In vivo dimerization of types 1, 2, and 3 iodothyronine selenodeiodinases

Cyntia Curcio-Morelli, Balazs Gereben, Ann Marie Zavacki, Brian W. Kim, Stephen Huang, John W. Harney, P. Reed Larsen, Antonio C. Bianco

Research output: Contribution to journalArticle

53 Citations (Scopus)

Abstract

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 75Se-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 (Mr) of 55,000 for D1, Mr 62,000 for D2, 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.

Original languageEnglish
Pages (from-to)937-946
Number of pages10
JournalEndocrinology
Volume144
Issue number3
DOIs
StatePublished - Mar 1 2003

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Iodide Peroxidase
Dimerization
Anti-Idiotypic Antibodies
Cysteine
Dithiothreitol
Enzymes
Immunoprecipitation
Disulfides
Alanine
Polyacrylamide Gel Electrophoresis
Western Blotting
Epithelial Cells
3-monoiodothyronine
Kidney
Proteins

ASJC Scopus subject areas

  • Endocrinology
  • Endocrinology, Diabetes and Metabolism

Cite this

Curcio-Morelli, C., Gereben, B., Zavacki, A. M., Kim, B. W., Huang, S., Harney, J. W., ... Bianco, A. C. (2003). In vivo dimerization of types 1, 2, and 3 iodothyronine selenodeiodinases. Endocrinology, 144(3), 937-946. https://doi.org/10.1210/en.2002-220960

In vivo dimerization of types 1, 2, and 3 iodothyronine selenodeiodinases. / Curcio-Morelli, Cyntia; Gereben, Balazs; Zavacki, Ann Marie; Kim, Brian W.; Huang, Stephen; Harney, John W.; Larsen, P. Reed; Bianco, Antonio C.

In: Endocrinology, Vol. 144, No. 3, 01.03.2003, p. 937-946.

Research output: Contribution to journalArticle

Curcio-Morelli, C, Gereben, B, Zavacki, AM, Kim, BW, Huang, S, Harney, JW, Larsen, PR & Bianco, AC 2003, 'In vivo dimerization of types 1, 2, and 3 iodothyronine selenodeiodinases', Endocrinology, vol. 144, no. 3, pp. 937-946. https://doi.org/10.1210/en.2002-220960
Curcio-Morelli C, Gereben B, Zavacki AM, Kim BW, Huang S, Harney JW et al. In vivo dimerization of types 1, 2, and 3 iodothyronine selenodeiodinases. Endocrinology. 2003 Mar 1;144(3):937-946. https://doi.org/10.1210/en.2002-220960
Curcio-Morelli, Cyntia ; Gereben, Balazs ; Zavacki, Ann Marie ; Kim, Brian W. ; Huang, Stephen ; Harney, John W. ; Larsen, P. Reed ; Bianco, Antonio C. / In vivo dimerization of types 1, 2, and 3 iodothyronine selenodeiodinases. In: Endocrinology. 2003 ; Vol. 144, No. 3. pp. 937-946.
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