泛素化修饰对Hedgehog受体Patched在原纤毛定位与肿瘤抑制活性的调控作用与机制

Sonic Hedgehog (Shh) defines one of the key systems that regulate developmental patterning and cell fate in vertebrates, making it necessary to tightly control every step of its signaling pathway. In a pilot screen for ubiquitin ligases that control Shh signaling, we identified Smurf1 and Smurf2 and found simultaneously knockdown of the two Smurfs lead to accumulation of Shh receptor Patched (Ptch) in the primary cilium and reduction in various Shh signaling responses, suggesting that Smurfs may regulate Ptch turnover and ciliary localization. Although individual Smurf1 or Smurf2 knockout showed no ostensible developmental phenotype in mice, complete removal of all Smurf activity is embryonically lethal, with about a third of embryos displaying defects consistent with a compromised Shh pathway. In this proposal, we plan to 1) search for additional Shh-induced ubiquitinated proteins by Ub-scan; 2) complete our ubiquitin ligase screen involved in Shh signaling; 3) characterize the mechanism of Shh signaling regulation by Smurfs via controlling ubiquitination and ciliary trafficking of Ptch; and 4) elucidate potential roles of Smurfs in cerebellum development and the tumorigenesis of medulloblastoma.

Sonic Hedgehog (Shh) 定义了脊椎动物中调控形态发生和细胞分化的一个重要控制系统，Shh信号转导的每一步都受严格调控。在筛选控制Shh信号转导泛素连接酶的预实验中，我们找到Smurf1和Smurf2，并发现同时敲低两个Smurf基因会造成Shh受体Patched (Ptch)在原纤毛内聚集，并消弱各种Shh信号转导反应，说明Smurfs可能调控Ptch降解代谢与原纤毛转运。尽管Smurf1或Smurf2单敲小鼠不呈现任何胚胎发育表型，但全部剔除Smurfs则会造成胚胎致死，近1/3小鼠的胚胎缺陷与Shh通路紊乱的情况吻合。本项目计划1）利用泛素扫描寻找受Shh信号控制的泛素化蛋白；2）完成影响Shh信号转导的E3 泛素连接酶筛选；3）阐明Smurfs通过调节Ptch泛素化和原纤毛转运影响Shh信号转导的机制；4）探索Smurfs对小脑发育和髓母细胞癌发生的调控作用与机理。

Sonic Hedgehog (Shh) 定义了脊椎动物中调控形态发生和细胞分化的一个重要控制系统，Shh信号转导的每一步都受严格调控。本研究旨在阐明Smurf泛素连接酶调节Shh信号转导的机制以及这一机制的生理意义。在基金委的资助下，我们按计划研究并发现：Shh信号诱导Ptch1受体内吞降解，这一作用依赖HECT家族E3泛素连接酶Smurf1和Smurf2与Ptch1受体PPXY信号序列的相互作用。Shh信号诱导Smurfs识别Ptch1，并与其一同富集在Caveolin-1脂筏进行内吞。Ptch1受体的内吞是细胞正常应答Shh信号和小脑正常发育的必要条件，提示Smurfs是潜在的治疗依赖Shh的肿瘤的新靶标。此外，我们针对一微型前脑无裂畸形的罕见病家系，对其Ptch1的新型突变展开机制研究。