Functional Analysis of the Von Hippel-Lindau Protein

描述（由申请人提供）：Von Hippel-Lindau病是一种遗传性癌症综合征，其特征为中枢神经系统肿瘤（血管母细胞瘤）、肾癌和肾上腺肿瘤（嗜铬细胞瘤）的风险增加。这种疾病发生在携带VHL肿瘤抑制基因缺陷拷贝的个体中。VHL失活在非遗传性肾癌和血管母细胞瘤中也很常见。VHL基因产物（pVHL）的最好理解的功能涉及“HIF”，其是参与适应缺氧的基因的主要转录调节因子。HIF由两种蛋白质组成，一个“α”亚基和一个“β”亚基。pVHL在氧气存在时靶向HIF α进行破坏。pVHL与HIF α的物理相互作用要求HIF α在两个脯氨酰残基之一上被EglN 1羟基化，EglN 1是一种氧依赖性酶。pVHL缺陷细胞不能破坏HIF α，因此过表达各种HIF应答基因。有3个HIF α家族成员，尽管HIF 2 α似乎与肾癌最相关。具体目标1将询问小鼠中的HIF α（尤其是HIF 2 α）稳定是否足以引起pVHL缺陷时观察到的病理变化。一些VHL突变导致非常不同的肾癌风险，尽管共享HIF α缺陷。具体目标2将尝试使用细胞生物学、基因组学和生物化学方法来理解这种差异风险的基础。一些VHL突变保留了调节HIF的能力，但引起嗜铬细胞瘤。我们最近发现，所有与家族性嗜铬细胞瘤（VHL，NF 1，c-Ret，SDHB，SDHC，SDHD）相关的基因调节交感神经前体细胞的凋亡后，神经生长因子撤退和嗜铬细胞瘤相关的等位基因允许细胞在这种情况下逃避细胞死亡。NGF撤除后的细胞凋亡需要EglN 3，这是HIF脯氨酰羟化酶EglN 1的一种蛋白。具体目标3将试图阐明EglN 3如何诱导细胞凋亡。许多人类癌症是由过度的细胞周期蛋白D1活性驱动的。在果蝇中，消除唯一的EglN家族成员会损害细胞周期蛋白D依赖性增殖。在目标4中，我们将继续我们的初步数据，即哺乳动物EglN家族的第三个成员EglN 2的缺失在体外和体内下调细胞周期蛋白D1。这项工作与许多人类癌症相关，并且已经激发了在肾癌中抑制HIF反应性生长因子（如VEGF）的药物的成功临床试验。这一领域的工作也为人类细胞如何“感知”和应对氧气供应不足提供了新的见解。这反过来又导致了新的药物，这些药物可能对心脏病发作和中风等疾病有用，这些疾病的特征是组织的氧气输送受损。

DESCRIPTION (provided by applicant): Von Hippel-Lindau disease is a hereditary cancer syndrome characterized by an increased risk of central nervous system tumors (hemangioblastomas), kidney cancer, and adrenal gland tumors (pheochromocytomas). This disorder arises in individuals who harbor a defective copy of the VHL tumor suppressor gene. VHL inactivation is also common in non-hereditary kidney cancers and hemangioblastomas. The best understood function of the VHL gene product (pVHL) relates to 'HIF', which is a master transcriptional regulator of genes involved in adaptation to hypoxia. HIF consists of two proteins, an 'alpha' subunit and a 'beta' subunit. pVHL targets HIFalpha for destruction when oxygen is present. The physical interaction of pVHL with HIFalpha requires that HIFalpha be hydroxylated on one of two prolyl residues by EglN1, which is an oxygen-dependent enzyme. pVHL-defective cells fail to destroy HIFalpha and consequently overexpress various HIF-responsive genes. There are 3 HIFalpha family members although HIF2alpha appears to be most relevant with respect to kidney cancer. Specific aim 1 will ask whether HIFalpha (especially HIF2alpha) stabilization in the mouse is sufficient to cause the pathological changes observed when pVHL is defective. Some VHL mutations cause very different risks of kidney cancer despite sharing HIFalpha defects. Specific aim 2 will attempt to understand the basis for this differential risk using cell biological, genomic, and biochemical approaches. Some VHL mutations preserve the ability to regulate HIF and yet cause pheochromocytoma. We recently found that all of the genes linked to familial pheochromocytoma (VHL, NF1, c-Ret, SDHB, SDHC, SDHD) regulate apoptosis of sympathoadrehal precursor cells following NGF withdrawal and that pheochromocytoma-associated alleles allow cells to escape cell death in this setting. Apoptosis after NGF withdrawal requires EglN3, a paralog of the HIF prolyl hydroxylase EglN1. Specific aim 3 will attempt to elucidate how EglN3 induces apoptosis. Many human cancers are driven by excessive Cyclin D1 activity. In the fly, elimination of the sole EglN family member impairs Cyclin D-dependent proliferation. In aim 4 we will pursue our preliminary data that loss of EglN2, the third member of the mammalian EglN family, downregulates Cyclin D1 in vitro and in vivo. This work is relevant to many human cancers and has already motivated successful clinical trials of agents that inhibit HIF-responsive growth factors (such as VEGF) in kidney cancer. Work in this area has also provided new insights into how human cells 'sense' and respond to inadequate oxygen availability. This, in turn, is leading to new drugs that might be useful in diseases such as heart attack and stroke that are characterized by impaired oxygen delivery to tissues.