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被整合到发育过程中基因表达的调控中。随着F-box蛋白的发现，我们分析这种整合机制的能力有了显著的进步，F-box蛋白作为特异性因子，针对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蛋白是如何被调节的，（ii）这些蛋白可以执行哪些类型的基因调节功能，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Partner of paired （Ppa）的功能，这是一个新发现的F-box蛋白，涉及果蝇胚胎发生过程中分割和核周期的调节。我们打算研究Ppa在果蝇和斑马鱼中的功能。我们的具体目的是：(1)确定Ppa是否具有转录抑制活性。我们将验证这一假设，即除了调节配对转录因子的降解外，果蝇Ppa还抑制配对激活靶基因转录的能力。我们将在体内测试Ppa缺失结构，并测试Ppa的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)表征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受到调控，从而在需要高水平降解底物的细胞中上调Ppa蛋白。通过异位表达研究、缺失分析和蛋白质合成抑制，我们将评估这种调控对底物的依赖性，以及它是否可能通过稳定Ppa蛋白而发生。我们还将测试一种底物对Ppa的上调是否会导致其他Ppa底物的表达变化。(3)鉴定新的Ppa底物。Ppa潜在的新底物将通过已知分割蛋白的双杂交试验和新候选蛋白的双杂交筛选来确定。潜在底物的ppa依赖性降解将通过体内表达试验进行测试（见目的1）。通过同时研究果蝇和斑马鱼的Ppa，我们将评估Ppa的哪些特性在不同的类群中是保守的，从而对f -box介导的蛋白质降解如何被整合到发育过程中的基因调控网络中提供更全面的理解。

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.