Characterizing the Funtion of Fibbrocystin and Fibbrocystin-L

描述(申请人提供)：常染色体隐性遗传性多囊肾病(ARPKD)是由大而复杂的基因PKHD1突变引起的。临床和分子研究已经揭示了与PKHD1突变相关的广泛的表型范围，从围产期死亡到成人发病的肝病。显著的等位基因异质性提供了进一步的复杂性，因此只有初步的基因型/表型相关性是可能的。这项建议的第一个目标将扩大这些突变研究(以及与表型的关联)，以进一步从分子上表征典型的和非典型的ARPKD人群，包括孤立的肝病。本课程将研究基因在决定表型中的作用，并强调在功能研究中用于测试的重要残基。ARPKD蛋白是一个大的膜结合蛋白，与其他PKD相关蛋白一样，定位于初生纤毛和基底体。Pkhdl缺陷动物纤毛变短、异常。在人类中发现了一种类似纤维囊藻蛋白的蛋白(-L，由PKHDL1编码)，尽管有可能的功能提示，但该蛋白的确切作用尚不清楚(它与ARPKD尚不清楚)。纤维囊藻蛋白-L是蛋白质家族中最古老的成员，与纤维囊藻蛋白不同，它存在于鱼类和衣藻中，在鱼类和衣藻中，沉默会由于鞭毛缺陷而导致运动能力减弱；这表明在其他生物中具有纤毛作用。第二个目标将使用单抗和表达构建物来确定(并确认)这两种蛋白的亚细胞定位，并观察纤维囊藻蛋白-L是否与睫状体轴/基底体轴相关。在极化的肾上皮细胞中，也将测试与shRNA单独或组合沉默这些基因相关的细胞表型。在第三个目标中，将使用原位杂交和吗啉方法来定位与斑马鱼发育中的击倒活动相关的表达部位和表型。前肾管的囊性表型是一种可能的结果，在一些综合征型PKD相关基因的斑马鱼变种中发现的会聚延伸运动缺陷也是一种可能的结果。最终目的是在小鼠体内，结构性地(如果必要的话，还可以有条件地在肾脏中)敲除PkhdH，以确定纤维囊藻毒素-L在哺乳动物中的作用。用现有的Pkhdl Dez小鼠进行繁殖将表明这两种纤维囊藻蛋白的丢失是否会导致相加的表型，这表明了相关的作用。总体而言，该项目将揭示有关纤维囊藻蛋白家族作用的功能信息，并确定涉及这些分子的信号/发育途径。

DESCRIPTION (provided by applicant): Autosomal recessive polycystic kidney disease (ARPKD) is caused by mutation to the large and complex gene, PKHD1. Clinical and molecular studies have revealed a wide phenotypic range associated with PKHD1 mutation, from perinatal death to adult onset liver disease. Further complexity is provided by marked allelic heterogeneity and so only preliminary genotype/phenotype correlations have been possible. The first aim of this proposal will extend these mutation studies (and associations with phenotype) to further molecularly characterize typical and atypical ARPKD populations, including isolated liver disease. The role of genotype in dictating phenotype will be examined, and important residues highlighted for testing in functional studies. The ARPKD protein is large and membrane bound, and in common with other PKD associated proteins, localized to primary cilia and the basal body. Pkhdl defective animals have shortened and abnormal cilia. A fibrocystin paralog is found in humans, fibrocystin-like (-L; encoded by PKHDL1), and although there are hints at possible function, the precise role of this protein is unclear (it is not known to be associated with ARPKD). Fibrocystin-L is the most ancient member of the protein family and, unlike fibrocystin, present in fish and Chlamydomonas, where silencing results in impaired motility due to defective flagella; suggesting a ciliary role in other organisms. The second aim will use monoclonal antibodies and expression constructs to determine (and confirm) the subcellular localization of both proteins, and see if fibrocystin-L is associated with the ciliary/basal body axis. The cellular phenotypes associated with silencing these genes by shRNA, singly or in combination, in polarized kidney epithelial cells, will also be tested. In the third aim, in situ hybridization and morpholino approaches will be used to localize the sites of expression and the phenotype associated with knocking down activity in the developing zebrafish. Cystic phenotypes in the pronephric duct are one possible outcome, as is defective convergent extension movements, as found in zebrafish morphants of some syndromic PKD associated genes. The final aim will knockout PkhdH in the mouse, constitutively (and, if necessary, conditionally in the kidney) to determine the role of fibrocystin-L in mammals. Breeding with the existing Pkhdl deZ mouse will show whether loss of both fibrocystins results in an additive phenotype, suggesting related roles. Overall the project will reveal functional information about the roles of the fibrocystin protein family and identify signaling/developmental pathways involving these molecules.