Functional Characterization of the Chlamydomonas CEP290/Nephrocystin-6 Homologue

描述(申请人提供)：本文提出的研究目标是利用纤毛藻衣藻的独特优势来鉴定纤毛蛋白CEP290/肾囊蛋白-6(以下简称CEP290)。CEP290基因突变会导致纤毛相关的人类疾病，包括Leber先天性黑发、Meckel综合征、Bardet-Biedl综合征(BBS)、Joubert综合征和High-Loken综合征。虽然这些疾病的表现各不相同，但所有这些疾病都具有与纤毛缺陷有关的特征：囊性肾脏疾病或肾小球肾炎、视网膜退化和失明、多指、中枢神经系统缺陷、肝纤维化，以及在BBS病例中的肥胖。肾癌(NPHP)是由9个不同的基因(肾囊素，NPHP1-9)中的任何一个突变引起的，是儿童和年轻人肾功能衰竭的最常见的遗传原因。目前尚不清楚CEP290基因缺陷是如何影响纤毛功能并导致纤毛相关疾病的。在包括衣藻在内的多种生物中的证据表明，CEP290定位于纤毛和鞭毛的过渡区--一个结构复杂的区域，人们对此知之甚少。这些研究将检验CEP290定位于过渡区的假设，在那里它调节蛋白质进入鞭毛的运动。这将通过对衣藻插入突变体中存在的纤毛表型的生化、遗传和细胞生物学分析来检验，该突变体含有大量的CEP290基因缺失。CEP290的精确定位将使用生化、遗传学和形态学方法来确定。用双向凝胶电泳法和质谱仪对CEP290突变体鞭毛和野生型鞭毛的蛋白质组成进行比较。衣藻提供的出色的生物化学将被用于纯化含有CEP290的复合体，与CEP290共同纯化的蛋白质将通过质谱学进行鉴定。虽然肾囊藻毒素蛋白具有共同的定位模式，而且肾囊藻毒素基因的缺陷都会导致NPHP，但目前尚不清楚肾囊藻毒素是否相互作用或调节彼此的定位。这一限制将使用另一个衣藻插入突变体来解决，该突变体对肾囊藻蛋白-4无效。这项研究有可能深入了解CEP290的基本功能，确定CEP290缺陷如何影响纤毛结构和/或功能，揭示过渡区的新功能，并提供有关纤毛基本生物学的新信息，因此对公共卫生具有极大的兴趣和相关性。

DESCRIPTION (provided by applicant): The goal of the research proposed here is to exploit the unique advantages of the ciliated alga Chlamydomonas to functionally characterize the ciliary protein CEP290/nephrocystin-6 (hereon referred to as CEP290). Mutations in CEP290 cause cilia-related human diseases, including Leber congenital amaurosis, Meckel syndrome, Bardet-Biedl syndrome (BBS), Joubert syndome, and Senior-Loken syndrome. Although disease manifestation varies in these disorders, all of these disorders present with characteristics that have been linked to defects in cilia: cystic kidney disease or nephronophthisis, retinal degeneration and blindness, polydactyly, defects in the central nervous system, liver fibrosis, and in the case of BBS,obesity. Nephronophthisis (NPHP), which results from mutations in any of nine distinct genes (nephrocystins, NPHP1-9), is the most common genetic cause of renal failure in children and young adults. It is currently unknown how defects in CEP290 affect cilia function and lead to cilia-related diseases. Evidence in multiple organisms, including Chlamydomonas, suggests that CEP290 localizes to the transition zone of cilia and flagella-a structurally complex region about which very little is known. These studies will test the hypothesis that CEP290 localizes to the transition zone where it regulates the movement of proteins into the flagella. This will be tested by biochemical, genetic, and cell biological analysis of ciliary phenotypes present in a Chlamydomonas insertional mutant harboring a large deletion of the CEP290 gene. The precise localization of CEP290 will be determined using biochemical, genetic, and morphological approaches. The protein composition of CEP290 mutant flagella will be compared to that of wild type using 2D gel electrophoresis and mass spectrometry. The outstanding biochemistry that Chlamydomonas offers will be utilized to purify complexes containing CEP290, and proteins co-purifying with CEP290 will be identified by mass spectrometry. Although nephrocystin proteins display common localization patterns and defects in the nephrocystin genes all result in NPHP, it is unknown if the nephrocystins interact with each other or regulate each other's localization. This limitation will be addressed using another Chlamydomonas insertional mutant that is null for nephrocystin-4. This research has great potential to add insight into the basic function of CEP290, determine how CEP290 defects affect cilia structure and/or function, reveal novel functions for the transition zone, and provide new information on the basic biology of cilia, and is therefore of utmost interest and relevance to public health.