Molecular mechanisms of PKA mutations underlying Cushing's syndrome

库欣综合征 PKA 突变的分子机制

• 批准号: 9904116
• 负责人: Mitchell Hamed Omar
• 金额: $ 6.53万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9904116
• 关键词: A kinase anchoring protein; Adrenal Gland Adenoma; Adrenal Gland Hyperfunction; Adrenal Glands; Architecture; Binding; Biochemistry; Biology; Biosensor; Blood; CRISPR/Cas technology; Catalytic Domain; Cells; Chemicals; Chronic; Cognitive; Collaborations; Corticotropin; Cushing Syndrome; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Development; Diabetes Mellitus; Diagnosis; Disease; Dissociation; Emotional; Endocrine Diagnostic Techniques; Endocrine System Diseases; Ensure; Environment; Enzymes; Face; Fluorescence Resonance Energy Transfer; Gene Proteins; Genes; Goals; Holoenzymes; Hormones; Hydrocortisone; Hypertension; Intervention; Island; Label; Lead; Link; Location; Macromolecular Complexes; Measurement; Microscopy; Modeling; Molecular; Mutation; Obesity; Pathologic; Pathway interactions; Patients; Pharmacology; Phosphorylation; Phosphotransferases; Physiological; Principal Investigator; Production; Proteins; Research; Research Personnel; Signal Transduction; Signaling Protein; Skin; Stress; Symptoms; Techniques; Testing; Thinness; Tissue Model; Training; Universities; Validation; Variant; Washington; Weight Gain; Work; base; body system; bone loss; experimental study; fluorophore; in vivo; innovation; live cell imaging; macromolecular assembly; mouse model; mutant; novel therapeutics; phosphoproteomics; prevent; protein complex; protein function; protein profiling; protein protein interaction; recruit; response; spatiotemporal; therapeutic candidate; tool

Project Summary Chronically high levels of the stress hormone cortisol are deleterious to many organs systems. ACTH- independent Cushing's syndrome is an endocrine disorder wherein the adrenal glands constitutively produce excess cortisol. Symptoms include obesity, thinning of the skin, cognitive and emotional problems, and bone loss. Recent studies have identified a mutation in the protein kinase A catalytic subunit (PKAc) in approximately 50% of these cases. This mutation, L205R, is on the face of PKAc that binds to its regulatory subunit, and is predicted to disrupt holoenzyme formation. Traditionally, the model for cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling has relied on evidence using supraphysiological levels of cAMP stimulation, which leads to full dissociation of PKAc from its regulatory subunit and from A-kinase anchoring proteins (AKAPs). Recent evidence shows that physiological levels of cAMP do not dissociate PKA holoenzymes, thereby redefining the model of PKA activation in cells. Based on this new information, the proposed experiments will test if Cushing's syndrome mutations in PKAc disrupt localization of active PKA and cause ectopic phosphorylation of substrates and downstream cortisol secretion. The hypothesis will be tested in two aims: Aim 1: Is the spatiotemporal profile of PKAc L205R altered in Cushing's syndrome? A combined strategy of CRISPR/Cas9 gene-editing and live-cell imaging with photoactivatable fluorophores and FRET-based biosensors will ascertain: 1) if mutant PKAc is recruited to AKAP-signaling islands, 2) whether these Cushing's mutants are more mobile inside cells, and 3) if PKAc activity aberrantly accumulates at subcellular regions. Aim 2: How does mutant PKAc cause excess cortisol production? I will use chemical biology techniques combined with cortisol measurement in NCI-H295R adrenal cells 1) to determine if mislocalization of PKAc activity is necessary and sufficient to boost cortisol release. Next, by combining miniTurbo proximity labeling with phospho-proteomics, I will establish 2) if the disease-causing L205R PKAc mutant displays altered substrate selectivity that adversely impacts downstream signaling. Additionally, a mouse model of adrenal PKAc L205R expression will be generated to evaluate candidate therapeutic intervention strategies resulting from this aim. The long-term goals of this research plan are to elucidate molecular mechanisms of the signaling underlying hypercortisolism in disease and to spur development of new therapeutic tools. The research will be conducted at the University of Washington in the Department of Pharmacology. This environment provides excellent training for academic-track postdoctoral researchers. Training benefits include strong collaboration within and among departments, approachable principal investigators performing innovative work, and frequent seminars from multiple departments featuring world experts in their respective fields.

项目摘要 长期过高的应激激素皮质醇水平对许多器官系统有害。ACTH- 独立库欣综合征是一种内分泌紊乱，肾上腺结构性地产生 皮质醇过多。症状包括肥胖、皮肤变薄、认知和情感问题以及骨骼。 损失。最近的研究发现了蛋白激酶A催化亚单位(PKAc)的突变。 其中约50%的病例。这种突变L205R位于PKAc的表面，与其调节作用相结合 亚基，预计会扰乱全酶的形成。 传统上，环磷酸腺苷(CAMP)-蛋白激酶A(PKA)信号转导模型 依赖于使用超生理学水平的cAMP刺激的证据，这导致PKAc完全解离 从其调节亚基和从A-激酶锚定蛋白(AKAP)。最近的证据表明， 生理水平的cAMP不会解离PKA全酶，因此重新定义了PKA的模型 细胞内的激活。基于这一新信息，拟议中的实验将测试库欣综合症 PKAc突变破坏了活性PKA的定位，并导致底物和 皮质醇的下游分泌。这一假设将在两个目标上得到检验： 目的1：库欣综合征患者PKAc L205R的时空分布是否发生改变？一种综合战略 CRISPR/Cas9基因编辑和活细胞成像的光可激活荧光团和基于FRET的 生物传感器将确定：1)突变的PKAc是否被招募到AKAP信令岛，2)这些库欣的 突变体在细胞内的流动性更强，以及3)如果PKAc活性在亚细胞区域异常积累。 目标2：突变的PKAc是如何导致皮质醇过度产生的？我将使用化学生物学技术 结合NCI-H295R肾上腺细胞皮质醇测定1)确定PKAc的错误定位 运动是促进皮质醇释放所必需的，也是充分的。接下来，通过结合mini Turbo邻近标记 通过磷酸蛋白质组学，我将确定2)如果导致疾病的L205R PKAc突变显示改变 对下游信号产生不利影响的底物选择性。另外，一种小鼠肾上腺模型 将生成PKAc L205R表达以评估候选治疗干预策略 从这个目标出发。 这项研究计划的长期目标是阐明潜在信号转导的分子机制。 疾病中的皮质醇过多，并刺激新的治疗工具的开发。将进行这项研究 在华盛顿大学药理学系。这种环境提供了极好的 培训学术跟踪的博士后研究人员。培训的好处包括内部和内部的强大协作 在各部门之间，负责创新工作的首席调查员平易近人，并经常举办研讨会 来自多个部门，拥有各自领域的世界专家。