Molecular-Genetic Analysis of 3p14 Genomic Stability

描述(申请人提供)：TRC8代表与VHL相互作用的任何蛋白质的第一次重排，其功能可能与肾细胞癌(RCC)的发展密切相关。TRC8基因是通过对一个遗传性肾细胞癌和非髓样甲状腺癌家系的t(3；8)易位进行定位克隆而确定的。虽然RCC的形态是相同的，但受影响的个体缺乏胚系VHL突变。VHL在干细胞因子相关(VCB)连接酶复合体中作为F-box蛋白发挥作用，该复合体针对HIFpha进行泛素化。TRC8位于内质网，含有一个具有E3-泛素连接酶活性的环-H2指。至少部分VHL的功能被认为涉及内质网的替代(非VCB)泛素连接酶复合体。为了确定TRC8的功能，我们分离了果蝇的同源物DTrc8，并利用了果蝇的遗传方法。DTrc8和DVhl的缺失导致相同的中线表型，并且Trc8在物理和遗传上与VHL相互作用。从双杂交筛选中，我们发现人和果蝇TRC8与JAB1/CSNS的MPN结构域相互作用，JAB1/CSNS是COP9信号体的一个组成部分，通过从Cullins中移除NEDD8来调节SCF功能。Jab1/Csn5具有脱氢酶活性，Jab 1还被证明调节核质转运和蛋白酶体介导的p27kip1的降解，这在肾细胞癌中是典型的异常，并与VHL突变有关。DTrc8的过表达具有几种不同的表型，对生长有抑制作用，而Trc8和VHL的过表达共同产生了一种独特的翅膀表型。在果蝇中进行的遗传杂交表明，Trc8的过度表达可以被细胞周期蛋白E纠正，并被类似p27kipl的分子Dacapo加剧。因此，我们假设Trc8过表达的表型依赖于Trc8与VHL和Jab1/Csn5的相互作用。我们还假设，人类TRC8的过度表达将在哺乳动物细胞中产生类似的后果。我们的总体战略是定义相互作用域，并引入扰乱它们的点突变。突变蛋白将在转基因果蝇中进行测试：1)Trc8过表达导致的表型变化；2)信号小体的生化特性(去动力和蛋白质复合体)；3)对确定的Jab1/Csn5突变表型的影响。来自RCC的自然发生的VHL突变将被测试它们对TRC8结合、ER的定位和功能以及VHL复合体的组装的影响。将在哺乳动物细胞中分析表达wt和突变的trc8的腺病毒对cullin脱染、VHL-GFP融合的泛素化、细胞周期参数和调控因子水平的影响。在我们的最终目标中，表达wt或突变型DTrc8的有丝分裂重组克隆将用于Trc8功能的独立分析(并将结果整合到哺乳动物分析中)。最后，由于wt DTrc8在成人感觉毛发中诱导出微小的表型，因此将测试eIF3h和eIF3f的MPN结构域是否相互作用，并进一步鉴定是否为阳性。

DESCRIPTION (provided by applicant): TRC8 represents the first rearrangement of any protein that interacts with VHL and its function may be germane to the development of renal cell carcinoma (RCC). The TRC8 gene was identified by positional cloning of a t(3;8) translocation in a family with hereditary RCC and non-medullary thyroid cancer. Affected individuals lacked germline VHL mutations although the morphology of the RCCs was identical. VHL functions as an F-box protein in an SCF-related (VCB) ligase complex that targets HIFalpha for ubiquitination. TRC8 is located in the ER and contains a RING-H2 finger with E3-ubiqutin ligase activity. At least part of the function of VHL is believed to involve the ER in an alternative (non-VCB) ubiquitin ligase complex. To identify a TRC8 function, we isolated the Drosophila homologue, DTrc8, and exploited genetic approaches in flies. Loss of DTrc8 and DVhl result in identical midline phenotypes, and Trc8 physically and genetically interacts with Vhl. From a 2-hybrid screen, we found that human and Drosophila TRC8 interact with the MPN domain of JAB1/CSNS, a component of the COP9 Signalosome which regulates SCF function by removing NEDD8 from Cullins. Jab1/Csn5 has deneddylase activity, and JAB 1 has also been shown to regulate nuclear-cytoplasmic transport and proteasome-mediated degradation of p27 kip1, which is characteristically abnormal in RCCs and which has been linked to VHL mutations. Overexpression of DTrc8 is growth suppressive with several distinct phenotypes, and overexpression of Trc8 and Vhl together produces a unique wing phenotype. Genetic crosses in flies demonstrate that the effects of Trc8 overexpression are corrected by cyclin E and exacerbated by the p27kipl-like molecule, Dacapo. Thus, we hypothesize that the Trc8 overexpression phenotypes are dependent upon the interactions of Trc8 with Vhl and Jab1/ Csn5. We also hypothesize that overexpression of human TRC8 will have similar consequences in mammalian cells. Our overall strategy is to define the interaction domains and introduce point mutations that disrupt them. Mutant proteins will be tested in transgenic flies for: 1) alterations in phenotypes resulting from Trc8 overexpression; 2) biochemical properties of the Signalosome (deneddylation and protein complexes) and, 3) effects on defined Jab1/Csn5 mutant phenotypes. Naturally occurring VHL mutations from RCCs will be tested for their effects on TRC8 binding, ER localization and function, and the assembly of VHL complexes. Adenoviruses expressing wt and mutant TRC8 will be analyzed in mammalian cells for effects on Cullin deneddylation, ubiquitination of a VHL-GFP fusion, cell-cycle parameters and levels of regulatory factors. In our final Aim, mitotic recombinant clones expressing wt or mutant DTrc8 will be used for an independent analysis of Trc8 function (and results integrated into the mammalian analysis). Lastly, because wt DTrc8 induces Minute-like phenotypes in adult sensory hairs, the MPN domains of eIF3h and eIF3f will be tested for interactions and further characterized if positive.