NEDD8-MODIFICATION IN VON HIPPEL-LINDAU SYNDROME

Von Hippel-Lindau综合征(VHL)是一种易患多种肿瘤的遗传性综合征，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相关肿瘤通常是多血管和过度产生血管生成多肽，如血管内皮生长因子(VEGF)，可能是因为VHL基因产物(PVHL)是低氧诱导的mRNAs的负调节因子。PVHL还通过细长蛋白B/C与人cullin-2(hCul-2)形成复合物(hCul-2-VBC)，在体内具有肿瘤抑制功能。有趣的是，在VHL综合征患者中，pVHL的一个频繁突变区域含有一个细长蛋白B/C的结合位点，该突变干扰了hCul-2-VBC复合体的形成，这表明无法形成hCul-2-VBC复合体在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NEDD8的单个分子共价修饰，NEDD8是一种新的泛素样蛋白，不以蛋白酶体降解的蛋白质为靶标。NEDD8对hCul-2的这种翻译后修饰可能调节hCul-2-VBC复合体的形成或功能。本研究以hCul-2为模型底物，对NEDD8修饰的机制和生物学功能进行了研究。特别是，我们将重点介绍NEDD8结合在VHL综合征发病机制中的作用。其目的是确定：1)NEDD8共价修饰的hCul-2的靶Lys残基；2)hCul-2的磷酸化与NEDD8结合的关系；3)NEDD8与hCul-2的结合对细胞周期进程的影响；4)NEDD8与hCul-2的结合在hCul-2-VBC复合体的形成和亚细胞定位中的作用以及在低氧诱导的mRNA调节中的作用，如血管内皮生长因子。

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.