Hedgehog信号途径转录因子Ci稳定性调节的研究

Hedgehog (Hh) signaling pathway is essential for cell growth control and differentiation both in vertebrate and invertebrate development.Disorder of Hh signaling can lead to a variety of human diseases, including cnacer and birth defect. The transcription factor of Hh signaling in Drosophila, Ci, and its vertebrate homologous Gli, contribute to regulating downstream gene expressions on a concentration dependent manner. The stability and activity of Ci/Gli are tightly controled by Hh gradient. It is well known that Cul1-Slimb and Cul3-Hib complexes play a very important role in the regulation of Ci stability. However, it is still largely unknown how these two ubiquitin E3 ligases regulate Ci155 processing to Ci75 or completely degradation. In this study, we will figure out the ubiquitin sites on Ci which are response to Ci processing or the completely degradation by using proteomics method. We will also investigate the effect of these sites on Ci stability, therefore on Hh signaling. In addition, we will seek the new Ci potential binding protein which will be involved in Ci processing and degradation. In this study, we will try to uncover the mechanism by which Ci processing and degradation are regulated by ubiquitiation and potential proteins. Considering the conservation between Ci and Gli, the mechanism study of Ci stability regulation might contribute to the clinical treatment of human diseases caused by Hh signaling disorder.

Hedgehog (Hh) 信号途径控制脊椎及非脊椎动物众多发育过程中细胞的生长及分化。该通路的错误调节可导致多种人类疾病。果蝇中的Ci蛋白是该途径的关键转录因子,与脊椎动物中的Gli蛋白同源。不同浓度的Hh通过对Ci/Gli活性及稳定性的调节严格控制下游基因的表达。虽然已知Cul1-Slimb与Cul3-HIB在该调节过程中起重要作用，但二者如何调控Ci155降解成抑制因子Ci76或者将Ci155完全降解仍是亟待解决的问题。本项目旨在探索不同Hh浓度下Ci的泛素化修饰位点，并验证该蛋白修饰的功能。本项目还将对控制Ci155降解为Ci76的结合蛋白进行寻找及其机制研究。鉴于Ci/Gli功能在进化上的保守性，对Ci活性调控机制的研究不仅会加深对Hh信号转导途径机理的理解，同时在由Hh信号转导途径异常导致的人类疾病的临床治疗上也会起到重要的指导性作用。

Hedgehog信号通路控制了脊椎动物以及非脊椎动物众多发育过程中细胞的生长及分化。该信号通路的关键转录因子Ci/Gli的活性及稳定性严格受到Hh信号的调节，并且直接影响下游信号通路的激活。已有的研究对Ci155加工成Ci75的具体分子机制以及Ci稳定性的调节机制的报道仍然有限。我们的研究工作以阐明这一基本问题为出发点，在果蝇细胞系中筛选出了能够与Ci结合并且可能参与调节Ci活性及稳定性的候选蛋白，通过深入研究，发现了Myosin调节轻链蛋白SQH（CG3595）在Hh信号通路中扮演的新角色——SQH在Hh信号通路中参与调控了Ci的稳定性。SQH参与保护Ci155及Ci75使之免于降解，除此之外，存在Hh的条件下，SQH能够促进Ci155加工成Ci75。研究还发现，T20S21两个位点的磷酸化对其功能的发挥至关重要，这一修饰是受Hh信号调控的。根据研究结果，我们提出了一个机制模型：SQH对Ci稳定性的调节是通过其自身磷酸化与非磷酸化修饰形式的动态平衡实现的。在Hh通路中，这一动态微调控机制避免了Hh信号通路的过度激活及抑制。存在Hh条件下，Ci主要以转录激活因子Ci155发挥功能，作为次要调节，磷酸化的SQH能够与Ci155结合，并可能招募其他降解相关蛋白共同参与了Ci155加工成Ci75的过程，并维持Ci75的少量存在，以限制下游基因的过度激活；不存在Hh条件下，Ci主要被加工成转录抑制因子Ci75，作为次要调节，此时SQH主要以非磷酸化形式存在，未被磷酸化修饰的SQH与Ci结合，并且部分抑制Ci155的降解，以此防止下游基因的过度抑制。这一研究结果在Hh信号通路研究领域第一次揭示了SQH参与调控了Ci的稳定性，有助于人们对Hh信号通路的理解以及相关人类疾病的研究。