AKAP Integration of Phosphorylation and Ubiquitin Signaling

磷酸化和泛素信号传导的 AKAP 整合

• 批准号: 10462195
• 负责人: Kerrie B Collins
• 金额: $ 7.03万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-16 至 2023-04-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10462195
• 关键词: A kinase anchoring protein; Affinity; Binding; Biochemical; Ca(2+)-Transporting ATPase; Calcium; Cardiac; Cardiac Myocytes; Cardiovascular Diseases; Cells; Cessation of life; Chemicals; Collaborations; Complex; Coupling; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Data; Deubiquitination; Development; Docking; Endoplasmic Reticulum; Environment; Enzymes; Event; Excision; Heart; Heart Diseases; Heart failure; Homeostasis; Image; Imaging Techniques; In Situ; Investigation; Island; Learning; Link; Macromolecular Complexes; Measurement; Measures; Mediating; Molecular; Myocardial Infarction; Myocardial dysfunction; Peptide Hydrolases; Peptides; Pharmacology; Phase; Phosphorylation; Physiology; Pilot Projects; Prevention; Primary Health Care; Principal Investigator; Process; Protein Kinase; Proteins; Recurrence; Relaxation; Research; Research Personnel; Resolution; Sarcoplasmic Reticulum; Signal Transduction; Site-Directed Mutagenesis; Solid; Stimulus; Stroke; Structural Models; Systolic heart failure; Testing; Training; Ubiquitin; Ubiquitination; United States National Institutes of Health; Universities; Washington; Work; biophysical techniques; cardioprotection; combat; crosslink; experimental study; genetic approach; in vivo; innovation; microscopic imaging; novel therapeutics; protein expression; protein protein interaction; reuptake; screening

PROJECT SUMMARY Every heartbeat involves the coordination of local cAMP and Ca2+ signaling events that elicit the contraction of cardiomyocytes. This recurrent process necessitates the reuptake of cytoplasmic Ca2+ through the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA2). Pathophysiological changes in SERCA2 protein expression following myocardial infarction have been linked to ubiquitin-mediated proteasomal degradation of the channel. Thus, molecular mechanisms that remove ubiquitin from SERCA2 have the potential to be cardioprotective. The Scott lab have discovered a macromolecular complex containing enzymes of the protein phosphorylation and protein ubiquitination machinery. This new signaling unit may modulate SERCA2 levels at the sarcoplasmic reticulum to facilitate healthy cardiomyocyte function. A more detailed investigation of these molecular and cellular findings is mandated by evidence that cardiovascular diseases account for 1 in 3 deaths in the US. My preliminary findings show that the A-Kinase Anchoring protein AKAP18 sequesters the cAMP-dependent protein kinase (PKA) and the ubiquitin-specific proteinase USP4 with SERCA2. Pilot studies in transfected cells suggest that anchored PKA phosphorylates USP4 to stimulate its deubiquitinase activity. This led to a working hypothesis that both enzymes, when associated with AKAP18, act cooperatively to protect against ubiquitin- mediated removal of SERCA2 from the sarcoplasmic reticulum. This exciting new cardioprotective paradigm will be tested by an experimental approach of two aims. Aim 1 will use state of the art biochemical and biophysical approaches to investigate the molecular organization of the USP4-AKAP18-PKA complex. Mechanistic studies in aim 2 will combine advanced imaging and in vivo genetic strategies to understand how these anchored enzymes coordinately control vital aspects of cardiac physiology, such as calcium handling. My 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. Moreover, the training potential of these studies is high. I will learn cutting edge expansion microscopy imaging techniques and will be introduced to measurement of calcium transients in live cardiomyocytes.

项目摘要 每一次心跳都涉及局部cAMP和Ca 2+信号事件的协调，这些事件引起心脏收缩。 心肌细胞这种反复发生的过程需要细胞质Ca 2+通过 肌质/内质网Ca ~（2+）ATP酶（SERCA 2）。SERCA 2蛋白的病理生理变化 心肌梗死后的表达与泛素介导的蛋白酶体降解有关， 频道。因此，从SERCA 2中去除泛素的分子机制有可能被 心脏保护。斯科特实验室发现了一种含有蛋白酶的大分子复合物 磷酸化和蛋白质泛素化机制。这种新的信号单元可以在一定程度上调节SERCA 2水平。 促进健康的心肌细胞功能。更详细的调查这些 有证据表明，心血管疾病占死亡人数的三分之一， 在美国. 我的初步研究结果表明，A-激酶转运蛋白AKAP 18隔离了cAMP依赖的 蛋白激酶（PKA）和泛素特异性蛋白酶USP 4与SERCA 2。转染细胞中的初步研究 提示锚定的PKA磷酸化USP 4以刺激其去泛素化酶活性。这导致了一个工作 假设这两种酶，当与AKAP 18结合时，协同作用以保护免受泛素- 介导的SERCA 2从肌浆网的去除。这种令人兴奋的新的心脏保护模式将 通过两个目标的实验方法进行测试。目标1将使用最先进的生物化学和生物物理 研究USP 4-AKAP 18-PKA复合物的分子结构的方法。机制研究 目标2将结合联合收割机先进的成像和体内遗传策略，以了解这些锚定 酶协调控制心脏生理学的重要方面，例如钙处理。 我的研究将在华盛顿大学的药理学系进行。这 环境为学术轨道博士后研究人员提供了良好的培训。培训益处包括 部门内部和部门之间的密切合作，平易近人的主要研究人员进行创新 工作，并经常从多个部门的研讨会，包括在各自领域的世界专家。 此外，这些研究的培训潜力很高。我将学习尖端的扩展显微成像 技术，并将被引入到测量钙瞬变活心肌细胞。