Functional characterization of the novel protein nephrocystin-5

描述(由申请人提供)： 肾单位病(NPHP)是一个重要的公共卫生问题，因为它是终末期肾病最常见的遗传原因，在生命的前30年。NPHP患者的肾脏表现出疤痕和囊性形成，这与许多其他类型的肾脏疾病相似。对肾囊素-5的研究将有助于深入了解NPHP中肾脏瘢痕形成和囊性形成的机制。这些机制可能与其他导致肾脏疾病和肾衰竭的原因相同。 赞助商的实验室已经通过位置克隆确定了引起NPHP的三个新基因(NPHP1、2和4)的突变。他们最近还发现反转蛋白基因的突变是导致NPHP 2型的原因，从而将肾囊性疾病与初级纤毛功能和左右轴决定联系在一起(Otto等人。《自然基因》34：413,2003)。最近，在NPHP 5型和视网膜色素变性患者中发现了另一种新基因NPHP5(Otto等人。大自然的吉内特。37：282-8,2005)。NPHP5的基因产物为肾囊蛋白-5。 这一应用的总体假设是，肾囊素-5参与了一个蛋白质复合体，该复合体可能包括其他肾囊藻毒素蛋白，并在形成紧密的细胞连接和心尖-基底极化方面发挥作用。 具体来说，我们会： 1.检测新蛋白--肾囊藻毒素-5的亚细胞定位。我们将使用激光扫描共聚焦显微镜来检查组织培养中的细胞以及小鼠组织切片。 2.研究肾囊藻毒素-5如何参与功能蛋白复合体。我们将用免疫共沉淀、2D-凝胶和质谱学方法检测肾囊藻毒素-5蛋白复合体。 3.研究NPHP5突变对肾囊素-5的亚细胞定位、上皮细胞单层完整性和细胞极化的影响。将产生表达截短形式的肾囊素-5的MDCK细胞。这些细胞将通过共聚焦显微镜检测肾囊藻毒素-5的定位，分析具有跨上皮电阻的紧密细胞连接的形成，并通过在胶原凝胶中生长来评估极化缺陷。

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.