Biochemical and structural characterization of mammalian deiodinases as key regulators of thyroid hormone metabolism

点击翻译按钮获取中文摘要

Thyroxine (T4) is the major secreted product of the thyroid gland. Through site specific deiodination, T4 is converted to T3, the receptor-binding hormone. An additional deiodination step from T3 to T2 leads to inactivation. Deiodination and decarboxylation of thyroid hormones (TH) further lead to formation of thyronamines, TAM. All these TH deiodinations are catalyzed by a family of three iodothyronine deiodinase (Dio) isoforms (Dio1, Dio2, Dio3), which differ in their regioselectivity among the two aromatic rings of TH. The deiodinases form an evolutionary family sharing significant sequence homology and most architectural and catalytic properties. They contain selenocysteine (Sec) in their active sites and an N-terminal transmembrane region that contributes to dimerization, which is essential for deiodinase activity. As key enzymes in TH metabolism, Dio enzymes appear to be attractive drug targets, and the development of isoenzyme-specific inhibitors would be desirable from the perspective of clinical use. Development of Dio-targeting drugs has been hampered by a lack of a molecular understanding of Dio structure and catalysis. The membrane association and selenoprotein properties of Dio both have hindered the efficient recombinant expression of mammalian Dio for structural and functional studies. We recently solved a crystal structure of an inactive and monomeric Dio3 catalytic domain by using an N-terminally deleted, soluble Sec->Cys mutant to obtain first insights into Dio catalysis. Structure and biochemical results identified an active site H-bond network, first substrate binding site details, and peroxiredoxins-resembling features suggesting a mechanism for Dio reduction after iodine release. We now plan to study further features of Dio structure and catalysis by recombinantly expressing full-length Dio. We will use the available catalytic domain proteins and the active and full-length samples for solving crystal structures of active Dio dimer, of oxidized catalytic domains, and of Dio ligand complexes to identify mechanistic details and ligand recognition features. We will further use these proteins for biochemical experiments, in particular activity studies and mass spectrometry analyses, to study the role of the active site H-bond network, the interplay of the conserved Cys and selenylsulfides/disulfides in enzyme reduction, and how this system is ultimately reduced by the thiol cofactor. We will then aim to exploit our structural and mechanistic insights for the improvement of existing Dio inhibitors and identification of novel ones for the development of potent and isoform-specific compounds. These drugs will be valuable as lead compounds for the development of therapeutics and as tools for physiological studies on Dio function.