Wills Edgar S, Te Morsche René H M, van Reeuwijk Jeroen, Horn Nicola, Geomini Iris, van de Laarschot Liyanne F M, Mans Dorus A, Ueffing Marius, Boldt Karsten, Drenth Joost P H, Roepman Ronald
Department of Gastroenterology and Hepatology, Radboud University Medical Center, 6500 HB Nijmegen, The Netherlands.
Department of Human Genetics and Radboud Insitute for Molecular Life Sciences, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands.
Hum Mol Genet. 2017 Nov 1;26(21):4190-4202. doi: 10.1093/hmg/ddx308.
Mutations in the PRKCSH, SEC63 and LRP5 genes cause autosomal dominant polycystic liver disease (ADPLD). The proteins products of PRKCSH (alias GIIB) and SEC63 function in protein quality control and processing in the endoplasmic reticulum (ER), while LRP5 is implicated in Wnt/β-catenin signaling. To identify common denominators in the PLD pathogenesis, we mapped the PLD interactome by affinity proteomics, employing both HEK293T cells and H69 cholangiocytes. Identification of known complex members, such as glucosidase IIA (GIIA) for PRKCSH, and SEC61A1 and SEC61B for SEC63, confirmed the specificity of the analysis. GANAB, encoding GIIA, was very recently identified as an ADPLD gene. The presence of GIIA in the LRP5 complex pinpoints a potential functional connection with PRKCSH. Interestingly, all three PLD-associated protein complexes included filamin A (FLNA), a multifunctional protein described to play a role in ciliogenesis as well as canonical Wnt signalling. As ciliary dysfunction may also contribute to hereditary liver cyst formation, we evaluated the requirement of PRKCSH and SEC63 for ciliogenesis and Wnt signaling. By CRISPR/Cas9 induced knockdown of both ADPLD genes in HEK293T cells and H69 cholangiocytes, we identified that their depletion results in defective ciliogenesis. However, only H69 knockouts displayed reduced Wnt3a activation. Our results suggest that loss of PRKCSH and SEC63 leads to general defects in ciliogenesis, while quenching of the Wnt signaling cascade is cholangiocyte-restricted. Interactions of all three PLD-associated protein complexes with FLNA may mark a common link between the ADPLD proteins and the cystogenic processes driving this disease.
PRKCSH、SEC63和LRP5基因的突变会导致常染色体显性多囊肝病（ADPLD）。PRKCSH（别名GIIB）和SEC63的蛋白质产物在内质网（ER）的蛋白质质量控制和加工过程中发挥作用，而LRP5则与Wnt/β-连环蛋白信号传导有关。为了确定PLD发病机制中的共同因素，我们通过亲和蛋白质组学绘制了PLD相互作用组图谱，使用了HEK293T细胞和H69胆管细胞。鉴定已知的复合物成员，如PRKCSH的葡糖苷酶IIA（GIIA）以及SEC63的SEC61A1和SEC61B，证实了分析的特异性。最近，编码GIIA的GANAB被鉴定为一个ADPLD基因。GIIA在LRP5复合物中的存在指出了与PRKCSH的潜在功能联系。有趣的是，所有三种与PLD相关的蛋白质复合物都包含细丝蛋白A（FLNA），这是一种多功能蛋白质，据描述在纤毛发生以及经典Wnt信号传导中发挥作用。由于纤毛功能障碍也可能导致遗传性肝囊肿形成，我们评估了PRKCSH和SEC63对纤毛发生和Wnt信号传导的需求。通过CRISPR/Cas9诱导敲低HEK293T细胞和H69胆管细胞中的两个ADPLD基因，我们发现它们的缺失会导致纤毛发生缺陷。然而，只有H69基因敲除显示Wnt3a激活减少。我们的结果表明，PRKCSH和SEC63的缺失会导致纤毛发生的普遍缺陷，而Wnt信号级联的淬灭则局限于胆管细胞。所有三种与PLD相关的蛋白质复合物与FLNA之间的相互作用可能标志着ADPLD蛋白质与驱动这种疾病的囊肿形成过程之间的共同联系。
