Modulating Nuclear Receptor Activity via Sumoylation

描述（由申请方提供）：通过类泛素化调节核受体活性类固醇生成因子-1（SF-1）是一种组成型活性核受体，调节肾上腺功能和下丘脑-垂体-性腺轴的关键方面。核受体包括SF-1和其他NR 5A受体的SUMO修饰被认为是抑制它们的转录活性。我们研究了SF-1类小泛素化在两个保守的赖氨酸Lys 119和Lys 194，分别位于相邻的DBD和LBD的功能和结构的后果。令人惊讶的是，虽然先前在细胞中的功能丧失研究预测在Lys 194处的类小泛素化将极大地影响SF-1功能，但完全类小泛素化的SF-1 LBD蛋白的构象和辅调节剂募集与非类小泛素化的蛋白相同;仅注意到Ser 203磷酸化的减少。相比之下，使用在Lys 119处小泛素化的DBD蛋白观察到在非典型SF-1靶标处的DNA结合的选择性损失。此外，通过Lys 119类小泛素化调节SF-1功能必须在DNA结合之前发生。我们认为转录因子的SUMO化是一个活跃的翻译后标记，它消除了对SUMO敏感的靶基因的识别，抑制了基因的表达。虽然这些分子和生物化学研究表明，sumoylation抑制SF-1的转录输出，这一假设需要在体内进行测试。为此，在SF-1等位基因中产生SUMO缺陷突变敲入小鼠，称为SF-12 KR/2KR。该突变小鼠在妊娠中期（E9.5-13.5）表现出胚胎致死性，考虑到SF-1缺失小鼠在出生后由于肾上腺功能不全而死亡，这是令人惊讶的。在这里，我们将测试的假设，正常的转录程序不适当地调节在一个sumo缺陷SF-1突变体使用整个动物，基因组和结构的方法。这种竞争性更新将解决关于SF-1类小泛素化作用的三个剩余问题：1）SF-12 KR/2 KR小鼠中哪些发育或激素途径被破坏，导致妊娠中期死亡。2)在胎盘和其他内分泌组织中，2KR SF-1突变不适当地调节了哪些特定的元件和基因？3)SF-1类小泛素化对DNA结合域的结构影响是什么？这些问题的答案应该提供在体内和结构机制的见解，以sumoylation如何调节基因表达。公共卫生相关性：翻译后修饰调节许多类型的细胞蛋白质的活性。蛋白质的类小泛素化涉及蛋白质缀合，并且通常抑制许多转录调节因子的活性。在这项研究中，我们将首次评估核受体的sumo缺陷突变如何影响内分泌组织的发育和功能。这项工作的结果将有可能确定重要的新基因靶点，这些靶点对sumoylated蛋白的相对库特别敏感。他们还将提供机制的见解sumoylation如何抑制和“微调”内分泌系统的转录程序。

DESCRIPTION (provided by applicant): Modulating Nuclear Receptor Activity via Sumoylation Steroidogenic factor-1 (SF-1) is a constitutively active nuclear receptor that regulates critical aspects of adrenal function and the hypothalamic-pituitary-gonadal axis. SUMO modification of nuclear receptors including SF-1 and other NR5A receptors is proposed to repress their transcriptional activity. We examined the functional and structural consequences of SF-1 sumoylation at two conserved lysines Lys119 and Lys194 that reside adjacent to the DBD and LBD, respectively. Surprisingly, while previous loss-of-function studies in cells predicted that sumoylation at Lys194 would greatly impact SF-1 function, the conformation and coregulator recruitment of fully sumoylated SF-1 LBD protein were identical to non-sumoylated protein; only a decrease in Ser203 phosphorylation was noted. By contrast, a selective loss of DNA binding at non-canonical SF-1 targets was observed using DBD protein sumoylated at Lys119. Moreover, regulating SF-1 function by Lys119 sumoylation must occur prior to DNA binding. We propose that sumoylation of transcription factors is an active posttranslational mark that eliminates recognition of "SUMO-sensitive" target genes and restrains gene expression. While these molecular and biochemical studies suggest that sumoylation restrains the transcriptional output of SF-1, this hypothesis needs to be tested in vivo. To this end, a SUMO-deficient mutant knock-in mouse was created in the SF-1 allele, referred to as SF-12KR/2KR. This mutant mouse exhibits embryonic lethality at mid-gestation (E9.5-13.5), which is surprising considering that SF-1 null mice die postnatally due to adrenal insufficiency. Here we will test the hypothesis that normal transcriptional programs are inappropriately regulated in a sumo-deficient SF-1 mutant using whole animal, genomic, and structural approaches. This competitive renewal will address three remaining questions as to the role of SF-1 sumoylation: 1) What developmental or hormonal pathways are subverted in SF-12KR/2KR mice that lead to mid-gestation lethality. 2) What specific elements and genes are inappropriately regulated by the 2KR SF-1 mutation in the placenta and other endocrine tissues? 3) What are the structural consequences of SF-1 sumoylation on the DNA binding domain. Answers to these questions should provide in vivo and structural mechanistic insights as to how sumoylation modulates gene expression. PUBLIC HEALTH RELEVANCE: Post-translational modifications regulate the activity of many types of cellular proteins. Sumoylation of proteins involves protein conjugation and in general dampens the activity of many transcriptional regulators. In this study we will assess for the first time, how a sumo-deficient mutation of the nuclear receptor affects endocrine tissue development and function. Results from this work will potentially identify important new gene targets that are especially sensitive to the relative pools of sumoylated protein. They will also provide mechanistic insight into how sumoylation restrains and "fine tunes" transcriptional programs of the endocrine system.