Regulation of Steroidogenic Genes by Trophic Hormones

描述（由申请人提供）：类固醇激素是多种生理过程的关键调节剂，包括钠稳态、生殖和第二性征的发展。这些分子通过充当核和质膜受体的配体，使组织以协调的方式对内外环境的变化作出反应。由于类固醇激素控制着几乎所有细胞类型中大量基因的表达，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源性激素促肾上腺皮质激素（ACTH）通过肾上腺皮质调节皮质醇产生的机制。我们的研究已经确定了脂质配体在调节核受体甾体生成因子-1 （SF-1）的转激活电位中的整体作用。此外，我们还发现PKA和PKC等信号介质可直接介导包括SF-1在内的几种核蛋白的暂时不同和可逆的翻译后修饰（PTMs）。这些PTMs通过控制修饰蛋白参与各种核过程（包括转录和剪接）的能力，作为蛋白质功能的主要调节剂。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，从而促进不同蛋白质-蛋白质、蛋白质- dna和蛋白质- rna复合物的组装。本研究项目将验证不同的信号级联促进多种调节CYP17转录和剪接的蛋白质的PTM的假设。此外，SF-1和NONO的信号依赖性PTMs调节蛋白质复合物的差异组装，促进多个核过程的耦合，包括抑制、转录起始、配体结合、转录延伸和终止以及RNA加工。此外，SF-1的信号依赖性PTMs和p54nrb等协同调节蛋白调节蛋白质复合物的差异组装，促进多种核过程的耦合，包括抑制、转录起始、配体合成、转录延伸和终止以及RNA加工。Specific Aim 1将通过质谱蛋白质组学方法分析p54nrb的蛋白复合物和PTMs，确定p54nrb桥接转录和剪接的机制。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。特异性目标2将定义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的配体结合口袋入口通道的柔性环路中的作用。我们提出ACTH/cAMP信号通过触发调节受体配体结合袋占用的PTMs来调节SF-1的交易激活电位。配体和磷酸化特异性抗体的质谱分析将定义SF-1与配体之间相互作用的信号依赖稳定化以及SF-1与协调节蛋白之间的关系。

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.