 processing in Hedgehog Signaling

描述（申请人提供）：Hedgehog （Hh）家族分泌信号分子在细胞命运规范、细胞增殖和分化中起重要作用。Hh信号的功能障碍导致人类胚胎发育异常和几种癌症；因此，了解Hh信号转导的分子机制对于预防和治疗Hh信号异常导致的出生缺陷和癌症具有重要意义。在小鼠中，Gli2和Gli3含锌指的转录因子是Hh信号的主要介质。在Hh信号缺失的情况下，大多数全长Gli3蛋白（Gli3FL）被蛋白水解加工成转录抑制因子（Gli3Rep）。Gli3的加工需要蛋白激酶A （PKA）、糖原合成酶激酶3 （GSK3）和酪蛋白激酶1 （CK1）的c端磷酸化，然后通过SCFTrCP E3泛素连接酶进行泛素化。多泛素化的Gli3随后被蛋白酶体处理。Gli3FL主要定位于细胞质中，穿梭于细胞质和细胞核之间，而Gli3Rep只存在于细胞核中。Hh信号通过一种未知的机制对抗Gli3加工，并将Gli3FL转化为Gli3Act激活剂，从而抑制Hh靶基因。Gli3和Gli2的稳定性也受到严格控制。迄今为止，在分子水平上对Hh信号如何调节Gli3加工、核易位或Gli2和Gli3的稳定性知之甚少。遗传和有限的生化证据表明，Hh信号通过各种效应分子调节这些分子事件，尽管这些效应分子与Gli蛋白之间的联系尚不清楚。这些效应物中的许多是脊椎动物特有的，尽管有些在果蝇中是保守的。我们的目标是确定这些效应物在Hh信号传导中的作用，以及它们调节Gli2和Gli3功能的分子机制。具体而言，Aim 1将了解Dzip1在Gli3功能调控中的作用，Aim 2将阐明利用纤颤内转运（IFT）和Dynein基因突变体加工Gli3的分子机制，Aim 3将了解Sufu突变体Hh通路激活的分子基础。这些研究的完成将大大促进我们对Gli3蛋白在Hh信号传导中的调控和功能的理解。公共卫生相关性：Hedgehog （Hh）家族分泌的信号蛋白在细胞命运规范和细胞增殖和分化中起着重要作用。Hh信号的缺失会导致多种出生缺陷，而Hh通路的异常激活会导致几种类型的人类癌症。了解Hh信号传导的分子机制可以为设计治疗药物以调节Hh通路活性提供见解，从而预防或治疗由Hh信号传导活性失调引起的出生缺陷和癌症。

DESCRIPTION (provided by applicant): The Hedgehog (Hh) family of secreted signaling molecules plays an important role in cell fate specification, cell proliferation and differentiation. Dysfunction in Hh signaling results in abnormal embryonic development and several types of cancer in human; thus, understanding the molecular mechanisms of Hh signal transduction is of great significance for the prevention and remedy of the birth defects and cancers caused by aberrant Hh signaling. In mice, Gli2 and Gli3 zinc-finger-containing transcription factors are the primary mediators of Hh signaling. In the absence of a Hh signal, most full-length Gli3 protein (Gli3FL) is proteolytically processed to a transcriptional repressor (Gli3Rep). Gli3 processing requires C-terminal phosphorylation by protein kinase A (PKA), glycogen synthase kinase 3 (GSK3), and casein kinase 1 (CK1), and subsequent ubiquitination by SCFTrCP E3 ubiquitin ligase. Polyubiquitinated Gli3 is then processed by the proteasome. Gli3FL is predominantly localized in the cytoplasm and shuttles between the cytoplasm and nucleus, while Gli3Rep is exclusively found in the nucleus. Hh signaling counters Gli3 processing through an unknown mechanism and converts Gli3FL into an activator, Gli3Act, thus derepressing Hh target genes. The stability of Gli3, as well as Gli2, is also tightly controlled. To date, little is known at the molecular level about how Hh signaling regulates Gli3 processing, nuclear translocation, or Gli2 and Gli3 stability. Genetic and limited biochemical evidence indicates that Hh signaling regulates these molecular events through various effector molecules, though the connections between these effectors and Gli proteins are unknown. Many of these effectors are vertebrate specific, although some are conserved in Drosophila. Our objectives are to determine the role of these effectors in Hh signaling and the molecular mechanisms by which they regulate Gli2 and Gli3 function. Specifically, Aim 1 will understand the role of Dzip1 in the regulation of Gli3 function, Aim 2 will elucidate the molecular mechanism of Gli3 processing using intraflagellar transport (IFT) and Dynein gene mutants, and Aim 3 will understand the molecular basis of the Hh pathway activation in Sufu mutant. The completion of the proposed studies will significantly advance our understanding of the regulation and function of Gli3 protein in Hh signaling. PUBLIC HEALTH RELEVANCE: The Hedgehog (Hh) family of secreted signaling proteins plays fundamental roles in cell fate specification and cell proliferation and differentiation. Loss of Hh signaling results in a wide range of birth defects, whereas aberrant activation of the Hh pathway causes several types of human cancer. Understanding the molecular mechanism of Hh signaling may provide insights into the design of therapeutic agents to modulate Hh pathway activity and therefore prevent or treat birth defects and cancers caused by misregulation of the Hh signaling activity.