Molecular-Genetic Analysis of 3p14 Genomic Stability

描述（由申请人提供）：TRC 8代表与VHL相互作用的任何蛋白质的首次重排，其功能可能与肾细胞癌（RCC）的发展密切相关。通过定位克隆一个遗传性RCC和非甲状腺髓样癌家族的t（3;8）易位，鉴定了TRC 8基因。受影响的个人缺乏生殖系VHL突变，虽然RCC的形态是相同的。VHL作为SCF相关（VCB）连接酶复合物中的F盒蛋白发挥作用，靶向HIF α进行泛素化。TRC 8位于ER中，含有具有E3-泛素连接酶活性的RING-H2指。据信VHL的至少部分功能涉及替代（非VCB）泛素连接酶复合物中的ER。为了鉴定TRC 8的功能，我们分离了果蝇同源物DTrc 8，并在果蝇中利用遗传方法。DTrc 8和DVhl的缺失导致相同的中线表型，并且Trc 8在物理上和遗传上与Vhl相互作用。通过双杂交筛选，我们发现人和果蝇TRC 8与JAB 1/CSNS的MPN结构域相互作用，JAB 1/CSNS是COP 9信号体的一个组分，其通过从Cullin中去除NEDD 8来调节SCF功能。Jab 1/Csn 5具有去核化酶活性，并且JAB 1也已显示出调节p27 kip 1的核质转运和蛋白酶体介导的降解，p27 kip 1在RCC中是特征性异常的并且与VHL突变相关。DTrc 8的过表达是生长抑制性的，具有几种不同的表型，并且Trc 8和Vhl的过表达一起产生独特的翅表型。果蝇中的遗传杂交表明，Trc 8过表达的影响被细胞周期蛋白E纠正，并被p27 kipl样分子Dacapo加剧。因此，我们假设Trc 8过表达表型依赖于Trc 8与Vhl和Jab 1/Csn 5的相互作用。我们还假设，人类TRC 8的过表达将在哺乳动物细胞中产生类似的后果。我们的总体策略是定义相互作用域并引入点突变来破坏它们。将在转基因果蝇中检测突变蛋白的以下方面：1）Trc 8过表达导致的表型改变; 2）信号体的生化特性（去卷曲和蛋白质复合物）; 3）对确定的Jab 1/Csn 5突变表型的影响。将测试来自RCC的天然存在的VHL突变对TRC 8结合、ER定位和功能以及VHL复合物组装的影响。将在哺乳动物细胞中分析表达野生型和突变型TRC 8的腺病毒对Cullin去螺旋化、VHL-GFP融合体的泛素化、细胞周期参数和调节因子水平的影响。在我们的最终目标中，表达wt或突变体DTrc 8的有丝分裂重组克隆将用于Trc 8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 DTrc 8在成年感觉毛中诱导Minute样表型，因此将测试eIF 3 h和eIF 3f的MPN结构域的相互作用，并且如果阳性则进一步表征。

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.