Modulating Nuclear Receptor Activity via Sumoylation

描述(由申请人提供)：通过总合作用调节核受体活性类固醇生成因子-1(SF-1)是一种结构性活性核受体，调节肾上腺功能和下丘脑-垂体-性腺轴的关键方面。对包括SF-1和其他NR5A受体在内的核受体进行相扑修饰，可以抑制它们的转录活性。我们研究了SF-1在两个保守的赖氨酸Lys119和Lys194上相加的功能和结构后果，这两个赖氨酸分别位于DBD和LBD附近。令人惊讶的是，尽管先前的细胞功能丧失研究预测，Lys194位的总和基化将极大地影响SF-1的功能，但完全总化的SF-1 LBD蛋白的构象和辅助调节因子募集与非总和蛋白相同；只注意到Ser203磷酸化的减少。相比之下，使用在Lys119处求和的DBD蛋白，可以观察到在非规范的SF-1靶点选择性地失去DNA结合。此外，Lys119对SF-1功能的调节必须发生在DNA结合之前。我们认为转录因子的相扑作用是一种活性的翻译后标记，它消除了对相扑敏感的靶基因的识别，抑制了基因的表达。虽然这些分子和生化研究表明，苏莫化作用抑制了SF-1的转录输出，但这一假说需要在体内进行验证。为此，在被称为SF-12KR/2KR的SF-1等位基因中创造了一个相扑缺陷突变敲入小鼠。这个突变的小鼠在怀孕中期(E9.5-13.5)表现出胚胎致死性，考虑到SF-1基因缺失的小鼠出生后死于肾上腺功能不全，这是令人惊讶的。在这里，我们将使用整体动物、基因组和结构方法测试正常转录程序在相扑缺陷SF-1突变体中受到不适当调控的假设。这一竞争性更新将解决关于SF-1 SUMO化作用的三个剩余问题：1)在SF-12KR/2KR小鼠中，哪些发育或激素途径被颠覆，从而导致中期妊娠死亡。2)胎盘和其他内分泌组织中的2KR SF-1突变对哪些特定的元件和基因进行了不适当的调控？3)SF-1在DNA结合域上的苏莫化有什么结构后果。对这些问题的回答应该提供关于苏莫化如何调节基因表达的体内和结构机制的见解。与公共健康相关：翻译后修饰调节多种类型的细胞蛋白的活性。蛋白质的苏莫化涉及蛋白质的结合，通常会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相扑缺陷突变如何影响内分泌组织的发育和功能。这项工作的结果可能会识别重要的新基因靶标，这些基因靶标对相对性的总和蛋白质池特别敏感。他们还将提供机械洞察力，了解苏莫化如何抑制内分泌系统的转录程序和“微调”。

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.