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.

项目概要/摘要 甲状腺激素 (TH) 缺乏会通过多种方式影响脊椎动物的生理机能。 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 成人的生长和听力有缺陷，无法发挥最大的潜在智力。 甲状腺功能减退症会导致高脂血症、生热作用改变以及体重增加，进而导致肥胖。 在整个脊椎动物亚门中，TH 是在前体蛋白甲状腺球蛋白 (Tg) 内合成的，编码 由单一TG基因，在促甲状腺激素（TSH）的调节下衍生而来。 主要来自 Tg N 末端（Tyr-5 位）的一个独特的、进化上保守的位点，而 三碘甲状腺原氨酸 (T3) 的主要形成位点源自 Tg 蛋白的碘化末端。 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 转化为 通过在各个器官中脱碘 T4），用 T4 治疗甲状腺功能减退症会导致许多患者持续存在甲状腺功能减退症。 甲状腺功能减退症状，而仅用 T3 直接治疗会导致甲状腺功能急剧上下波动 我一直有兴趣知道，如果甲状腺本身，会产生什么后果， 受TSH时刻调节，在生理状态下能选择性产生体内全部T3吗？ 由 TSH（Tg 的羧基末端 ChEL 结构域）调节 — 基本上只提供 T3 对身体来说——要么足以，要么不足以维持所有关键的发育和代谢 为了测试这一点，我建议使用 CRISPR/Cas9 诱变来开发前两个功能。 具有纯合 TG 敲入突变体的基因编辑小鼠模型，编码：a) Tg-Y5F 取代（消除 Tg 的主要 T4 形成位点）或 b) 仅编码分泌性 ChEL 结构域 保留 Tg 的主要 T3 形成位点 — 在这两种情况下，Tg 的主要 T3 形成位点都保持完整 甲状腺在生理上受 TSH 调节。有了这些新动物，我将研究发育， 我将寻求严格的验证，包括生长、行为、听力、身体成分和体重以及体温调节。 通过平行细胞培养实验进行体内激素生成研究，以表达相同的 Tg 变体，以及 使用 Tg 来表征他们 T4 生产的数量不足，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分析。 这个为期 5 年的 K01 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职业所需的尖端生化技能。 该提案对于了解 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，同时深入了解 TH 合成的进化起源 脊椎动物，并为未来改进人类管理开辟新的可能性 甲状腺功能减退症。