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

哺乳动物脱碘酶作为甲状腺激素代谢关键调节剂的生化和结构特征

• 批准号: 280029505
• 负责人: Professor Dr. Ulrich Schweizer
• 金额: --
• 依托单位: Institut für Biochemie und Molekularbiologie (IBMB)
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2020-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/280029505?language=en
• 关键词: Biochemical; structural; characterization; mammalian; deiodinases

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.

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T3，受体结合激素。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原氨酸（TAM）的形成。所有这些TH脱碘都是由三种碘甲腺原氨酸脱碘酶（Dio）同种型（Dio 1，Dio 2，Dio 3）催化的，它们在TH的两个芳环之间的区域选择性不同。脱碘酶形成一个进化家族，具有显著的序列同源性和大多数结构和催化性质。它们在其活性位点含有硒代半胱氨酸（Sec）和有助于二聚化的N-末端跨膜区，这是脱碘酶活性所必需的。作为TH代谢的关键酶，Dio酶似乎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将是可取的。由于缺乏对Dio结构和催化作用的分子理解，Dio靶向药物的开发受到阻碍。Dio的膜结合和硒蛋白性质都阻碍了Dio在哺乳动物中的高效重组表达，以用于结构和功能的研究。我们最近通过使用N-末端缺失的可溶性Sec->Cys突变体解决了无活性和单体Dio 3催化结构域的晶体结构，以获得对Dio催化的初步了解。结构和生化结果确定了一个活性位点的H-键网络，第一个底物结合位点的细节，和过氧化物氧还蛋白类似的功能，表明碘释放后的Dio还原机制。我们现在计划通过重组表达全长Dio来研究Dio结构和催化的进一步特征。我们将使用现有的催化结构域蛋白质和活性和全长样品来解决活性二聚体，氧化催化结构域和二配体复合物的晶体结构，以确定机制细节和配体识别功能。我们将进一步使用这些蛋白质的生化实验，特别是活性研究和质谱分析，研究活性位点H-键网络的作用，保守的半胱氨酸和硒基硫化物/二硫化物在酶还原中的相互作用，以及该系统是如何最终减少巯基辅因子。然后，我们的目标是利用我们的结构和机制的见解，改善现有的Dio抑制剂和识别新的有效的和异构体特异性化合物的发展。这些药物将是有价值的先导化合物的治疗发展和作为工具的生理研究Dio功能。