Functional Dissection of an F-box Protein in Development

描述（由申请人提供）：蛋白质降解与转录和其他转录后调控一起整合到发育期间的基因表达调控中。随着F-box蛋白的发现，我们剖析这种整合机制的能力已经大大提高，F-box蛋白作为特异性因子，靶向泛素介导的降解的特定底物。我们的主要目标是了解（i）F-box蛋白是如何调节的，（ii）这些蛋白可以执行什么样的基因调控功能，以及（iii）共享一个共同的F-box蛋白的降解底物如何在功能上相互关联。作为一个模型系统，我们正在解剖的伴侣配对（Ppa），一个新发现的F-box蛋白参与调控果蝇胚胎发育过程中的分割和核周期的功能。我们建议在果蝇和斑马鱼中检查Ppa功能。我们的具体目的是：（1）确定Ppa是否具有转录抑制活性。我们将测试的假设，除了调节配对转录因子的降解，果蝇Ppa也抑制配对的能力，激活靶基因的转录。我们将在体内测试Ppa缺失构建体，并测试Ppa的假定转录抑制结构域是否具有异源DNA结合活性。我们还将确定斑马鱼Ppa是否具有抑制活性。(2)描述Ppa的转录后调控。我们的初步表达分析表明，果蝇Ppa蛋白的表达在转录后水平进行调节，以上调Ppa蛋白在细胞中具有高水平的底物需要降解。使用异位表达研究、缺失分析和蛋白质合成抑制，我们将评估这种调节的底物依赖性，以及它是否可能通过Ppa蛋白的稳定而发生。我们还将测试是否Ppa上调一个基板导致其他Ppa基板的表达的变化。(3)确定新的Ppa底物。潜在的新底物的PPA将被确定使用已知的分割蛋白和新的候选人的双杂交筛选的双杂交测试。将使用体内表达试验检测潜在底物的PPA依赖性降解（见目的1）。通过研究果蝇和斑马鱼Ppa在平行，我们将评估Ppa的属性是保守的类群，从而提供了一个更全面的了解如何F-盒介导的蛋白质降解可以整合到基因调控网络的发展。

DESCRIPTION (provided by applicant): Protein degradation is integrated, alongside transcriptional and other post-transcriptional regulation, into the regulation of gene expression during development. Our ability to dissect the mechanisms underlying this integration has advanced significantly with the discovery of F-box proteins, which act as specificity factors that target particular substrates for ubiquitin-mediated degradation. Our primary goals are to understand (i) how F-box proteins are regulated, (ii) what kinds of gene regulatory functions can be performed by these proteins, and (iii) how degradation substrates that share a common F-box protein are functionally related to each other. As a model system, we are dissecting the function of Partner of paired (Ppa), a newly-identified F-box protein implicated in the regulation of Drosophila segmentation and nuclear cycle during embryogenesis. We propose to examine Ppa function in Drosophila and zebrafish. Our specific aims are: (1) To determine whether Ppa has transcriptional repression activity. We will test the hypothesis that, in addition to regulating degradation of the Paired transcription factor, Drosophila Ppa also represses the ability of Paired to activate transcription of target genes. We will test Ppa deletion constructs in vivo, and test if Ppa's putative transcriptional repression domain functions with a heterologous DNA-binding activity. We will also determine whether zebrafish Ppa has repression activity. (2) To characterize post-transcriptional regulation of Ppa. Our preliminary expression analysis suggests that Drosophila Ppa protein expression is regulated at the post-transcriptional level in order to up-regulate Ppa protein in cells with high levels of substrate requiring degradation. Using ectopic expression studies, deletion analysis, and protein-synthesis inhibition, we will assess the substrate dependence of this regulation, and whether it might occur through stabilization of the Ppa protein. We will also test whether Ppa up-regulation by one substrate leads to changes in expression of other Ppa substrates. (3) To identify new Ppa substrates. Potential new substrates of Ppa will be identified using two-hybrid tests of known segmentation proteins and a two-hybrid screen for new candidates. Ppa-dependent degradation of potential substrates will be tested using in vivo expression assays (see Aim 1). By examining Drosophila and zebrafish Ppa in parallel, we will assess which properties of Ppa are conserved across taxa, and thereby provide a more general understanding of how F-box-mediated protein degradation can be integrated into gene regulatory networks in development.