Functional characterization of the novel protein nephrocystin-5

描述（由申请人提供）： 肾结核（NPHP）是一个重要的公共卫生问题，因为它是生命最初三十年中终末期肾病最常见的遗传原因。NPHP患者的肾脏表现出疤痕和囊肿形成，与许多其他类型的肾脏疾病共有的特征。对肾囊素-5的研究将有助于深入了解NPHP肾瘢痕形成和囊肿形成的机制。这些机制可能与其他肾脏疾病和肾衰竭的原因相同。 申办方实验室已通过定位克隆在三个新基因（NPHP 1、2和4）中发现了导致NPHP的突变。他们最近还鉴定了导致NPHP 2型的反转蛋白基因中的突变，从而将肾囊肿疾病与初级纤毛和左右轴决定的功能联系起来（Otto等，Nature Genet 34：413，2003）。最近，在患有NPHP 5型和色素性视网膜炎的患者中鉴定了另一种新基因NPHP 5（Otto等人，Nature Genet. 37：282-8，2005）。NPHP 5的基因产物是肾囊蛋白-5。 本申请的总体假设是肾囊蛋白-5参与蛋白质复合物，其可能包括其它肾囊蛋白质，并且在形成紧密细胞连接和顶端-基底极化中起作用。 具体而言，我们将： 1.检查新蛋白质肾囊蛋白-5的亚细胞定位。我们将采用激光扫描共聚焦显微镜检查组织培养中的细胞以及小鼠组织切片。 2.检查肾囊蛋白-5如何参与功能性蛋白质复合物。我们将研究肾囊蛋白-5蛋白复合物与免疫共沉淀，二维凝胶和质谱。 3.表征NPHP 5突变对肾囊蛋白-5的亚细胞定位、上皮单层的完整性和细胞极化的影响。将产生表达肾囊蛋白-5的截短形式的MDCK细胞。将用共聚焦显微镜评价这些细胞的肾囊蛋白-5定位，用跨上皮电阻测定细胞紧密连接的形成，并通过在胶原凝胶中生长来评价极化缺陷。

DESCRIPTION (provided by applicant): Nephronophthisis (NPHP) is an important public health issue because it is the most common genetic cause of end-stage renal disease in the first three decades of life. The kidneys of patients with NPHP demonstrate scarring and cyst formation, features shared with many other types of kidney diseases. The study of nephrocystin-5 will provide insight into the mechanisms for kidney scarring and cyst formation in NPHP. These mechanisms may be common to other causes of kidney disease and kidney failure. The sponsor's lab has identified by positional cloning mutations in three novel genes (NPHP1, 2, and 4) to cause NPHP. They also recently identified mutations in the inversin gene as causing NPHP type 2, thus linking renal cystic disease to the function of primary cilia and left-right axis determination (Otto et al. Nature Genet 34:413, 2003). Very recently another novel gene, NPHP5, was identified in patients with NPHP type 5 and retinitis pigmentosa (Otto, et al. Nature Genet. 37:282-8, 2005). The gene product of NPHP5 is nephrocystin-5. The overall hypothesis of this application is that nephrocystin-5 participates in a protein complex, which likely includes the other nephrocystin proteins, and functions in the formation of tight cell junctions and apical-basal polarization. Specifically we will: 1. Examine the subcellular localization of the novel protein, nephrocystin-5. We will employ laser scanning confocal microscopy to examine cell in tissue culture as well as murine tissue sections. 2. Examine how nephrocystin-5 participates in a functional protein complex. We will examine the nephrocystin-5 protein complex with co-immunoprecipitations, 2D-gels and mass spectrometry. 3. Characterize the effect of NPHP5 mutations on the subcellular localization of nephrocystin-5, the integrity of the epithelial monolayer and cell polarization. MDCK cells expressing truncated forms of nephrocystin-5 will be generated. These cells will be evaluated for the localization of nephrocystin-5 with confocal microscopy, assayed for the formation of tight cell junctions with transepithelial electrical resistance and evaluated for polarization defects by growing them in a collagen gel.