Characterizing the Funtion of Fibbrocystin and Fibbrocystin-L

描述（由申请方提供）：常染色体隐性遗传性多囊肾病（ARPKD）是由大而复杂的基因PKHD 1突变引起的。临床和分子生物学研究揭示了PKHD 1突变相关的广泛表型范围，从围产期死亡到成人发病性肝病。进一步的复杂性是由显著的等位基因异质性提供的，因此只有初步的基因型/表型相关性是可能的。该提案的第一个目的是扩展这些突变研究（以及与表型的关联），以进一步从分子上表征典型和非典型ARPKD人群，包括孤立性肝病。基因型在决定表型中的作用将被检查，重要的残基突出显示，用于功能研究中的测试。ARPKD蛋白是大的和膜结合的，并且与其他PKD相关蛋白共同定位于初级纤毛和基体。Pkhdl缺陷动物的纤毛缩短和异常。在人类中发现了一种纤维囊蛋白，纤维囊蛋白样（-L;由PKHDL 1编码），尽管有可能的功能提示，但这种蛋白质的确切作用尚不清楚（尚不清楚与ARPKD相关）。纤维囊蛋白-L是蛋白质家族中最古老的成员，与纤维囊蛋白不同，存在于鱼类和衣原体中，其中沉默导致由于有缺陷的鞭毛而导致的运动受损;这表明在其他生物体中的纤毛作用。第二个目标将使用单克隆抗体和表达构建体来确定（并确认）两种蛋白质的亚细胞定位，并查看纤维囊蛋白-L是否与睫状体/基底体轴相关。还将在极化的肾上皮细胞中测试与通过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，原位杂交和吗啉代方法将被用来本地化的网站的表达和表型与敲低活动在发展中的斑马鱼。原肾管中的囊性表型是一种可能的结果，因为是有缺陷的会聚延伸运动，如在斑马鱼中发现的一些综合征PKD相关基因的变形。最终的目标将敲除小鼠中的PkhdH，组成性地（并且如果必要的话，在肾脏中有条件地）以确定纤维囊蛋白-L在哺乳动物中的作用。用现有的Pkhdl deZ小鼠育种将显示两种纤维囊蛋白的损失是否导致加性表型，表明相关的作用。总的来说，该项目将揭示纤维囊蛋白家族的功能信息，并确定涉及这些分子的信号传导/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.