Thyroid Hormone Receptor Isoform-Specific Actions

描述（申请人提供）：甲状腺激素是哺乳动物正常发育、生长、神经分化和代谢调节所必需的，也是两栖动物变态所必需的。有两种甲状腺激素受体（TR）基因，TRα和TRβ，在发育和成人组织中具有不同的表达模式。TRβ基因和TRα基因显性负突变杂合子家族中报告的不同表型强烈支持TR亚型特异性的相关性。然而，TR异构体特异性作用的机制还不清楚。核受体的翻译后修饰被认为是基因调控的重要机制。SUMO修饰的蛋白质主要是转录因子和核激素受体。SUMO化需要SUMO与蛋白质的共有识别基序N-Lys-X-Glu（N-Glu是一种大的疏水氨基酸）内的赖氨酸缀合，其快速且动态地修饰参与细胞过程的蛋白质。我们确定，通过SUMO与TRα在赖氨酸283和389处以及TRβ在赖氨酸50、146和443处的缀合，TR的翻译后修饰在三碘甲状腺原氨酸（T3）诱导的基因诱导和阻遏以及TR亚型特异性中起着重要作用。TRα-SUMO缀合是配体非依赖性的，并利用E3连接酶PIASxb。TRβ-SUMO是配体依赖性的，主要利用E3连接酶PIAS 1。如瞬时转染试验和内源性基因调控研究所示，T3依赖性基因调控需要SUMO 1和SUMO 3与TR偶联。SUMO 1和SUMO 3在瞬时功能测定中的T3诱导中的作用与TR和辅因子结合在内源性基因调节中的模式密切匹配，如通过染色质免疫沉淀（ChIP）测定所确定的。辅阻遏物在甲状腺激素作用中起重要作用，TR类小泛素化对于辅阻遏物的募集很重要。我们已经证明，TR类小泛素化对于甲状腺激素调节的代谢作用（包括脂肪细胞分化）以及与重要代谢调节因子（包括LXR和PPARα）的相互作用非常重要。具体目标包括：1。利用体外模型确定TR类小泛素化和TRα/TRβ亚型特异性作用在代谢调节中的作用。2.确定sumoylation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，代谢信号通路，以及识别和结合T3调节的基因。3.在小鼠模型中评价TRα和TRβ类小泛素化位点突变对甲状腺激素代谢调节的影响。我们将评估引入TRα（K389 Q）和TRβ（K443 Q）基因类小泛素化突变对甲状腺激素轴、组织水平甲状腺状态和代谢调节的影响。了解TR类小泛素化在代谢调节和TR亚型特异性作用中的作用，可能会导致识别与代谢疾病（包括血脂异常和肥胖症）治疗相关的新型甲状腺激素信号转导靶点。

DESCRIPTION (provided by applicant): 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, TRα and TRβ, 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 TRβ gene and TRα 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 ψ-Lys-X-Glu (ψ 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 TRα at lysines 283 and 389, and TRβ at lysines 50, 146 and 443, plays an essential role in triiodothyronine (T3)-induced gene induction and repression as well as TR isoform-specificity. TRα-SUMO conjugation is ligand-independent and utilizes the E3 ligase PIASxb. TRβ-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 PPARα. Specific aims that will be pursued include; 1. Utilize in vitro models to determine the role of TR sumoylation and TRα/TRβ 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 TRα and TRβ sumoylation site mutations on thyroid hormone regulation of metabolism in a mouse model. We will assess the impact of introducing TRα (K389Q) and TRβ (K443Q) gene sumoylation mutations 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.