NEDD8-MODIFICATION IN VON HIPPEL-LINDAU SYNDROME

von Hippel-Lindau (VHL) 综合征是一种遗传性综合征，使受影响的患者容易发生多种肿瘤，包括散发性肾细胞癌、嗜铬细胞瘤和中枢神经系统血管母细胞瘤。 VHL 相关肿瘤通常血管丰富，并过度产生血管生成肽，例如血管内皮生长因子 (VEGF)，可能是因为 VHL 基因产物 (pVHL) 是缺氧诱导 mRNA 的负调节因子。 pVHL 还已知通过 elongins B/C 与人 cullin-2 (hCul-2) 形成复合物 (hCul-2-VBC)，并在体内具有肿瘤抑制功能。有趣的是，VHL综合征患者的pVHL频繁突变区域包含elongins B/C的结合位点，并且该突变干扰hCul-2-VBC复合物的形成，表明无法形成hCul-2-VBC复合物在VHL综合征的发病机制中起着关键作用。 最近，我们发现 hCul-2 被单分子 NEDD8 共价修饰，NEDD8 是一种新型泛素样蛋白，不靶向被蛋白酶体蛋白水解降解的蛋白质。 NEDD8 对 hCul-2 的这种翻译后修饰可以调节 hCul-2-VBC 复合物的形成或功能。该提案旨在以hCul-2作为模型底物研究NEDD8修饰的机制和生物学功能。 我们将特别关注 NEDD8- 结合在 VHL 综合征发病机制中的作用。 目的是定义：1) hCul-2 的目标赖氨酸残基被 NEDD8 共价修饰，2) hCul-2 的磷酸化和 NEDD8 缀合之间的关系，3) NEDD8 与 hCul-2 缀合对细胞周期进程的影响，4) NEDD8 与 hCul-2 缀合在细胞周期形成和亚细胞定位中的作用。 hCul-2-VBC 复合物和缺氧诱导 mRNA（例如 VEGF）的调节。

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.