Selective proteolysis of human type 2 deiodinase: a novel ubiquitin-proteasomal mediated mechanism for regulation of hormone activation.
We investigated the mechanism by which T4 regulates its activation to T3 by the type 2 iodothyronine deiodinase (D2). D2 is a short- lived (t1/2 50 min), 31-kDa endoplasmic reticulum (ER) integral membrane selenoenzyme that generates intracellular T3. Inhibition of the ubiquitin (Ub) activating enzyme, E1, or MG132, a proteasome blocker, inhibits both the basal and substrate-induced acceleration of D2 degradation. Using a catalytically active transiently expressed FLAG-tagged-NH2-D2, we found rapid synthesis of high molecular mass (100-300 kDa) Ub-D2 conjugates that are catalytically inactive. Ub-D2 increases when cells are exposed to D2 substrate or MG132 and disappears rapidly after E1 inactivation. Fusion of FLAG epitope to the COOH terminus of D2 prolongs its half-life approximately 2.5-fold and increases the levels of active and, especially, Ub-D2. This indicates that COOH-terminal modification interferes with proteasomal uptake of Ub-D2 that can then be deubiquitinated. Interestingly, the type 1 deiodinase, a related selenoenzyme that also converts T4 to T3 but with a half-life of >12 h, is inactivated but not ubiquitinated or degraded after exposure to substrate. Thus, ubiquitination of the ER-resident enzyme D2 constitutes a specific posttranslational mechanism for T4 regulation of its own activation in the central nervous system and pituitary tissues in which D2-catalyzed T4 to T3 conversion is the major source of intracellular T3.
Gereben
B
B
Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
Goncalves
C
C
Harney
J W
JW
Larsen
P R
PR
Bianco
A C
AC
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D000939:Epitopes / Q000235:genetics; D006728:Hormones / Q000378:metabolism*; D006801:Humans; D007453:Iodide Peroxidase / Q000235:genetics / Q000378:metabolism*; D008025:Ligases / Q000378:metabolism; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism*; D009842:Oligopeptides; D010455:Peptides / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013974:Thyroxine / Q000378:metabolism; D014284:Triiodothyronine / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism*; D000097564:Iodothyronine Deiodinase Type II
D003546:Cysteine Endopeptidases; D000939:Epitopes; D006728:Hormones; D007453:Iodide Peroxidase; D008025:Ligases; D009097:Multienzyme Complexes; D009842:Oligopeptides; D010455:Peptides; D046988:Proteasome Endopeptidase Complex; D011994:Recombinant Proteins; D013974:Thyroxine; D014284:Triiodothyronine; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins; D000097564:Iodothyronine Deiodinase Type II; C085706:FLAG peptide; C109812:iodothyronine deiodinase type I