Real-Time Imaging of Hypoxia based on VHL Activity

描述(由申请人提供)： Von Hippel-Lindau病(VHL)是一种由VHL抑癌基因胚系突变引起的遗传性癌症综合征。VHL基因产物pVHL是多蛋白复合体的一部分，该复合体在氧气存在下泛素化称为HIF(缺氧诱导因子)的异源二聚体转录因子的α亚基，从而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，称为氧依赖降解结构域(ODD)。这种相互作用受到氧依赖的、酶促的羟化作用的调节，该羟化作用是在这个HIF 20聚体内保守的脯氨酸残基上进行的。我将这个20mer与萤火虫荧光素酶(奇数荧光素酶)融合在一起。在初步实验中，我建立了奇数荧光素酶在体外以羟基化依赖的方式与pVHL结合。在转基因实验中，在模拟缺氧处理后，诱导了奇数荧光素酶嵌合体，而不是野生型荧光素酶。最后，我对肿瘤异种移植的初步数据表明，可以用Xenogen相机在活体动物中检测到奇数荧光素酶活性，并且仅限于肿瘤的中心区域，已知这些区域是低氧的。我建议使用奇数荧光素酶或类似的融合蛋白，奇数绿色荧光蛋白，作为氧气生物传感器。使用这些生物传感器，我计划开发基于细胞的分析，可以用来识别直接或间接破坏VHL/HIF相互作用的化合物。此外，我将开发一种表达奇荧光素酶的转基因小鼠。这样的小鼠可以用来研究以低氧细胞存在为特征的疾病，如癌症，也可以用来监测改变组织氧合和/或调节HIF的药物的药效学效应。

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.