Characterization of PKD Protein-Positive Exosome-Like Vesicles
Characterization of PKD Protein-Positive Exosome-Like Vesicles
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DOI：
10.1681/asn.2008060564
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发表时间：
2009-02-01
影响因子：
13.6
通讯作者：
Ward, Christopher J.
中科院分区：
文献类型：
--
作者：
Hogan, Marie C.;Manganelli, Luca;Ward, Christopher J.
与常染色体显性和常染色体隐性多囊肾病相关的蛋白质（多囊蛋白-1、多囊蛋白-2和纤维囊蛋白）定位于各种亚细胞区室，但其功能位点被认为位于初级纤毛上。在 Pkhd1(de12/d12) 小鼠中，PC1 + 囊泡围绕着纤毛，这使我们能够详细分析这些结构。我们对尿外泌体样囊泡 (ELV) 进行细分，并分离出富含多囊蛋白-1、纤囊蛋白（裂解形式）和多囊蛋白-2 的亚群。这去除了主要污染物 Tamm-Horsfall 蛋白，并将 ELV 细分为至少三个不同的群体，通过水通道蛋白-2、多囊蛋白-1 和 Podocin 的存在进行划分。 PKD ELV 的蛋白质组学分析鉴定出 552 种蛋白质（其中 232 种尚未进入尿蛋白质组数据库），其中许多与信号传导有关，包括分子 Smoothened。我们还检测到与囊性疾病相关的基因的另外两种蛋白质产物：胱氨酸（Cystin）（小鼠 cpk 基因座的产物）和 ADP-核糖基化因子样 6（人类 Bardet-Biedl 综合征基因 (BBS3) 的产物）。我们的蛋白质组学分析证实，多囊蛋白-1和纤维囊蛋白的裂解在体内发生，其方式与多囊蛋白-1中的GPS位点和纤维囊蛋白中的前蛋白转化酶位点的裂解一致。在体外，这些 PKD ELV 优先以快速且高度特异性的方式与肾和胆管上皮细胞的初级纤毛相互作用。这些数据表明 PKD 蛋白在尿液中以膜颗粒形式脱落，并且这些颗粒与初级纤毛相互作用。
Proteins associated with autosomal dominant and autosomal recessive polycystic kidney disease (polycystin-1, polycystin-2, and fibrocystin) localize to various subcellular compartments, but their functional site is thought to be on primary cilia. PC1 + vesicles surround cilia in Pkhd1(de12/d12) mice, which led us to analyze these structures in detail. We subfractionated urinary exosome-like vesicles (ELVs) and isolated a subpopulation abundant in polycystin-1, fibrocystin (in their cleaved forms), and polycystin-2. This removed Tamm-Horsfall protein, the major contaminant, and subfractionated ELVs into at least three different populations, demarcated by the presence of aquaporin-2, polycystin-1, and podocin. Proteomic analysis of PKD ELVs identified 552 proteins (232 not yet in urinary proteomic databases), many of which have been implicated in signaling, including the molecule Smoothened. We also detected two other protein products of genes involved in cystic disease: Cystin, the product of the mouse cpk locus, and ADP-ribosylation factor-like 6, the product of the human Bardet-Biedl syndrome gene (BBS3). Our proteomic analysis confirmed that cleavage of polycystin-1 and fibrocystin occurs in vivo, in manners consistent with cleavage at the GPS site in polycystin-1 and the proprotein convertase site in fibrocystin. In vitro, these PKD ELVs preferentially interacted with primary cilia of kidney and biliary epithelial cells in a rapid and highly specific manner. These data suggest that PKD proteins are shed in membrane particles in the urine, and these particles interact with primary cilia.