Regulatory mechanism of urate transport function by urate transporter binding protein PDZK1

尿酸盐转运蛋白PDZK1对尿酸盐转运功能的调控机制

• 批准号: 15590233
• 负责人: ANZAI Naohiko
• 金额: $ 1.34万
• 依托单位: Kyorin University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15590233/
• 关键词: Yeast Two-hybrid assay; Urate transporter; PDZ proteins; Co-immunoprecipitation; GST pull dow assay; Surface plasmon resonance; Surface biotinylation assay; 近位尿細管

Based on our preliminary results that the identification of intracellular binding protein PDZK1 for renal urate transporter URAT1 by yeast two-hybrid (Y2H) screening, we were planning to extend this research to clarify the functional regulatory mechanism of urate transport via urate transporter URAT1 by its binding protein PDZK1.In 2003, we clarified these three points. (1)We carried out the in vitro biochemical binding experiments such as co-immunoprecipitation assay and GST-pull down assay to confirm the URAT1-PDZK1 interaction identified by Y2H experiments. Wild type URAT1 proteins could bind with PDZK1, but URAT1 lacking the last three amino acid residues including PDZ motif (T-Q-F) failed to bind with PDZK1. (2)We established URAT1-expressing HEK293 cells (HEK-URAT1) by lipofection method and performed urate transport kinetic study with or without PDZK1 cotransfection. PDZK1 transfection into HEK-URAT1 cell increased Vmax without changing Km. (3) We detected the colocalization of … More both URAT1 and PDZK1 at the apical membrane of renal proximal tubules by immunohistochemical analysis using human kidney serial sections. In addition, coimmunoprecipitation experiments using human kidney total protein demonstrated the binding between URAT1 and PDZK1. These two findings showed the physiological importance of this interaction.In 2004, we further examined this URAT1-PDZK1 interaction. (1)We prepared the GST fused URAT1 C-terminal and MBP fused individual PDZ domains of PDZK1 and performed Surface Plasmon Resonance assay (Biacore) to check their binding affinities. The bindings were confirmed in PDZK1 PDZ domain 1, 2 and 4 by SPR. Their dissociation constant (K_D) is 1.9 - 516 nM. These values were compatible as PDZ interactions. (2)By the cell surface biotinylation experiments, we recognize that, at least, the enhancement of urate transport activity after the transfection of PDZK1 is due to the increased expression of URAT1 proteins at the plasma membrane of HEK293 cells, may be through the stabilization of URAT1 proteins by PDZK1 interaction. (3)By the database searches, we have already found two SNPs of human PDZK1. Y145H is located in the middle of the second PDZ domain of PDZK1 and V372E is located at the C-terminal side in the linker between PDZ domain 3 and 4. In the Y2H assay, we could not find the difference among three clones (wild type, Y145H, and V372E), whereas SPR experiments clearly exhibited the reduced affinity for Y145H mutants. These two PDZK1 SNP mutants did not affect the urate transport activity via URAT1. Less

基于我们通过酵母二杂交（Y2H）筛选鉴定肾尿酸转运蛋白URAT1的细胞内结合蛋白PDZK1的初步结果，我们计划扩展这项研究，以阐明其结合蛋白PDZK1通过尿酸转运蛋白URAT1对尿酸转运的功能调节机制。2003年，我们阐明了这三点。 (1)我们进行了免疫共沉淀实验和GST-pull down实验等体外生化结合实验来证实Y2H实验鉴定的URAT1-PDZK1相互作用。野生型URAT1蛋白可以与PDZK1结合，但缺少包括PDZ基序(T-Q-F)在内的最后三个氨基酸残基的URAT1无法与PDZK1结合。 (2)通过脂转染法建立表达URAT1的HEK293细胞(HEK-URAT1)，并在有或没有PDZK1共转染的情况下进行尿酸盐转运动力学研究。 PDZK1 转染 HEK-URAT1 细胞可增加 Vmax，但不改变 Km。 (3) 我们使用人肾连续切片进行免疫组织化学分析，检测到 URAT1 和 PDZK1 在肾近曲小管顶膜上的共定位。此外，使用人肾总蛋白的免疫共沉淀实验证明了URAT1和PDZK1之间的结合。这两项发现表明了这种相互作用的生理重要性。2004年，我们进一步研究了这种URAT1-PDZK1相互作用。 (1)我们制备了融合了URAT1 C端的GST和融合了PDZK1的各个PDZ结构域的MBP，并进行了表面等离子共振测定(Biacore)以检查它们的结合亲和力。通过 SPR 证实了 PDZK1 PDZ 结构域 1、2 和 4 中的结合。它们的解离常数 (K_D) 为 1.9 - 516 nM。这些值与 PDZ 相互作用是兼容的。 (2)通过细胞表面生物素化实验，我们认识到，转染PDZK1后尿酸转运活性的增强至少是由于HEK293细胞质膜上URAT1蛋白表达增加，可能是通过PDZK1相互作用稳定URAT1蛋白所致。 (3)通过数据库检索，我们已经发现了人类PDZK1的两个SNP。 Y145H位于PDZK1的第二个PDZ结构域的中间，V372E位于PDZ结构域3和4之间的连接子的C端侧。在Y2H测定中，我们无法发现三个克隆（野生型、Y145H和V372E）之间的差异，而SPR实验清楚地表现出对Y145H突变体的亲和力降低。这两个 PDZK1 SNP 突变体不影响通过 URAT1 的尿酸盐转运活性。较少的

项目成果

Babu Ellapan: "Identification of a novel system L amino acid transporter structurally distinct from heterodimeric amino acid transporters"J Biol Chem. 278・44. 43838-43845 (2003)

Babu Ellapan：“结构上与异二聚体氨基酸转运蛋白不同的新型系统 L 氨基酸转运蛋白的鉴定”J Biol Chem 278・44（2003）。

Takeshi Sakata: "Novel single nucleotide polymorphisms of organic cation transporter 1 (SLC22A1) affecting transport functions"Biochem Biophys Res Commun. 313・3. 789-793 (2004)

Takeshi Sakata：“影响运输功能的有机阳离子转运蛋白 1 (SLC22A1) 的新型单核苷酸多态性”Biochem Biophys Res Commun. 313・3 (2004)。

The multivalent PDZ domain-containing protain PDZK1 regulates transport activity of renal urate-anion exchanger URAT1 via its C-terminal.

含有多价 PDZ 结构域的蛋白 PDZK1 通过其 C 末端调节肾尿酸阴离子交换剂 URAT1 的转运活性。

• 发表时间: 2004
• 期刊: J Biol Chem 279
• 作者: Y.Yajima;M.Narita;A.Usui;C.Kaneko;M.Miyatake;M.Narita;T.Yamaguchi;H.Tamaki;H.Wachi;Y.Seyama;T.Suzuki;Anzai N.
• 通讯作者: Anzai N.

Mutations in SLC6A19, encoding BOAT1, cause Hartnup disorder.

编码 BOAT1 的 SLC6A19 突变会导致 Hartnup 障碍。

• 发表时间: 2004
• 期刊: Nat Genet. 36
• 作者: Kleta R.

Expression of human organic anion transporters in the choroid plexus and their interactions with neurotransmitter metabolites

• DOI: 10.1254/jphs.93.430
• 发表时间: 2003-12-01
• 期刊: JOURNAL OF PHARMACOLOGICAL SCIENCES
• 影响因子: 3.5
• 作者: Alebouyeh, M;Takeda, M;Endou, H
• 通讯作者: Endou, H