The Role of Thyroglobulin in Thyroid Hormone Synthesis

项目摘要/摘要 甲状腺激素(TH)缺乏以多种方式影响脊椎动物的生理。儿科 先天性甲状腺功能减退症(全世界最常见的先天性内分泌疾病)的后果包括 发育和听力有缺陷，无法实现最大的潜在智力。在成年人中， 甲状腺功能减退会导致高脂血症、产热改变和体重增加，从而导致肥胖。贯穿始终 整个脊椎动物亚门，它们是在前体蛋白甲状腺球蛋白(TG)内合成的，编码 由单个TG基因调控，受促甲状腺激素(TSH)调节。甲状腺素(T4)来源于 主要来自Tg N端的一个独特的、进化上保守的位点(在Tyr-5位置上)，而 三碘甲腺原氨酸(T3)的主要形成部位来自TG蛋白的另一端。甘油三酯的碘化 是体内T4的唯一来源，但只是循环T3的部分贡献者(剩余的T3被转换 通过在不同的器官中脱碘T4)。甲状腺功能减退症的T4治疗使许多患者持续 甲状腺功能减退症状，而单独使用T3的直接治疗会导致患者剧烈的上下波动 血液中的三碘甲状腺原氨酸水平。我一直很想知道，如果甲状腺本身， 受TSH时时刻刻调控，能选择性地产生体内所有的T3吗？在生理状态下 由TSH调节，TG的羧基末端螯合域-通过提供基本上唯一的T3 对身体来说--要么将不足以维持所有关键的发育和代谢 支持的功能。为了测试这一点，我建议使用CRISPR/Cas9-突变来开发前两个 具有纯合子TG敲入突变体的基因编辑的小鼠模型，编码以下任一种：a)TG-Y5F 替换(消除Tg的主要T4形成部位)或b)仅编码分泌的CHEL结构域 在这两种情况下都保留了Tg-的主要T3形成部位，在 TSH对甲状腺的生理调节作用。有了这些新奇的动物，我将研究发展， 生长、行为、听力、身体成分和体重，以及体温调节。我会寻求严格的验证 通过平行细胞培养实验进行体内激素遗传学研究，以表达相同的TG变体，以及 用TG定量表征他们T4产量的不足但T3产量的能力 体外碘化，然后进行纳米液相色谱串联质谱分析。中概述的计划 这个为期5年的K01奖将使我在整个动物甲状腺病理生理学方面获得更多的专业知识 以及我未来独立科学生涯所需的尖端生化技能。此外，这一点 这一建议对于了解T4是否有任何特殊作用具有重要的临床意义 从新生儿到成人，同时提供了对TH合成的进化起源的深刻洞察 脊椎动物，并为未来人类管理的精细化打开了新的可能性 甲状腺功能减退。

PROJECT SUMMARY/ ABSTRACT Thyroid hormone (TH) deficiency impacts vertebrate physiology in a myriad of ways. The pediatric consequences of congenital hypothyroidism (the most common congenital endocrinopathy worldwide) include defective growth and hearing, and an inability to achieve maximal potential intelligence. In adults, hypothyroidism leads to hyperlipidemia, altered thermogenesis, and weight gain promoting obesity. Throughout the entire vertebrate subphylum, THs are synthesized within the precursor protein thyroglobulin (Tg), encoded by the single TG gene, under the regulation of thyroid-stimulating hormone (TSH). Thyroxine (T4) derives primarily from a unique, evolutionarily conserved site at the N-terminus of Tg (on position Tyr-5), whereas the primary formation site for triiodothyronine (T3) derives from the opposite end of the Tg protein. Iodination of Tg is the sole source of T4 in the body, but only a partial contributor to circulating T3 (the remaining T3 is converted by deiodinating T4 in various organs). Treatment of hypothyroidism with T4 leaves many patients with persistent hypothyroid symptoms, whereas direct treatment exclusively with T3 results in dramatic up-down swings in blood levels of T3. I have been interested to know, what would be the consequences if the thyroid gland itself, regulated moment-to-moment by TSH, could selectively produce all of the body’s T3? Under physiological regulation by TSH, the carboxyl-terminal ChEL domain of Tg — by providing essentially exclusively T3 to the body — either will or will not be sufficient to sustain all critical developmental and metabolic functions supported by TH. To test this I propose to use CRISPR/Cas9-mutagenesis to develop the first two genetically-edited mouse models with homozygous TG knock-in mutants encoding either: a) a Tg-Y5F substitution (eliminating the primary T4-forming site of Tg) or b) encoding only the secretory ChEL domain that preserves the primary T3-forming site of Tg — in both cases leaving the primary T3-forming site of Tg intact in a thyroid gland physiologically regulated by TSH. With these novel animals in hand, I will study development, growth, behavior, hearing, body composition and weight, and thermoregulation. I will seek rigorous validation of in vivo hormonogesis studies with parallel cell culture experiments to express the same Tg variants, and characterize quantitatively their deficiency of T4 production but competence for T3 production, using Tg iodination in vitro, followed by nano-liquid chromatography tandem-mass spectrometry. The plan outlined in this 5-year K01 Award will permit me to develop additional expertise in whole animal thyroid pathophysiology as well as cutting edge biochemical skills needed for my future independent scientific career. Furthermore, this proposal is of significant clinical relevance to understand if there is any special role of T4 from neonates to adults, while providing deep insight into the evolutionary origins of TH synthesis in vertebrates, and opening new possibilities for future refinement in the management of human hypothyroidism.