Modulation of the Human Glucuronosyltransferase UGT1A Pathway by Splice Isoform Polypeptides Is Mediated through Protein-Protein Interactions
Modulation of the Human Glucuronosyltransferase UGT1A Pathway by Splice Isoform Polypeptides Is Mediated through Protein-Protein Interactions
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DOI：
10.1074/jbc.m109.083139
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发表时间：
2010-02-05
影响因子：
4.8
通讯作者：
Guillemette, Chantal
中科院分区：
文献类型：
--
作者：
Bellemare, Judith;Rouleau, Melanie;Guillemette, Chantal
本研究调查了新发现的 45 kDa 酶促失活 UGT1A 剪接多肽（称为同工型 i2）对 UGT1A 介导的葡萄糖醛酸化作用的调节作用的分子机制。最初，使用模拟人体组织中 UGT1A1 亚型 1 和 2 相对丰度的诱导系统，葡萄糖醛酸苷的形成率显着降低。然后，我们使用组成型表达 i1 和 i2 两种异构体的异源系统，深入研究剪接 i2 的存在对葡萄糖醛酸化动力学的影响。 UGT1A1、UGT1A7 和 UGT1A8 被选为这些研究的候选者。在所有情况下，HEK293 细胞中 i1 和 i2 的共表达会导致葡萄糖醛酸化反应速度显着降低，而不影响所有测试底物的亲和力 (K-m (app)) 以及共底物 UDP-葡萄糖醛酸的 K-m。该数据与显性失活抑制模型一致，但不支持通过底物或共底物竞争性结合而介导的 UGT1A_i2 介导的抑制。相反，免疫共沉淀实验的数据表明存在同源寡聚（i1-i1 或 i2-i2）和异源寡聚（i1-i2）复合物，其中 i2-i2 和 i1-i2 亚基无活性。因此，蛋白质-蛋白质相互作用可能是i2剪接多肽抑制活性UGT1A_i1的原因。这种新的调节机制可以选择性地调节细胞对内源/外源刺激的反应。
This study investigated the molecular mechanisms underlying the regulatory effect of the newly discovered 45-kDa enzymatically inactive UGT1A spliced polypeptides, named isoform i2, upon UGT1A-mediated glucuronidation. Initially, using an inducible system that mimics the relative abundance of isoforms 1 and 2 of UGT1A1 in human tissues, the rates of formation of glucuronides were significantly reduced. We then used a heterologous system constitutively expressing both isoforms i1 and i2 for an in- depth investigation of the presence of spliced i2 on glucuronidation kinetics. UGT1A1, UGT1A7, and UGT1A8 were selected as candidates for these studies. In all cases, co-expression of i1 and i2 in HEK293 cells leads to a significant reduction of the velocity of the glucuronidation reaction without affecting the affinity (K-m (app)) for all substrates tested and the K-m for the co-substrate, UDP-glucuronic acid. The data are consistent with a dominant-negative model of inhibition but do not sustain with an UGT1A_i2-mediated inhibition by competitive binding for substrate or the co-substrate. In contrast, the data from the co-immunoprecipitation experiments are indicative of the existence of a mixture homo-oligomeric (i1-i1 or i2-i2) and hetero-oligomeric (i1-i2) complexes in which the i2-i2 and i1-i2 subunits would be inactive. Thus, protein-protein interactions are likely responsible for the inhibition of active UGT1A_i1 by i2 spliced polypeptides. This new regulatory mechanism may alternatively modulate cellular response to endo/xeno stimulus.