Regulation of Steroidogenic Genes by Trophic Hormones

描述(由申请人提供)：类固醇激素是一系列不同生理过程的关键调节器，包括钠稳态、生殖和第二性征的发展。这些分子通过充当核受体和质膜受体的配体，使组织能够以协调的方式对内外环境的变化做出反应。由于类固醇激素控制着几乎所有细胞类型中许多基因的表达，类固醇生成细胞利用多种机制确保严格控制这些分子的合成。我们研究的一个主要目标是了解垂体衍生激素促肾上腺皮质激素(ACTH)调节肾上腺皮质皮质醇产生的机制。我们的研究已经确定了脂质配体在调节核受体类固醇生成因子-1(SF-1)的反式激活潜力中起着不可或缺的作用。此外，我们还发现，PKA和PKC等信号介体直接对包括SF-1在内的几种核蛋白进行时间上不同和可逆的翻译后修饰(PTM)。这些PTM通过控制修饰的蛋白质参与包括转录和剪接在内的各种核过程的能力来发挥蛋白质功能的主要调节作用。我们认为ACTH通过调节靶蛋白的PTM来控制类固醇激素的生物合成，从而促进不同的蛋白质-蛋白质、蛋白质-DNA和蛋白质-RNA复合体的组装。这项研究项目将检验这一假说，即不同的信号级联促进调节CYP17转录和剪接的多个蛋白质的PTM。此外，SF-1和NONO的信号依赖的PTMS调节蛋白质复合体的差异组装，促进多个核过程的耦合，包括抑制、转录起始、配体结合、转录本的延长和终止以及RNA处理。此外，SF-1和p54nrb等共调控蛋白的信号依赖的PTM调节蛋白质复合体的差异组装，促进多个核过程的耦合，包括抑制、转录起始、配体合成、转录本的延长和终止以及RNA处理。具体目标1将通过使用质谱蛋白质组学方法分析p54nrb的蛋白质复合体和PTM来确定p54nrb在转录和剪接之间架起桥梁的机制。这些研究还将确定ACTH/cAMP刺激的PTM如何调节p54nrb控制各种核过程的能力。具体目标2将确定PTM控制SF-1功能的机制。我们已经确定了信号依赖的PTM在SF-1配体结合口袋入口处的灵活环中的作用。我们认为，ACTH/cAMP信号通过触发PTM调节受体配体结合口袋的占用来调节SF-1的反式激活潜能。配体和磷酸特异性抗体的质谱分析将确定SF-1与配体之间相互作用的信号依赖稳定性与SF-1与共调节蛋白之间的关系。 与公共卫生相关：了解基因是如何在制造类固醇激素的细胞中调节的，将有助于深入了解皮质醇和肾上腺雄激素的病理生理浓度产生的机制。这项工作将提供对多种内分泌疾病的洞察，包括肾上腺增生、多囊卵巢综合征和库欣病。

DESCRIPTION (provided by applicant): Steroid hormones are key regulators of a diverse array of physiological processes, including sodium homeostasis, reproduction, and the development of secondary sex characteristics. These molecules allow tissues to respond in a coordinated manner to changes in the internal and external environments by functioning as ligands for both nuclear and plasma membrane receptors. Because steroid hormones control the expression of numerous genes in virtually all cell types, steroidogenic cells utilize multiple mechanisms that ensure tight control of the synthesis of these molecules. A major goal of our research is to understand the mechanisms by which the pituitary-derived hormone adrenocorticotropin (ACTH) regulates cortisol production by the adrenal cortex. Our research has identified an integral role lipid ligands in regulating the transactivation potential of the nuclear receptor steroidogenic factor-1 (SF-1). Further, we have found that signaling mediators such as PKA and PKC direct temporally distinct and reversible post-translational modifications (PTMs) of several nuclear proteins, including SF-1. These PTMs serve as master regulators of protein function by controlling the ability of modified proteins to participate in varied nuclear processes, including transcription and splicing. We propose that ACTH controls steroid hormone biosynthesis by modulating the PTM of target proteins, thus facilitating the assembly of distinct protein-protein, protein-DNA, and protein-RNA complexes. This research project will test the hypothesis that distinct signaling cascades promotes the PTM of multiple proteins that regulate the transcription and splicing of CYP17. Further, signal-dependent PTMs of SF-1 and NONO, modulate the differential assembly of protein complexes that facilitate the coupling of multiple nuclear processes, including repression, transcriptional initiation, ligand binding, transcript elongation and termination, and RNA processing. Further, signal-dependent PTMs of SF-1 and coregulatory proteins such as p54nrb, modulate the differential assembly of protein complexes that facilitate the coupling of multiple nuclear processes, including repression, transcriptional initiation, ligand synthesis, transcript elongation and termination, and RNA processing. Specific Aim 1 will determine the mechanism by which p54nrb bridges transcription and splicing by employing mass spectrometric proteomic approaches to analyze of protein complexes and PTMs of p54nrb. These studies will also define how ACTH/cAMP- stimulated PTM regulates the ability of the p54nrb to control varied nuclear processes. Specific Aim 2 will define the mechanism by which PTM controls SF-1 function. We have identified a role for signal- dependent PTM in a flexible loop at the entryway to the ligand-binding pocket of SF-1. We propose that ACTH/cAMP signaling regulates SF-1 transactivation potential by triggering PTMs that regulate occupancy of the receptor's ligand binding pocket. Mass spectrometric analysis of ligands and phospho-specific antibodies will define the relationship between signal-dependent stabilization of the interactions between SF-1 and ligand and SF-1 and coregulatory proteins. PUBLIC HEALTH RELEVANCE: Understanding how genes are regulated in cells that make steroid hormones will provide insight into the mechanisms by which pathophysiological concentrations of cortisol and adrenal androgens are produced. This work will provide insight into multiple endocrine disorders, including adrenal hyperplasia, polycystic ovary syndrome, and Cushing's disease.