Unraveling the molecular events driven by CaMKII in Ca2+-coupled cells

项目摘要 细胞与细胞之间的通讯对于所有多细胞生物体的功能至关重要。细胞间的一个关键因素 通讯受Ca ~（2+）浓度的调节。Ca 2 +/钙调蛋白依赖性蛋白激酶II（CaMKII）是一种钙离子依赖性蛋白激酶。 在哺乳动物中由α、β、γ和δ四种基因编码的Ca 2+敏感酶。有大量的 这四种脊椎动物CaMKII基因的多样性。选择性剪接产生多达386个转录物， 这导致了386种蛋白质的产生，然后这些蛋白质被差异性地后修饰， 形成异源寡聚复合物，最终形成数千种化学上不同的CaMKII 蛋白质型我们对CaMKII在长期记忆形成中的关键作用特别感兴趣 （神经元：α，β），受精（卵母细胞：γ）和心脏生理学（心肌细胞：δ）。有趣的是，这些细胞 它们使用Ca 2+振荡进行通信，但时间尺度（分钟到毫秒）截然不同。一个人如何 酶是否具有这种多功能性？我们假设，选择性剪接和修饰创造了一个 在特定细胞类型中表达的独特的CaMKII变体组，从而导致差异功能输出。 要充分阐明这些复杂的生物学作用，需要更深入地了解CaMKII的变异， 序列和蛋白质水平，这些变化的结构和构象分支，以及这些变化如何影响我们的生活。 变量影响细胞内的CaMKII相互作用。在这一建议中，我们寻求扩大我们对以下问题的理解： 使用测序、生物化学、结构生物学和分子生物学的组合方法， 细胞分析。完成拟议的工作将使我们能够揭示许多作用的分子基础 神经元、心肌细胞和卵母细胞中CaMKII的表达-对治疗干预具有深远意义 治疗神经系统疾病心脏功能障碍和不孕不育

PROJECT SUMMARY Cell to cell communication is critical for function in all multicellular organisms. A key factor for intercellular communication is regulation by Ca2+ concentration. Ca2+/calmodulin dependent protein kinase II (CaMKII) is a Ca2+ sensitive enzyme that is encoded by four genes in mammals: α, β, γ, and δ. There is an incredible amount of diversity generated from the four vertebrate CaMKII genes. Alternative splicing produces up to 386 transcripts, which leads to the production of 386 proteins that are then differentially post-translationally modified, and mix to form hetero-oligomeric complexes, ultimately culminating in thousands of chemically distinct CaMKII proteoforms. We are specifically interested in the crucial roles CaMKII plays in long-term memory formation (neurons: α, β), fertilization (oocytes: γ), and cardiac physiology (cardiomyocytes: δ). Intriguingly, these cells all communicate using Ca2+ oscillations but on vastly different timescales (minutes to milliseconds). How does one enzyme accommodate this multifunctionality? We hypothesize that selective splicing and modification creates a unique set of CaMKII variants expressed in specific cell types, thereby leading to differential functional outputs. Fully elucidating these complex biological roles requires a deeper understanding of CaMKII variation at the sequence and protein level, structural and conformational ramifications of these variations, and how these variables affect CaMKII interactions within the cell. In this proposal, we seek to expand our understanding of CaMKII function inside cells using a combinatorial approach of sequencing, biochemistry, structural biology, and cellular assays. Completion of the proposed work will allow us to uncover the molecular basis for the many roles of CaMKII in neurons, cardiomyocytes, and oocytes – with far-reaching implications on therapeutic intervention for neurologic disease, cardiac dysfunction, and infertility.