In vivo dimerization of types 1, 2, and 3 iodothyronine selenodeiodinases.
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.
Curcio-Morelli
Cyntia
C
Thyroid Section, Division of Endocrinology, Diabetes and Hypertension, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
Gereben
Balazs
B
Zavacki
Ann Marie
AM
Kim
Brian W
BW
Huang
Stephen
S
Harney
John W
JW
Larsen
P Reed
PR
Bianco
Antonio C
AC
D015153:Blotting, Western; D002384:Catalysis; D002460:Cell Line; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006801:Humans; D007163:Immunosorbent Techniques; D007453:Iodide Peroxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007553:Isotope Labeling; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012644:Selenium Radioisotopes; D013329:Structure-Activity Relationship; D014162:Transfection; D000097564:Iodothyronine Deiodinase Type II
D004220:Disulfides; D004229:Dithiothreitol; D007453:Iodide Peroxidase; D011994:Recombinant Proteins; D012644:Selenium Radioisotopes; D000097564:Iodothyronine Deiodinase Type II; C109812:iodothyronine deiodinase type I; C117274:iodothyronine deiodinase type III