FUBP1对PKD2蛋白表达的调控及其在遗传性多囊肾发展的作用

全世界有数千万人受遗传性多囊肾疾病（ADPKD）困扰，患者因缺乏治疗药物要靠透析维持或肾移植。ADPKD还导致肝和胰腺囊肿、心脑血管瘤及高血压等。ADPKD源于PKD1或PKD2的一个等位基因种系突变及随后经历“二次打击”造成的，包括蛋白表达减少或另一等位基因产生后天突变。囊肿病变细胞中只有20-43%具有后天突变，其余则源于蛋白量下降。最近我们报道了细胞合成因子eIF2α通过PKD2 RNA的5’端调控其蛋白表达，预实验也发现RNA结合蛋白 FUBP1和PKD2 3’端能负调控PKD2蛋白翻译及斑马鱼的相关疾病，但机制还不清楚。本课题首先用细胞模型揭示FUBP1和PKD2 3’端调控PKD2蛋白翻译的机制,其次用斑马鱼研究FUBP1如何调控PKD2相关的疾病,最后用双基因敲除小鼠研究FUBP1调控PKD2+/-鼠肾及肝囊肿发展的机理并筛选抑制短肽，为获得治疗ADPKD短肽药物提供新依据。

Autosomal dominant polycystic kidney disease (ADPKD) is an inherited disorder that affects 0.2% of the population worldwide, resulting in billions of dollars each year in direct medical costs. Hemodialysis and transplantation are the only current treatments for ADPKD. ADPKD patients have one allele with mutations in PKD1, a large membrane receptor protein, or PKD2, a Ca-permeable cation channel. Progression to cyst formation follows a 'second hit', ie, somatic mutations or dosage reduction in the second allele. Cystogenesis can be due to either loss- or gain-of-function of PKD1 or PKD2; thus understanding how their cellular levels are regulated is critically important. . We recently reported the up-regulation of PKD2 translation by eIF2α through the 5' untranslated region (5'UTR) of PKD2 mRNA.Our preliminary experiments identified a 3’UTR fragment called 3FI and its binding protein called far upstream element-binding protein 1 (FUBP1) that together inhibit PKD2 translation and regulate PKD2-associated disorders in zebrafish, but the underlying mechanism is unclear. Here we will furst study the molecular pathway underlying this regulation using cultured cells. We will then use zebrafish and mouse models to investigate the cellular pathway that underlies how PKD2-associated cystic diseases are regulated by 3FI and FUBP1. .RESEARCH PLAN. Aim 1. Investigate the mechanism underlying the regulation of PKD2 translation by FUBP1 in cultured cells. We will first determine the importance of the 3FI-FUBP1 and FUBP1-4EBP1 bindings for the regulation of PKD2 translation by FUBP1, through which we will also identify FUBP1 peptides that reduce the 3FI-FUBP1 and FUBP1-4EBP1 bindings. .Aim 2: Examine how FUBP1 regulates PKD2-associated disorders in zebrafish. We will determine whether disruption of complex 3FI-FUBP1-4EBP1, by FUBP1 reduction or blocking peptides, reduces the disease severity in larval PKD2+/- zebrafish, through which we will also identify short peptide inhibitors with therapeutic potentials. FUBP1 and PKD2 knockout fish lines will be obtained using CRISPRs. .Aim 3. Examine regulation of PKD2-associated renal and hepatic cystogenesis by FUBP1 in mice. We will generate PKD2+/-/FUBP1+/- double knockout mice through cross-breeding between our PKD2+/- and FUBP1+/- mice and examine whether FUBP1 dosage reduction increases PKD2 expression and reduces cystic severity associated with PKD2+/-. We will also examine whether membrane-permeable peptide inhibitors reduce the cystic severity through increasing PKD2 protein expression. .SIGNIFICANCE. This project is novel in studying the regulation of PKD2 dosage and associated pathogenesis via a cellular pathway linking PKD2 3’UTR, FUBP1 and the translation initiation complex. By mapping the involvement of this pathway in the regulation of ADPKD by FUBP1, we will gain insights into the mechanism of renal and hepatic cystogenesis, and identify therapeutic peptide inhibitors against mouse ADPKD.