Real-Time Imaging of Hypoxia based on VHL Activity

描述（由申请人提供）： Von Hippel-Lindau（VHL）病是由VHL肿瘤抑制基因的生殖系突变引起的遗传性癌症综合征。VHL基因产物pVHL是多蛋白复合物的一部分，该复合物在氧存在下聚泛素化异源二聚体转录因子HIF（缺氧诱导因子）的α亚基，这导致它们被蛋白酶体破坏。PVHL直接与位于HIF区域内的共线20个氨基酸残基序列结合，该区域称为氧依赖性降解结构域（ODD）。这种相互作用是严格调节氧依赖性，酶促羟基化的保守脯氨酸残基在这个HIF 20聚体。我将这20聚体与萤火虫荧光素酶（ODD-荧光素酶）融合。在初步实验中，我建立了ODD-荧光素酶结合pVHL在体外的羟基化依赖性的方式。在转染实验中，用低氧模拟物处理后诱导了ODD-荧光素酶嵌合体，而不是野生型荧光素酶。最后，我对肿瘤异种移植物的初步数据表明，ODD-荧光素酶活性可以在活体动物中用Xenogen相机检测到，并且仅限于肿瘤的中心区域，已知这些区域是缺氧的。我建议使用ODD-荧光素酶或类似的融合蛋白，ODD-GFP，作为氧生物传感器。利用这些生物传感器，我计划开发基于细胞的检测方法，用于鉴定直接或间接破坏VHL/HIF相互作用的化合物。此外，我将开发一种表达ODD-荧光素酶的转基因小鼠。这样的小鼠可用于研究以缺氧细胞的存在为特征的疾病，例如癌症，并且还可用于监测改变组织氧合和/或调节HIF的药剂的药效学效应。

DESCRIPTION (provided by applicant): Von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome caused by germline mutation of the VHL tumor suppressor gene. The VHL gene product, pVHL, is part of a multiprotein complex that polyubiquitinates the alpha subunits of the heterodimeric transcription factor called HIF (hypoxia-inducible factor) in the presence of oxygen, which results in their destruction by the proteasome. PVHL binds directly to a colinear 20 amino acid residue sequence located within a region of HIF called the Oxygen-Dependent Degradation Domain (ODD). This interaction is strictly regulated by oxygen-dependent, enzymatic hydroxylation of a conserved proline residue within this HIF 20 mer. I fused this 20 mer to firefly luciferase (ODD-Luciferase). In pilot experiments I established that ODD-luciferase binds to pVHL in vitro in a hydroxylation-dependent manner. In transfection experiments the ODD-luciferase chimera, but not wild-type luciferase, was induced following treatment with hypoxia mimetics. Finally, my preliminary data with tumor xenografts indicate that ODD-luciferase activity can be detected with a Xenogen camera in living animals and is restricted to the central regions of tumors, which are known to be hypoxic. I propose to use ODD-luciferase or an analogous fusion protein, ODD-GFP, as oxygen biosensors. Using these biosensors, I plan to develop cell based assays that might be used to identify compounds that either directly or indirectly disrupt the VHL/HIF interaction. Furthermore, I will develop a transgenic mouse expressing ODD-luciferase. Such mice could be used to study diseases characterized by the presence of hypoxic cells, such as cancer, and could also be used to monitor pharmacodynamic effects of agents that alter tissue oxygenation and/or modulate HIF.