Thyroid Hormone Receptor Isoform-Specific Actions

项目摘要 甲状腺激素对正常发育、生长、神经分化和代谢调节至关重要 在哺乳动物中，并且是两栖动物变态所必需的。有两种甲状腺激素受体（TR） 基因TR <$和TR <$，在发育和成体组织中具有不同的表达模式。的相关性 的TR亚型特异性的强有力的支持，在家庭杂合子报告的不同表型， TR基因和TR基因的显性负突变。TR亚型特异性作用的机制， 然而，这一点并没有得到很好理解。核受体的翻译后修饰越来越受到人们的重视 被认为是基因调控的重要机制。大多数蛋白质被小的类泛素修饰， SUMO修饰子是转录因子和核激素受体。类小泛素化需要缀合 SUMO与赖氨酸内的共有识别基序<$-Lys-X-Glu（<$是一种大的疏水性氨基酸） 一种蛋白质，可快速动态地修饰参与细胞过程的蛋白质。我们确定 通过SUMO与TR <$在赖氨酸283和389处的缀合，以及TR <$在 赖氨酸50、146和443在三碘甲腺原氨酸（T3）诱导的基因诱导和抑制中起重要作用 以及TR同种型特异性。TR？-SUMO偶联不依赖于配体，并利用E3连接酶 PIASxb. TR-SUMO是配体依赖性的，主要利用E3连接酶PIAS 1。SUMO 1和SUMO 3 T3依赖性基因调控需要与TR偶联，如瞬时转染中所示 内源基因调控的测定和研究。SUMO 1和SUMO 3在T3诱导中的作用 功能测定与TR和辅因子结合在内源性免疫调节中的模式密切匹配。 基因，如通过染色质免疫沉淀（ChIP）测定。辅阻遏物在 在甲状腺激素作用中，TR类小泛素化对于辅阻遏物的募集是重要的。我们有 证明TR类小泛素化对于甲状腺激素调节的代谢作用是重要的， 脂肪细胞分化和与重要的代谢调节因子（包括LXR和PPAR）的相互作用。具体 将追求的目标包括：1.利用体外模型确定TR类小泛素化的作用， TR <$/TR <$异构体在代谢调节中的特异性作用。2.确定类小泛素化如何调节TR- 介导的基因调控，包括TR与转录辅因子的相互作用，代谢信号传导途径， 以及识别和结合T3调节的基因。3.评价TR <$和TR <$sumoylation位点的影响 突变对甲状腺激素调节代谢的小鼠模型。我们将评估 在甲状腺激素轴、组织上引入TR ²（K389 Q）和TR ²（K443 Q）基因类小化突变 甲状腺水平和代谢调节。了解TR类小泛素化在代谢调节中的作用 和TR-亚型特异性作用可能导致识别新的甲状腺激素信号转导相关靶点。 涉及代谢疾病包括血脂异常和肥胖症的治疗。

PROJECT SUMMARY Thyroid hormone is essential for normal development, growth, neural differentiation, and metabolic regulation in mammals, and is required for amphibian metamorphosis. There are two thyroid hormone receptor (TR) genes, TR¿ and TR¿, with different patterns of expression in development and in adult tissues. The relevance of TR isoform specificity is strongly supported by the distinct phenotypes reported in families heterozygous for dominant negative mutations of the TR¿ gene and TR¿ gene. The mechanism of TR isoform-specific action, however, is not well understood. Posttranslational modification of nuclear receptors is being increasingly recognized as an important mechanism of gene regulation. Most proteins modified by small ubiquitin-like modifier (SUMO) are transcription factors and nuclear hormone receptors. Sumoylation requires conjugation of SUMO to a lysine within the consensus recognition motif ¿-Lys-X-Glu (¿ is a large hydrophobic amino acid) of a protein, which rapidly and dynamically modifies proteins involved in cellular processes. We determined that posttranslational modification of TR by conjugation of SUMO to TR¿ at lysines 283 and 389, and TR¿ at lysines 50, 146 and 443, plays an essential role in triiodothyronine (T3)-induced gene induction and repression as well as TR isoform-specificity. TR¿-SUMO conjugation is ligand independent and utilizes the E3 ligase PIASxb. TR¿-SUMO is ligand dependent and utilizes predominantly the E3 ligase PIAS1. SUMO1 and SUMO3 conjugation to TR are required for T3-dependent gene regulation, as demonstrated in transient transfection assay and studies of endogenous gene regulation. The role of SUMO1 and SUMO3 in T3-induction in transient functional assays is closely matched to the pattern of TR and co-factor binding in regulation of endogenous genes, as determined by Chromatin Immunoprecipitation (ChIP) assays. Co-repressors play an essential role in thyroid hormone action, and TR sumoylation is important for co-repressor recruitment. We have demonstrated that TR sumoylation is important for thyroid hormone-regulated metabolic actions including adipocyte differentiation and cross-talk with important metabolic regulators including LXR and PPAR¿. Specific aims that will be pursued include; 1. Utilize in vitro models to determine the role of TR sumoylation and TR¿/TR¿ isoform-specific actions in metabolic regulation. 2. Determine how sumoylation modulates TR- mediated gene regulation including TR interaction with transcription co-factors, metabolic signaling pathways, and recognition and binding to T3-regulated genes. 3. Evaluate the influence of TR¿ and TR¿ sumoylation site mutations on thyroid hormone regulation of metabolism in a mouse model. We will assess the impact of introducing TR¿ (K389Q) and TR¿ (K443Q) gene sumoylation mutantions on the thyroid hormone axis, tissue level thyroid status, and metabolic regulation. Understanding the role of TR sumoylation in metabolic regulation and TR-isoform specific action may lead to the identification of novel thyroid hormone signaling targets relevant to therapies for metabolic diseases, including dyslipidemia and obesity.