Functional Analysis of the Von Hippel-Lindau Protein

描述(申请人提供)：von Hippel-Lindau病是一种遗传性癌症综合征，其特征是患中枢神经系统肿瘤(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人身上。VHL失活在非遗传性肾癌和血管母细胞瘤中也很常见。最被理解的VHL基因产物(PVHL)的功能与HIF有关，HIF是参与适应低氧的基因的主要转录调节因子。HIF由两种蛋白质组成，一个是‘阿尔法’亚基，一个是‘贝塔’亚基。PVHL在氧气存在的情况下以HIFAlpha为目标进行破坏。PVHL与HIFAlpha的物理相互作用要求HIFAlpha在两个脯氨酸残基中的一个被EGLN1羟化，EGLN1是一种氧依赖酶。PVHL缺陷细胞不能破坏HIFα，因此过度表达各种HIF反应基因。有3个HIFα家族成员，尽管HIF2α似乎与肾癌的相关性最大。具体目标1将询问小鼠体内HIFAlpha(尤其是HIF2Alpha)的稳定性是否足以引起pVHL缺陷时观察到的病理变化。一些VHL突变会导致非常不同的肾癌风险，尽管它们都有HIFpha缺陷。具体目标2将尝试使用细胞生物学、基因组和生化方法来了解这种不同风险的基础。一些VHL突变保留了调节HIF的能力，但仍会导致嗜铬细胞瘤。我们最近发现，所有与家族性嗜铬细胞瘤相关的基因(VHL、NF1、c-Ret、SDHB、SDHC、SDHD)都调节NGF停用后交感神经前体细胞的凋亡，在这种情况下，嗜铬细胞瘤相关的等位基因可以让细胞逃脱细胞死亡。停用NGF后的细胞凋亡需要EglN3，这是HIF Prolyl羟基酶EGLN1的类似序列。具体目标3将试图阐明EglN3是如何诱导细胞凋亡的。许多人类癌症是由过度的细胞周期蛋白D1活性引起的。在果蝇中，唯一的EglN家族成员的消除会损害Cyclin D依赖的增殖。在目标4中，我们将继续我们的初步数据，丢失哺乳动物EglN家族的第三个成员EglN，在体外和体内下调Cyclin D1。这项工作与许多人类癌症相关，并已经推动了抑制肾癌HIF反应生长因子(如血管内皮生长因子)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。这反过来又导致了可能对心脏病发作和中风等疾病有用的新药，这些疾病的特点是向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.