Identification of the key residues responsible for the assembly of selenodeiodinases.

Type I deiodinase is the best characterized member of a small family of selenoenzymes catalyzing the bioactivation and disposal of thyroid hormone. This enzyme is an integral membrane protein composed of two 27-kDa subunits that assemble into a functional enzyme after translation using a highly conserved sequence of 16 amino acids in the C-terminal half of the polypeptide, (148)DFLXXYIXEAHXXDGW(163). In this study, we used alanine scanning mutagenesis to identify the key residues in this domain required for holoenzyme assembly. Overexpression of sequential alanine-substituted mutants of a dimerization domain-green fluorescent protein fusion showed that sequence (152)IYI(154) was required for type I enzyme assembly and that a catalytically active monomer was generated by a single I152A substitution. Overexpression of the sequential alanine-substituted dimerization domain mutants in type II selenodeiodinase-expressing cells showed that five residues ((153)FLIVY(157)) at the beginning and three residues ((164)SDG(166)) at the end of this region were required for the assembly of the type II enzyme. In vitro binding analysis revealed a free energy of association of -60 +/- 5 kJ/mol for the noncovalent interaction between dimerization domain monomers. These data identify and characterize the essential residues in the dimerization domain that are responsible for the post-translational assembly of selenodeiodinases.

Neuroanatomical pathways for thyroid hormone feedback in the human hypothalamus.

CONTEXT: Recent findings point to an increasing number of hypothalamic proteins involved in the central regulation of thyroid hormone feedback. The functional neuroanatomy of these proteins in the human hypothalamus is largely unknown at present. OBJECTIVE: The aim of this study was to report the distribution of type II and type III deiodinase (D2 and D3) as well as the recently identified T(3) transporter, monocarboxylate transporter 8 (MCT8), in the human hypothalamus. DESIGN: The study included enzyme activity assays, immunocytochemical studies, and mRNA in situ hybridizations in postmortem human hypothalamus (n = 9). RESULTS: D2 immunoreactivity is prominent in glial cells of the infundibular nucleus/median eminence, blood vessels, and cells lining the third ventricle. By contrast, both D3 and MCT8 are expressed by neurons of the paraventricular (PVN), supraoptic, and infundibular nucleus (IFN). In support of these immunocytochemical data, D2 and D3 enzyme activities are detectable in the mediobasal human hypothalamus. Combined D2, D3, MCT8, and thyroid hormone receptor immunohistochemistry and TRH mRNA in situ hybridization clearly showed that D3, MCT8, and thyroid hormone receptor isoforms are all expressed in TRH neurons of the PVN, whereas D2 is not. CONCLUSIONS AND IMPLICATIONS: Based on these findings, we propose three possible routes for thyroid hormone feedback on TRH neurons in the human PVN: 1) local thyroid hormone uptake from the vascular compartment within the PVN, 2) thyroid hormone uptake from the cerebrospinal fluid in the third ventricle followed by transport to TRH neurons in the PVN or IFN neurons projecting to TRH neurons in the PVN, and 3) thyroid hormone sensing in the IFN of the mediobasal hypothalamus by neurons projecting to TRH neurons in the PVN.

Hypothalamic type II iodothyronine deiodinase: a light and electron microscopic study.

In the central nervous system, the active form of thyroid hormone, T3, derives from the cellular uptake and intracellular 5'-monodeiodination of T4 by type II 5'-monodeiodinase (DII). Here, we report that using an antiserum raised against the C-terminus of the full-length SeDII, immunolabeled cells were found in the rat hypothalamus in agreement with the DII mRNA distribution. Light and electron microscopy shows that DII is localized in astrocytes and tanycytes, supporting the hypothesis that these cells play an important role in the mediation peripheral signals, such as thyroid hormones, on hypothalamic functions.