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

甲状腺素（T4）是甲状腺的主要分泌产物。通过位点特异性脱碘，T4转化为受体结合激素T3。从T3到T2的额外脱碘步骤导致失活。甲状腺激素（TH）的脱碘和脱羧进一步导致甲状腺胺（TAM）的形成。所有这些脱碘都是由一个家族的三个碘甲状腺原氨酸脱碘酶（Dio）异构体（Dio1, Dio2, Dio3）催化的，它们在TH的两个芳香环上的区域选择性不同。脱碘酶形成一个进化家族，具有重要的序列同源性和大多数结构和催化性质。它们的活性位点含有硒代半胱氨酸（Sec）和一个n端跨膜区域，有助于二聚化，这是脱碘酶活性所必需的。Dio酶作为TH代谢的关键酶，是有吸引力的药物靶点，从临床应用的角度来看，开发同工酶特异性抑制剂是可取的。由于缺乏对Dio结构和催化的分子理解，Dio靶向药物的开发一直受到阻碍。Dio的膜结合性和硒蛋白特性阻碍了Dio在哺乳动物中的高效重组表达，不利于其结构和功能的研究。我们最近通过使用n端缺失的可溶性Sec->Cys突变体解决了非活性和单体Dio3催化结构域的晶体结构，从而首次了解了Dio催化作用。结构和生化结果鉴定了活性位点氢键网络、第一底物结合位点细节和类似过氧化物还原素的特征，表明碘释放后Dio还原的机制。我们现在计划通过重组表达全长Dio进一步研究其结构特征和催化作用。我们将使用可用的催化结构域蛋白和活性和全长样品来求解活性Dio二聚体、氧化催化结构域和Dio配体复合物的晶体结构，以确定机理细节和配体识别特征。我们将进一步将这些蛋白质用于生化实验，特别是活性研究和质谱分析，以研究活性位点h -键网络的作用，保守的Cys和硒基硫化物/二硫化物在酶还原中的相互作用，以及该系统最终如何被硫醇辅助因子还原。然后，我们的目标是利用我们的结构和机制见解来改进现有的Dio抑制剂，并鉴定新的抑制剂，以开发有效的和同型特异性的化合物。这些药物将作为先导化合物用于治疗药物的开发和Dio功能生理学研究的工具。

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.