Regulation of Steroid Hormone Production by Inter-Organelle Substrate Exchange

细胞器间底物交换对类固醇激素产生的调节

• 批准号: 8535148
• 负责人: Marion B. Sewer
• 金额: $ 30.94万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-20 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8535148
• 关键词: Address; Adrenal Cortex; Adrenal Glands; Anabolism; Androgens; Attenuated; Binding; Binding Proteins; Biochemical Reaction; Biological Process; Cells; Cholesterol; Complex; Corticotropin; Cortodoxone; Data; Endocrine System Diseases; Endoplasmic Reticulum; Ensure; Enzymes; Family; Genes; Genetic Transcription; Glucocorticoids; Guanosine Triphosphate Phosphohydrolases; Hormones; Human; Hydrocortisone; Inner mitochondrial membrane; Kinesin; Knowledge; Lipid Binding; Mediating; Metabolic; Metabolism; Microtubule Polymerization; Microtubules; Mitochondria; Monomeric GTP-Binding Proteins; Movement; Nuclear Receptors; Organelles; Output; Pathway interactions; Phospho-Specific Antibodies; Phosphorylation; Physiological; Pituitary-dependent Cushing' s disease; Play; Polycystic Ovary Syndrome; Post-Translational Protein Processing; Production; Proteins; Reaction; Regulation; Research; Role; Series; Signal Transduction; Site; Steroid biosynthesis; Structure; Testing; Tubulin; Work; adrenal hyperplasia; base; dehydroepiandrosterone; insight; member; mutant; overexpression; oxysterol binding protein; response; rho; small hairpin RNA; stable cell line; steroid hormone; trafficking; transcription factor; uptake

DESCRIPTION (provided by applicant): Steroid hormones such as cortisol are key regulators of a diverse array of biological processes that evoke physiological changes by binding to members of the nuclear receptor super family of transcription factors. Because glucocorticoids control the expression of numerous genes, multiple mechanisms are used to ensure tight control over hormone production. Our preliminary findings provide evidence for the role of adrenocorticotropin (ACTH)-stimulated, microtubule-dependent mitochondrial trafficking in maintaining optimal glucocorticoid output. We have also identified several proteins, including the GTPase RhoA and the Rho effector diaphanous 1 (DIAPH1), that act to facilitate this movement. ACTH signaling controls the ability of DIAPH1 to interact with RhoA and several other binding partners. However, the functional significance of these interactions has not fully been elucidated, nor have we defined if ACTH-stimulated mitochondrial movement enables cortisol production by facilitating the delivery of substrate between the ER and mitochondria. We hypothesize that ACTH-stimulated increases in the rate of mitochondrial trafficking are required for inter- organelle substrate exchange and glucocorticoid biosynthesis. To test this hypothesis we will define the mechanism by which ACTH signaling controls DIAPH1 complex assembly (Specific Aim 1). We have generated a stable cell line that expresses shRNA against DIAPH1 to define the role of the protein in mitochondrial movement and hormone production. We will also assess the functional significance of the interactions between DIAPH1 and several binding partners, including tubulin, kinesin, and the oxysterol- binding-protein related protein 2 (ORP2). Studies will also be performed to define the role of phosphorylation in controlling DIAPH1 function (Specific Aim 2). We have found that ACTH signaling stimulates the phosphorylaton of DIAPH1. The role of this post-translational modification on DIAPH1 function will be examined using phospho-mutants and a phospho-specific antibody. Finally, we will determine the mechanism by which 11-deoxycortisol is transported between the ER and mitochondria (Specific Aim 3). We have found that ORP2 co-purifies with DIAPH1 and hypothesize that this lipid binding protein may facilitate substrate transfer between the ER and mitochondria. We will characterize the role of ORP2 in cortisol biosynthesis and also determine if fusion between ER and mitochondria play a role in inter-organelle substrate delivery. The knowledge gained from these studies will provide valuable information about a largely unexplored facet of steroid hormone synthesis in the adrenal cortex. Our findings may also provide insight into the mechanism by which substrates are transferred between the ER and mitochondria in other metabolic processes.

描述（由申请人提供）：类固醇激素（例如皮质醇）是各种生物学过程的关键调节剂，它们通过与转录因子的核受体超级家族的成员结合来引起生理变化。由于糖皮质激素控制着许多基因的表达，因此使用多种机制来确保对激素产生的严格控制。我们的初步发现为脂质性皮质激素（ACTH）刺激的微管依赖性线粒体运输在维持最佳糖皮质激素输出中的作用提供了证据。我们还鉴定了几种蛋白质，包括GTPase RhoA和Rho效应子透射1（diaph1），这些蛋白质可促进这种运动。 ACTH信号传导控制Diaph1与RhoA和其他几个结合伴侣相互作用的能力。但是，这些相互作用的功能意义尚未完全阐明，我们也没有确定ACTH刺激的线粒体运动是否通过促进ER和线粒体之间的底物的递送来使皮质醇产生。我们假设ACTH刺激的线粒体运输速率增加是细胞器底物交换和糖皮质激素生物合成所必需的。为了检验该假设，我们将定义ACTH信号控制Diaph1复合物组件的机制（特定目标1）。我们已经产生了稳定的细胞系，该细胞系表达shRNA针对Diaph1，以定义蛋白质在线粒体运动和激素产生中的作用。我们还将评估diaph1与几个结合伴侣之间相互作用的功能意义，包括微管蛋白，驱动蛋白和氧蛋白酶结合蛋白相关蛋白2（ORP2）。还将进行研究以定义磷酸化在控制diaph1功能中的作用（特定目标2）。我们发现，ACTH信号传导刺激了Diaph1的磷酸laton。这种翻译后修饰在diaph1功能上的作用将使用磷酸突变剂和磷酸化特异性抗体进行检查。最后，我们将确定在ER和线粒体之间运输11-脱氧皮质醇的机制（特定目标3）。我们发现，ORP2与Diaph1共同纯化，并假设该脂质结合蛋白可以促进ER和线粒体之间的底物转移。我们将表征ORP2在皮质醇生物合成中的作用，并确定ER和线粒体之间的融合是否在轨道间底物递送中起作用。从这些研究中获得的知识将提供有关肾上腺皮质中类固醇激素合成的很大程度上未探索的方面的宝贵信息。我们的发现还可以洞悉其他代谢过程中ER和线粒体之间底物转移的机制。