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

项目总结 细胞间的交流对于所有多细胞生物体的功能都是至关重要的。细胞间的一个关键因素 交流是由钙浓度调节的。钙/钙调蛋白依赖的蛋白激酶II(CaMKII)是一种 哺乳动物对钙敏感的酶，由四个基因编码：α，β，γ和δ。有一个难以置信的数量 四种脊椎动物CaMKII基因产生的多样性。选择性剪接产生多达386个转录本， 这导致了386种蛋白质的产生，这些蛋白质随后被差异翻译后修饰，并混合到 形成杂低聚络合物，最终形成数千种化学上不同的CaMKII 蛋白质形式。我们特别感兴趣的是CaMKII在长期记忆形成中扮演的关键角色 (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 使用钙离子振荡进行交流，但时间尺度(几分钟到几毫秒)截然不同。一个人怎么能 酵素能适应这种多功能吗？我们假设选择性的剪接和修饰创建了一个 在特定细胞类型中表达的一组独特的CaMKII变体，从而导致不同的功能输出。 要充分阐明这些复杂的生物学作用，需要更深入地了解CaMKII在 序列和蛋白质水平，这些变异的结构和构象分支，以及这些 变量影响细胞内的CaMKII相互作用。在这项建议中，我们寻求扩大我们对 CaMKII在细胞内的功能使用测序、生物化学、结构生物学和 细胞分析。拟议工作的完成将使我们能够揭示许多角色的分子基础 CaMKII在神经元、心肌细胞和卵母细胞中的表达--对治疗干预的深远影响 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.