Functional analysis of PKD proteins in urinary exosomes

描述（由申请人提供）：以前，尿外泌体已被确定为含有肾脏蛋白质的排泄囊泡（50-100nm），因此是肾脏生物标志物的可获取来源。啮齿类动物ARPKD模型和患者样本的分析显示，初级纤毛周围积聚了外泌体样囊泡（ELVs），这是所有形式PKD发病机制的核心细胞器。进一步分析尿液elv显示，它们富含3种PKD相关蛋白，即多囊蛋白-1和-2 （ADPKD）和纤维囊蛋白-多导蛋白（ARPKD），表明PKD蛋白在肾脏elv中具有功能作用。蛋白质组学分析证实了三种主要的人类囊基因产物的存在，并提供了552种蛋白质的列表，其中一些具有有趣的生物学功能（例如，平滑蛋白相对丰富）。此外，我们能够显示elv和初级纤毛之间的物理相互作用，使用细胞和整个小管系统。我们建议在这些初步观察的基础上进行后续研究，以充分表征这些囊泡的结构和功能，并确定它们在PKD发展中的重要性。第一个目的是研究存在于PKD-ELV上且与囊性疾病有关的具有大细胞外区域的膜蛋白在PKD-ELV/原发性纤毛相互作用中的作用。我们将使用Pkhd1pk(+)\pk（+）小鼠作为表位标记pkd - elv的来源，跟踪相互作用和含有来自PC1、MEGF8、纤维囊素/多ductin和FAT4的结构域的fc嵌合体来阻断相互作用。在第二个目标中，我们将分析PKD-ELV对细胞信号传导的影响，通过测量能够整合PKD-ELV信号与那些被阻断的细胞的mRNA和miRNA谱。我们将使用能够阻断相互作用的fc嵌合体，例如多囊蛋白-1的LRR_WSC结构域，来抑制PKD-ELV与初级纤毛的结合，我们还将从Pkhd1基因内含子2中具有Cre可移除的转录STOP盒的小鼠中获得配对的初级细胞群。分离的细胞一半用Cre腺病毒处理，另一半用酶活性的Cre腺病毒处理。从fc嵌合体处理的细胞和对照以及两个配对的细胞群体中（在单独的实验中）分离mRNA和miRNA， mRNA将使用长SAGE和高通量DNA测序进行分析，miRNA将用于再次建立高通量测序文库。因此，我们将能够识别受PKD-ELV/原纤毛相互作用缺失（fc嵌合体阻断）调控的基因，并在转录停止盒缺失后恢复其纤维囊蛋白功能（因此PKD-ELV/原纤毛相互作用）的细胞中确认这些基因。

DESCRIPTION (provided by applicant): Previously, urinary exosomes have been identified as excreted vesicles (50-100nm) that contain a selection of kidney proteins, and hence are an accessible source of kidney biomarkers. Analysis of rodent models of ARPKD and patient samples revealed the accumulation of exosome-like vesicles (ELVs) around primary cilia, an organelle central to the pathogenesis in all forms of PKD. Further analysis of urinary ELVs showed they are highly enriched for three PKD-related proteins, polycystin-1 and -2 (ADPKD) and fibrocystin-polyductin (ARPKD), suggesting a functional role for the PKD proteins in kidney ELVs. Proteomic analysis confirmed the presence of the three major human cystogene products and supplied a list of 552 proteins, some with intriguing biological functions (for example smoothened protein was relatively abundant). In addition we were able to show physical interaction between ELVs and primary cilia, using cellular and whole tubule systems. We propose here to follow up on these preliminary observations to fully characterize the structure and function of these vesicles and determine their importance to the development of PKD. The first aim will investigate the role of membrane proteins with large extracellular regions that are present on PKD-ELVs and that are implicated in cystic disease, in the PKD-ELV/primary cilium interaction. We will use the Pkhd1pk(+)\pk(+) mouse as a source of epitope tagged PKD-ELVs to follow the interaction and Fc-chimeras containing domains derived from PC1, MEGF8, fibrocystin/polyductin and FAT4 to block the interaction. In the second aim we will analyze the effect of PKD-ELVs on cell signaling by surveying the mRNA and miRNA profiles of cells which can integrate a PKD-ELV signal with those that are blocked. We will use Fc-chimeras capable of blocking the interaction, such as the LRR_WSC domain from polycystin-1, to inhibit PKD-ELV binding to the primary cilium and we will also make paired primary cell populations from a mouse with a Cre removable transcriptional STOP cassette in intron 2 of the Pkhd1 gene. One half of the isolated cells will be treated with a Cre adenovirus and the other with an enzymically inactive Cre adenovirus. mRNA and miRNA will be isolated, from Fc-chimera treated cells and controls and the two paired cell populations (in separate experiments), the mRNA will be analyzed using long SAGE and high throughput DNA sequencing, the miRNA will be used to make a library again for high throughput sequencing. We will therefore be able to identify genes regulated by loss of PKD-ELV/primary cilium interactions (Fc-chimera block) and confirm these in the cells which regain their fibrocystin function (and hence PKD-ELV/primary cilium interactions) after the deletion of the transcriptional STOP cassette.