Functional analysis of PKD proteins in urinary exosomes

尿液外泌体中 PKD 蛋白的功能分析

• 批准号: 8326613
• 负责人: CHRISTOPHER J WARD
• 金额: $ 34.3万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2013-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8326613
• 关键词: Adenoviruses; Affect; American; Autosomal Dominant Polycystic Kidney; Autosomal Recessive Polycystic Kidney; Bacterial Adhesins; Biliary; Binding; Biological; Biological Assay; Biological Markers; Biological Process; Cade; Cataloging; Catalogs; Cells; Chimera organism; Cilia; Complex; Cyclic AMP; DNA Sequence; Data; Development; Disease; Epitopes; Erinaceidae; Event; Extracellular Domain; Genes; Genetic; Human; Integral Membrane Protein; Introns; Kidney; Kidney Failure; Knockout Mice; Libraries; Membrane; Membrane Proteins; Messenger RNA; MicroRNAs; Mouse Strains; Mus; Mutation; N-terminal; Nature; Online Mendelian Inheritance In Man; Organelles; PKD1 gene; PKD2 gene; PKD2 protein; Pathogenesis; Pathway interactions; Patients; Polycystic Kidney Diseases; Population; Proprotein Convertase 1; Proteins; Proteome; Proteomics; Renal tubule structure; Rodent Model; Role; Sampling; Signal Pathway; Signal Transduction; Source; Structure; Surveys; System; Urine; Vesicle; base; extracellular; follow-up; human SMO protein; insight; novel; polycystic kidney disease 1 protein; protein kinase D; research study; response; serial analysis of gene expression; theories; urinary

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.

描述（由申请人提供）：以前，尿外泌体已被鉴定为含有选择性肾脏蛋白的排泄囊泡（50- 100 nm），因此是肾脏生物标志物的可获得来源。对ARPKD啮齿动物模型和患者样本的分析显示，初级纤毛周围积聚了外来体样囊泡（ELV），初级纤毛是所有形式PKD发病机制的核心细胞器。对尿ELV的进一步分析表明，它们高度富集三种PKD相关蛋白，多囊蛋白-1和-2（ADPKD）和纤维囊蛋白-多导管蛋白（ARPKD），表明PKD蛋白在肾ELV中的功能作用。蛋白质组学分析证实了三种主要的人类cystogene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smoothened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。我们建议在这里跟进这些初步观察，充分表征这些囊泡的结构和功能，并确定其对PKD发展的重要性。第一个目标将调查的PKD-ELV上存在的，并在囊性疾病牵连，在PKD-ELV/初级纤毛相互作用的大细胞外区域的膜蛋白的作用。我们将使用Pkhd 1 pk（+）\pk（+）小鼠作为表位标记的PKD-ELV的来源来跟踪相互作用，并使用含有来自PC 1、MEGF 8、纤维囊蛋白/多导管蛋白和FAT 4的结构域的Fc嵌合体来阻断相互作用。在第二个目标中，我们将通过调查可以整合PKD-ELV信号的细胞的mRNA和miRNA谱来分析PKD-ELV对细胞信号传导的影响。我们将使用能够阻断相互作用的Fc嵌合体，例如来自多囊蛋白-1的LRR_WSC结构域，来抑制PKD-ELV与初级纤毛的结合，并且我们还将从Pkhd 1基因的内含子2中具有Cre可移除转录终止盒的小鼠中制备配对的初级细胞群。一半分离的细胞将用Cre腺病毒处理，另一半用无酶活性的Cre腺病毒处理。将从Fc-嵌合体处理的细胞和对照以及两个配对的细胞群体（在单独的实验中）分离mRNA和miRNA，将使用长SAGE和高通量DNA测序分析mRNA，将使用miRNA再次制备文库用于高通量测序。因此，我们将能够鉴定由PKD-ELV/初级纤毛相互作用的丧失（Fc-嵌合体阻断）调节的基因，并在删除转录STOP盒后重新获得其纤维囊蛋白功能（并因此获得PKD-ELV/初级纤毛相互作用）的细胞中确认这些基因。