In vivo dimerization of types 1, 2, and 3 iodothyronine selenodeiodinases
In vivo dimerization of types 1, 2, and 3 iodothyronine selenodeiodinases
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DOI：
10.1210/en.2002-220960
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发表时间：
2003-03-01
期刊：
影响因子：
4.8
通讯作者：
Bianco, AC
中科院分区：
文献类型：
--
作者：
Curcio-Morelli, C;Gereben, B;Bianco, AC
本研究的目的是检验 1、2 和 3 型碘甲状腺原氨酸硒代碘酶（D1、D2 和 D3）可以形成同型二聚体的假设。该策略包括在人胚胎肾上皮细胞中瞬时共表达野生型（wt）脱碘酶（靶标）和带有 FLAG 标记的丙氨酸或半胱氨酸突变体（诱饵）。使用抗 FLAG 抗体对 Se-75 标记的细胞裂解物进行免疫沉淀沉淀的 SDS-PAGE 显示了各个 wt 酶的正确大小的条带，其对应于细胞裂解物中总脱碘酶蛋白的大约 2-5%。使用抗 FLAG 抗体对瞬时表达单个 FLAG 标记的半胱氨酸脱碘酶的细胞裂解物进行蛋白质印迹分析，显示每种脱碘酶都有特定的单体条带，以及 D1 的相对分子质量 (M-r) 为 55,000、132 的 M 62,000 和 D3 的 Mr 65,000 的其他次要条带，这些条带在 100 ℃下被 100 mm 二硫苏糖醇消除。 C.抗FLAG抗体免疫耗尽来自共表达无活性FLAG标记的Ala突变体和相应wt酶(D1或D2)的细胞裂解物的10% D1和38% D2活性，但未能免疫耗尽wtD3活性。 D1 或 D2 活性存在于这些各自的颗粒中。我们得出结论：1）过表达的硒代碘化酶可能通过二硫桥形成同二聚体； 2)至少对于D1和D2，单体形式具有催化活性，证明这两种脱碘酶的催化活性仅需要一种wt单体配偶体。
The goal of the present investigation was to test the hypothesis that types 1, 2, and 3 iodothyronine selenodeiodinases (D1, D2, and D3) can form homodimers. The strategy included transient coexpression of wild-type (wt) deiodinases (target), and FLAG-tagged alanine or cysteine mutants (bait) in human embryonic kidney epithelial cells. SDS-PAGE of the immunoprecipitation pellet of Se-75-labeled cell lysates using anti-FLAG antibody revealed bands of the correct sizes for the respective wt enzymes, which corresponded to approximately 2-5% of the total deiodinase protein in the cell lysate. Western blot analysis with anti-FLAG antibody of lysates of cells transiently expressing individual FLAG-tagged-cysteine deiodinases revealed specific monomeric bands for each deiodinase and additional minor bands of relative molecular mass (M-r) Of 55,000 for D1, M 62,000 for 132, and Mr 65,000 for D3, which were eliminated by 100 mm dithiothreitol at 100 C. Anti-FLAG antibody immunodepleted 10% of D1 and 38% of D2 activity from lysates of cells coexpressing inactive FLAG-tagged Ala mutants and the respective wt enzymes (D1 or D2) but failed to immunodeplete wtD3 activity. D1 or D2 activities were present in these respective pellets. We conclude 1) that over-expressed selenodeiodinases can homodimerize probably through disulfide bridges; and 2) at least for D1 and D2, monomeric forms are catalytically active, demonstrating that only one wt monomer partner is required for catalytic activity of these two deiodinases.