Molecular mechanisms of PKA mutations underlying Cushing's syndrome

项目摘要 长期高水平的应激激素皮质醇对许多器官系统有害。促肾上腺皮质激素 独立性库欣综合征是一种内分泌紊乱，其中肾上腺组成性地产生 过量的皮质醇症状包括肥胖、皮肤变薄、认知和情绪问题以及骨质疏松。 损失最近的研究已经确定了蛋白激酶A催化亚基（PKAc）的突变， 大约50%的病例。这种突变，L205 R，是在PKAc的表面，结合到它的调控蛋白。 亚基，并预测破坏全酶的形成。 传统上，环磷酸腺苷（cAMP）-蛋白激酶A（PKA）信号传导的模型具有以下优点： 依赖于使用超生理水平的cAMP刺激的证据，其导致PKAc的完全解离 来自其调节亚基和来自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，从而重新定义PKA模型 激活细胞。基于这一新的信息，拟议的实验将测试库欣综合征是否 PKAc的突变破坏活性PKA的定位并引起底物的异位磷酸化， 下游皮质醇分泌。该假设将在两个目标中进行检验： 目的1：PKAc L205 R的时空分布在库欣综合征中是否改变？综合战略 CRISPR/Cas9基因编辑和活细胞成像与光活化荧光团和基于FRET 生物传感器将确定：1）突变的PKAc是否被募集到AKAP信号岛，2）这些库欣蛋白是否被激活， 突变体在细胞内更移动的，和3）如果PKAc活性异常地在亚细胞区域积累。 目的2：PKAc突变如何导致皮质醇过量产生？我会用化学生物学技术 结合NCI-H295 R肾上腺细胞中的皮质醇测量1）以确定PKAc是否错误定位 活动是必要的，足以促进皮质醇的释放。接下来，通过结合miniTurbo邻近标记， 与磷酸蛋白质组学，我将建立2）如果致病L205 R PKAc突变显示改变， 底物选择性对下游信号传导产生不利影响。此外，肾上腺皮质激素的小鼠模型 将产生PKAc L205 R表达，以评价候选治疗干预策略， 从这个目标。 这项研究计划的长期目标是阐明潜在的信号转导的分子机制， 这将有助于研究疾病中的皮质醇增多症，并促进新治疗工具的开发。研究将在 在华盛顿大学的药理学系。这种环境提供了很好的 培养博士后研究人员。培训的好处包括内部的密切协作， 部门之间，平易近人的首席研究员进行创新工作，并经常举行研讨会 来自多个部门的专家在各自的领域。

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.