AKAP Integration of Phosphorylation and Ubiquitin Signaling

磷酸化和泛素信号传导的 AKAP 整合

基本信息

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
项目类别：
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

项目摘要

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.

项目概要每次心跳都涉及局部 cAMP 和 Ca2+ 信号传导事件的协调，从而引发心脏收缩心肌细胞。这个反复发生的过程需要通过以下途径重新摄取细胞质 Ca2+：肌浆/内质网 Ca2+ ATP 酶 (SERCA2)。 SERCA2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 SERCA2 中去除泛素的分子机制有可能是心脏保护作用。斯科特实验室发现了一种含有蛋白质酶的大分子复合物磷酸化和蛋白质泛素化机制。这个新的信号单元可以调节 SERCA2 水平肌浆网促进健康的心肌细胞功能。对这些内容进行更详细的调查分子和细胞研究结果有证据表明心血管疾病导致三分之一的死亡在美国。我的初步发现表明 A 激酶锚定蛋白 AKAP18 隔离了 cAMP 依赖性蛋白激酶 (PKA) 和泛素特异性蛋白酶 USP4 以及 SERCA2。转染细胞的初步研究表明锚定的 PKA 磷酸化 USP4 以刺激其去泛素酶活性。这导致了工作假设这两种酶与 AKAP18 结合时，协同作用以防止泛素化介导 SERCA2 从肌浆网的去除。这种令人兴奋的新心脏保护范例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 USP4-AKAP18-PKA 复合物分子组织的方法。机理研究目标 2 将结合先进的成像和体内遗传策略来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药理学系进行。这环境为学术型博士后研究人员提供了良好的培训。培训好处包括部门内部和部门之间的强有力的合作，平易近人的主要研究人员进行创新工作，以及来自多个部门的频繁研讨会，其中包括各自领域的世界专家。此外，这些研究的培训潜力很高。我将学习最先进的膨胀显微镜成像技术并将被引入活心肌细胞中钙瞬变的测量。