Curcio-Morelli Cyntia, Gereben Balazs, Zavacki Ann Marie, Kim Brian W, Huang Stephen, Harney John W, Larsen P Reed, Bianco Antonio C
Thyroid Section, Division of Endocrinology, Diabetes and Hypertension, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
Endocrinology. 2003 Mar;144(3):937-46. doi: 10.1210/en.2002-220960.
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 (75)Se-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(r) 62,000 for D2, and M(r) 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 overexpressed 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.
本研究的目的是验证1型、2型和3型碘甲状腺原氨酸脱碘酶（D1、D2和D3）能够形成同型二聚体这一假说。研究策略包括在人胚肾上皮细胞中瞬时共表达野生型（wt）脱碘酶（靶标）以及带有FLAG标签的丙氨酸或半胱氨酸突变体（诱饵）。使用抗FLAG抗体对（75）Se标记的细胞裂解物进行免疫沉淀沉淀的SDS-PAGE分析显示，各wt酶出现了正确大小的条带，其在细胞裂解物中脱碘酶总蛋白中所占比例约为2%-5%。对瞬时表达单个带有FLAG标签的半胱氨酸脱碘酶的细胞裂解物进行抗FLAG抗体的蛋白质印迹分析，结果显示每种脱碘酶都有特定的单体条带，以及相对分子质量（M(r)）分别为55,000（D1）、62,000（D2）和65,000（D3）的额外小条带，这些条带在100℃下经100 mM二硫苏糖醇处理后消失。抗FLAG抗体从共表达无活性的带有FLAG标签的Ala突变体和相应wt酶（D1或D2）的细胞裂解物中免疫去除了10%的D1活性和38%的D2活性，但未能免疫去除wtD3活性。这些相应沉淀中存在D1或D2活性。我们得出结论：1）过表达的脱碘酶可能通过二硫键形成同型二聚体；2）至少对于D1和D2而言，单体形式具有催化活性，这表明这两种脱碘酶的催化活性仅需要一个wt单体伴侣。
