蛋白类泛素化途径（Neddylation）对原纤毛形成的调控机理探究

Primary cilia receive and transduce extracellular signals that are essential for normal development and tissue homeostasis. Mutations disrupting primary cilia cause many human genetic disease termed as “ciliopathy”. In addition, it is speculated that cilia might function during tumorigenesis as a regulatory mechanism to control developmental signals. Mislocalization or loss of essential centrosomal / ciliary proteins have been found underlie the structural or functional defects of primary cilia. Primary ciliogenesis is controlled by a remarkably complex signaling network, yet, little is known about the crucial regulatory mechanism switching on/off this program. Our preliminary data showed Neddylation strongly promotes primary ciliogenesis, indicating that: Neddylation of ciliary proteins might contribute to this process; alternatively, Neddylation may enhance the activity of CRL (Cullin-RING Ligase) to degrade inhibitors or disassembly factors. Besides, key regulators of Neddylation might switching this program on by activating essential Neddylation-related proteins. Using biochemical, cell biology and bioinformatical techniques, we will dissect the interaction network of Nedd8 by candidate or unbiased approaches to identify essential regulators for ciliogenesis. We will also determine the role of these regulators in mediating the assembly and disassembly of cilia. Finally, we will fully elucidate the molecular mechanisms underlying this selective regulation of these players by the Neddylation machinery. These studies will provide new insights into the mechanism of Neddylation-regulated ciliogenesis and may lead to novel mechanistic and therapeutic insights for ciliopathies and cancer.

原纤毛接收和转导多种胞外信号保证生物体的正常发育并维持机体内稳态。原纤毛异常会引发多种遗传性疾病，并与癌症的发生、发展密切相关。很多中心体蛋白和纤毛蛋白的缺失及突变，或者上述蛋白在细胞内的错定位都会导致原纤毛结构或功能的异常。原纤毛的形成机制复杂，尤其是调控机理尚待深入研究。我们的研究显示，Neddylation能有效促进原纤毛的形成：说明Neddylation可能直接修饰纤毛蛋白并提高其活性；或者通过活化CRL泛素连接酶，降解关键的抑制因子或促纤毛解聚因子；又或者有关键的Neddylation调节因子通过活化Neddylation相关蛋白，参与原纤毛形成的调控。在本研究中，我们将结合生物化学、细胞生物学、生物信息学等方法，分离鉴定关键纤毛调控因子，系统阐明Neddylation对上述蛋白的调节机理及其在原纤毛形成中的重要作用，并为纤毛病变性疾病与肿瘤的防治提供理论依据和潜在的治疗靶点。

纤毛是一种主要以微管为结构基础的细胞器，它通过信号通路调节发育和组织内稳态。虽然纤毛形成与解聚跟细胞周期相关，但仍缺乏分子机制的研究。我们的研究发现，Nedd 8激活酶（NAE）小分子抑制剂MLN4924，抑制原纤毛形成并加速其解聚。机制研究发现Akt1特异性调节上述过程，药物抑制（MK2206）或Akt1的敲降，逆转MLN4924对纤毛形成的抑制，但Akt2或Akt3却无类似效果。有趣的是，pAkt 1-Ser473似乎以MLN4924依赖的方式，通过Cep97和NDE1调节原纤毛的形成与解聚，而pAkt-Thr308则以VHL依赖的方式调节纤毛的长度。最后，我们证明了MLN4924抑制了小鼠毛发的再生，该过程需要原纤毛及其调控的Hh信号通路。综上所述，本研究表明蛋白Neddylation修饰通过Akt1和HVL在调节纤毛生成过程中起了意想不到的作用，并为MLN4924在治疗肿瘤等纤毛相关疾病中的潜在应用提供了理论依据与分子基础。此外，我们还发现Neddylation小分子抑制剂MLN4924通过调控细胞周期、自噬、衰老等方式，抑制胃癌细胞的增殖、存活与转移，证实了MLN4924在胃癌的治疗中有一定的应用前景。