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。甲状腺激素受体（thyroid hormone receptor， TR）有两个基因，分别是TRα和TRβ，在发育和成体组织中有不同的表达模式。在TRβ基因和TRα基因显性负突变的杂合家族中报道的不同表型有力地支持了TR异构体特异性的相关性。然而，TR异构体特异性作用的机制尚不清楚。核受体的翻译后修饰作为一种重要的基因调控机制正日益被人们所认识。小泛素样修饰物修饰的蛋白质多为转录因子和核激素受体。Sumoylation需要将SUMO偶联到蛋白质的共识识别基序ψ- lys - x - glu （ψ是一个大的疏水氨基酸）中的赖氨酸上，从而快速和动态地修饰参与细胞过程的蛋白质。我们发现，通过SUMO与赖氨酸283和389位点的TRα和赖氨酸50、146和443位点的TRβ结合对TR进行翻译后修饰，在三碘甲状腺原氨酸（T3）诱导的基因诱导和抑制以及TR亚型特异性中起重要作用。TRα-SUMO偶联与配体无关，利用E3连接酶PIASxb。tr - β- sumo是配体依赖性的，主要利用E3连接酶PIAS1。瞬态转染实验和内源基因调控研究表明，SUMO1和SUMO3与TR的结合是t3依赖性基因调控所必需的。通过染色质免疫沉淀（Chromatin Immunoprecipitation, ChIP）检测，SUMO1和SUMO3在瞬时功能实验中诱导t3的作用与内源基因调控中TR和辅因子结合的模式密切相关。辅助抑制因子在甲状腺激素的作用中起着至关重要的作用，而TR酰化对辅助抑制因子的募集至关重要。我们已经证明，TR summoylation对于甲状腺激素调节的代谢活动（包括脂肪细胞分化）和与重要代谢调节因子（包括LXR和PPARα）的串扰很重要。将追求的具体目标包括：1. 利用体外模型确定TR summoylation和TRα/TRβ亚型特异性作用在代谢调节中的作用。2. 确定sumo化如何调节TR介导的基因调控，包括TR与转录辅助因子的相互作用、代谢信号通路以及对t3调控基因的识别和结合。3. 在小鼠模型中评估TRα和TRβ summoylation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 (K389Q)和TRβ (K443Q)基因summoylation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酰化在代谢调节中的作用和TR异构体的特异性作用可能会导致发现与代谢疾病（包括血脂异常和肥胖）治疗相关的新的甲状腺激素信号靶点。

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.