Division of Genetics and Cell Biology, Dulbecco Telethon Institute at Dibit, San Raffaele Scientific Institute, Milan, Italy.
PLoS One. 2010 Sep 14;5(9):e12719. doi: 10.1371/journal.pone.0012719.
Mutations in PKD1, the gene encoding for the receptor Polycystin-1 (PC-1), cause autosomal dominant polycystic kidney disease (ADPKD). The cytoplasmic C-terminus of PC-1 contains a coiled-coil domain that mediates an interaction with the PKD2 gene product, Polycystin-2 (PC-2). Here we identify a novel domain in the PC-1 C-terminal tail, a polyproline motif mediating an interaction with Src homology domain 3 (SH3). A screen for interactions using the PC-1 C-terminal tail identified the SH3 domain of nephrocystin-1 (NPHP1) as a potential binding partner of PC-1. NPHP1 is the product of a gene that is mutated in a different form of renal cystic disease, nephronophthisis (NPHP). We show that in vitro pull-down assays and NMR structural studies confirmed the interaction between the PC-1 polyproline motif and the NPHP1 SH3 domain. Furthermore, the two full-length proteins interact through these domains; using a recently generated model system allowing us to track endogenous PC-1, we confirm the interaction between the endogenous proteins. Finally, we show that NPHP1 trafficking to cilia does not require PC-1 and that PC-1 may require NPHP1 to regulate resistance to apoptosis, but not to regulate cell cycle progression. In line with this, we find high levels of apoptosis in renal specimens of NPHP patients. Our data uncover a link between two different ciliopathies, ADPKD and NPHP, supporting the notion that common pathogenetic defects, possibly involving de-regulated apoptosis, underlie renal cyst formation.
PKD1 基因突变导致常染色体显性多囊肾病(ADPKD),该基因编码多囊蛋白-1(PC-1)。PC-1 的细胞质 C 端含有一个卷曲螺旋结构域,介导与 PKD2 基因产物多囊蛋白-2(PC-2)的相互作用。在此,我们在 PC-1 C 端尾部发现了一个新的结构域,一个多脯氨酸基序,介导与Src 同源结构域 3(SH3)的相互作用。使用 PC-1 C 端尾部进行相互作用筛选,鉴定出肾病性胱氨酸病(NPHP)不同形式的突变基因产物 nephrocystin-1(NPHP1)是 PC-1 的潜在结合伴侣。NPHP1 是一个基因的产物,该基因在另一种肾囊性疾病肾单位肾痨(NPHP)中发生突变。我们表明,体外下拉实验和 NMR 结构研究证实了 PC-1 多脯氨酸基序与 NPHP1 SH3 结构域之间的相互作用。此外,这两种全长蛋白通过这些结构域相互作用;使用最近生成的允许我们追踪内源性 PC-1 的模型系统,我们证实了内源性蛋白之间的相互作用。最后,我们表明,NPHP1 向纤毛的运输不需要 PC-1,而 PC-1 可能需要 NPHP1 来调节对细胞凋亡的抗性,但不需要调节细胞周期进程。与此一致,我们在 NPHP 患者的肾标本中发现高水平的细胞凋亡。我们的数据揭示了两种不同纤毛病(ADPKD 和 NPHP)之间的联系,支持了这样一种观点,即常见的致病缺陷,可能涉及不受调节的细胞凋亡,是导致肾脏囊肿形成的原因。