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.

项目摘要/摘要 甲状腺龙（Th）缺乏以多种方式影响脊椎动物生理。小儿 先天性甲状腺功能减退症的后果（全世界最常见的先天性内分泌病）包括 有缺陷的增长和听力，无法实现最大的潜在智力。在成年人中 甲状腺功能减退症会导致高脂血症，体重变化和体重增加促进肥胖症。自始至终 整个脊椎动物亚球，Ths在编码的前体蛋白甲状腺球蛋白（TG）中合成 通过单个TG基因，在甲状腺刺激的马龙（TSH）的调节下。甲状腺素（T4）衍生 主要来自TG的N末端的独特，进化配置的位点（在Tyr-5位置），而 三碘甲状腺素（T3）的主要地层位点源自TG蛋白的另一端。 TG的碘化 是体内T4的唯一来源，但只有部分贡献者循环T3（其余T3被转换 通过在各种器官中除外T4）。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T3则导致急剧上下摇摆 T3的血液水平。我一直想知道，如果甲状腺本身会带来什么后果 TSH受调节的瞬间，可以有选择地生产身体的所有T3吗？在生理下 TSH的调节，TSH（TG的羧基末端切子结构域）通过基本上提供T3来调节 对身体 - 要么将不足以维持所有关键的发展和代谢 TH支持的功能。为了测试这一点，我建议使用CRISPR/CAS9-Mutagenesis开发前两个 带有纯合TG敲入突变体的基因编辑的小鼠模型，它们都编码：a）TG-Y5F 替换（消除TG的主要T4形成位点）或b）仅编码秘密的chel域 保留TG的主要T3形成位点 - 在这两种情况下 甲状腺由TSH物理调节。借助这些新颖的动物，我将研究发展， 生长，行为，听力，身体组成和体重以及温度调节。我将寻求严格的验证 通过平行细胞培养实验的体内乳舌研究研究，以表达相同的TG变体，并且 使用TG定量地表征其T4生产不足，但具有T3的能力 体外碘化，然后进行纳米液色谱串联质量光谱法。概述的计划 这项为期5年的K01奖将使我能够在全动物甲状腺病理生理学方面发展更多专业知识 以及我未来独立科学职业所需的尖端生化技能。此外，这个 提案具有重要的临床意义，以了解T4是否有任何特殊作用 对成年人的新生儿，同时深入了解了TH综合的进化起源 脊椎动物，并为人类管理中的未来改进开辟了新的可能性 甲状腺功能减退症。