Characterization of the protein dimerization domain responsible for assembly of functional selenodeiodinases.
Thyroid hormone metabolism is catalyzed by a small family of selenoenzymes. Type I deiodinase (D1) is the best characterized family member and is an integral membrane protein composed of two 27-kDa subunits that assemble to a functional holoenzyme after translation. To characterize the protein domain(s) responsible for this post-translational assembly event, we used deletion/truncation analysis coupled with immune depletion assays to map the dimerization domain of D1. The results of our studies show that a highly conserved sequence of 16 amino acids in the C-terminal half of the D1 subunit, -D148FL-YI-EAH-DGW163-, serves as the dimerization domain. Based on the high conservation of this domain, we synthesized a novel bait peptide-green fluorescent protein fusion probe (DDD(GFP)) to examine holoenzyme assembly of other family members. Overexpression of either the DDD(GFP) or an inert D1 subunit (M4) into SeD2 (accession number U53505)-expressing C6 cells specifically led to the loss of >90% of the catalytic activity. Catalytically inactive D2 heterodimers composed of SeD2: DDD(GFP) subunits were rescued by specific immune precipitation with anti-SeD2 IgG, suggesting that SeD2 requires two functional subunits to assemble a catalytically active holoenzyme. These findings identify and characterize the essential dimerization domain responsible for post-translational assembly of selenodeiodinases and show that family members can intermingle through this highly conserved protein domain.
Leonard
Jack L
JL
Molecular Endocrinology Laboratory, Department of Cell and Molecular Physiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA. jack.leonard@umassmed.edu
Simpson
Gregory
G
Leonard
Deborah M
DM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D019281:Dimerization; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007453:Iodide Peroxidase / Q000737:chemistry*; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D051381:Rats; D013379:Substrate Specificity; D000097564:Iodothyronine Deiodinase Type II
D007453:Iodide Peroxidase; D000097564:Iodothyronine Deiodinase Type II; C109812:iodothyronine deiodinase type I