NEDD8-MODIFICATION IN VON HIPPEL-LINDAU SYNDROME

von Hippel-Lindau（VHL）综合征是一种遗传性综合征，易使受影响的患者发生各种肿瘤，包括散发性肾细胞癌、嗜铬细胞瘤和CNS血管母细胞瘤。 VHL相关肿瘤通常是血管过多的，并过度产生血管生成肽，如血管内皮生长因子（VEGF），这可能是因为VHL基因产物（pVHL）是缺氧诱导mRNA的负调节因子。 还已知pVHL通过延伸蛋白B/C与人cullin-2（hCul-2）形成复合物（hCul-2-VBC），并且在体内具有肿瘤抑制功能。有趣的是，VHL综合征患者中pVHL的频繁突变区域含有延伸蛋白B/C的结合位点，并且突变干扰hCul-2-VBC复合物的形成，表明不能形成hCul-2-VBC复合物在VHL综合征的发病机制中起关键作用。 最近，我们发现hCul-2被一个单一分子的NEDD 8共价修饰，NEDD 8是一种新型的泛素样蛋白，其不靶向蛋白质被蛋白酶体蛋白水解降解。 NEDD 8对hCul-2的翻译后修饰可以调节hCul-2- VBC复合物的形成或功能。本研究以hCul-2为模型底物，研究NEDD 8修饰的机制和生物学功能。 特别是，我们将重点关注NEDD 8结合在VHL综合征发病机制中的作用。 目的是界定：1）被NEDD 8共价修饰的hCul-2的靶Lys残基，2）磷酸化与hCul-2的NEDD 8-缀合之间的关系，3）NEDD 8-缀合至hCul-2对细胞周期进程的影响，4）NEDD 8-缀合至hCul-2在hCul-2-VBC复合物的形成和亚细胞定位中的作用以及在低氧诱导的mRNA的调节中的作用，例如VEGF。

The von Hippel-Lindau (VHL) syndrome is a hereditary syndrome that predisposes affected patients to develop a variety of neoplasms including sporadic renal cell carcinomas, pheochromocytomas, and CNS hemangioblastomas. VHL-associated neoplasms are typically hypervascular and overproduce angiogenic peptides, such as vascular endothelial growth factor (VEGF), probably because the VHL gene product (pVHL) is a negative regulator of hypoxia-inducible mRNAs. pVHL is also known to form a complex (hCul-2-VBC) with human cullin-2 (hCul-2) through elongins B/C and possesses a tumor suppressor function in vivo. Interestingly, a frequently mutated region of pVHL in patients with VHL syndrome contains a binding site for elongins B/C and the mutations interfere with the formation of the hCul-2-VBC complex, suggesting that the inability to form the hCul-2-VBC complex plays a critical role in the pathogenesis of VHL syndrome. Recently, we found that hCul-2 is covalently modified by a single molecule of NEDD8, a novel ubiquitin-like protein which does not target proteins for proteolytic degradation by proteasome. This post-translational modification of hCul-2 by NEDD8 may regulate the formation or the function of the hCul-2- VBC complex. This proposal is designed to study the mechanism and biological function of NEDD8-modification, using hCul-2 as a model substrate. In particular, we will focus on the role of NEDD8- conjugation in the pathogenesis of VHL syndrome. The aims are to define: 1) the target Lys residue of hCul-2 which is covalently modified by NEDD8, 2) the relationship between phosphorylation and NEDD8-conjugation of hCul-2, 3) the effect of NEDD8- conjugation to hCul-2 on cell-cycle progression, 4) the role of NEDD8-conjugation to hCul-2 in the formation and subcellular localization of hCul-2-VBC complex and in the regulation of hypoxia-inducible mRNA, such as VEGF.