The Role of Thyroglobulin in Thyroid Hormone Synthesis

甲状腺球蛋白在甲状腺激素合成中的作用

• 批准号: 10552539
• 负责人: Cintia E. Citterio
• 金额: $ 7.71万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-19 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10552539
• 关键词: Adult; Affect; Alleles; Animal Model; Animals; Behavior; Biochemical; Biological Assay; Blood; Body Composition; Body Weight; Breeding; C-terminal; CRISPR/Cas technology; Cell Culture Techniques; Childhood; Clinical; Competence; Coupling; Cretinism; Data; Defect; Development; Event; Functional disorder; Future; Gene Fusion; Genes; Growth; Hand; Hearing; Hormones; Human; Hyperlipidemia; Hypothyroidism; In Vitro; Intelligence; Iodination reaction; Knock-in; Mediating; Mentored Research Scientist Development Award; Metabolic; Modeling; Monoiodotyrosine; Mus; Mutagenesis; N-terminal; Names; Obesity; Organ; Patients; Phenotype; Physiologic Thermoregulation; Physiological; Physiology; Play; Population; Positioning Attribute; Production; Protein Precursors; Proteins; Reaction; Recombinants; Regulation; Role; Site; Source; Symptoms; Testing; Thermogenesis; Thyroglobulin; Thyroid Gland; Thyroid Hormones; Thyrotropin; Thyrotropin Receptor; Thyroxine; Time; Transfection; Triiodothyronine; Validation; Variant; Vertebrates; Weight Gain; body system; career; clinically relevant; deiodination; experience; experimental study; hormone deficiency; hormone regulation; in vivo; insight; interest; mouse model; mutant; nano-liquid chromatography; neonate; novel; preservation; skills; success; tandem mass spectrometry

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.

项目总结/摘要 甲状腺激素（TH）缺乏症以多种方式影响脊椎动物的生理学。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病）的后果包括 有缺陷的生长和听力，以及无法实现最大的潜在智力。在成人中， 甲状腺功能减退导致高脂血症、产热改变和体重增加，从而促进肥胖。在整个 在整个脊椎动物亚门中，TH在前体蛋白甲状腺球蛋白（Tg）内合成， 由单个TG基因在促甲状腺激素（TSH）的调控下产生。甲状腺素（T4）衍生物 主要来自Tg的N-末端（在位置Tyr-5上）的独特的进化保守位点，而 三碘甲状腺原氨酸（T3）的主要形成位点来自Tg蛋白的相对端。Tg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被转化为 通过使各种器官中的T4脱碘）。用T4治疗甲状腺功能减退症使许多患者持续 甲状腺功能减退症状，而直接治疗完全与T3的结果在戏剧性的上下波动， 血液中的T3水平我很想知道，如果甲状腺本身， 每时每刻都受到TSH的调节，可以选择性地产生身体所有的T3？在生理 通过TSH调节，Tg的羧基末端ChEL结构域-通过基本上专门提供T3 对身体-无论是将或将不足以维持所有关键的发展和代谢 功能由TH支持。为了测试这一点，我建议使用CRISPR/Cas9诱变来开发前两个 具有纯合TG敲入突变体的基因编辑的小鼠模型，所述突变体编码： 取代（消除Tg的初级T4形成位点）或B）仅编码分泌性ChEL结构域， 保留了Tg的主要T3形成位点-在两种情况下，使Tg的主要T3形成位点在一种 甲状腺由TSH生理调节。有了这些新奇的动物在手，我将学习发展， 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 使用平行细胞培养实验进行体内造血研究，以表达相同的Tg变体，以及 用Tg定量表征它们T4生产的不足，但T3生产的能力， 体外碘化，然后进行纳米液相色谱串联质谱。该计划概述于 这个为期5年的K 01奖将使我能够在整个动物甲状腺病理生理学方面发展更多的专业知识 以及我未来独立科学事业所需的尖端生物化学技能。而且这 这项提议对于了解T4是否有任何特殊作用具有重要的临床意义， 新生儿到成人，同时提供深入了解TH合成的进化起源， 脊椎动物，并开辟新的可能性，为未来的完善管理人类 甲状腺功能减退症。