Significance of the novel Bok-IP3 receptor interaction

肌醇 1,4,5-三磷酸受体 (IP3R) 在脊椎动物细胞内信号传导中发挥着关键作用。 它们是三个非常相似的蛋白质家族，在内质网中形成四聚体 Ca2 通道 （ER）膜。在过去的二十年里，我一直在研究 IP3R 的分子生物学，最近 发现 Bok 是一种人们知之甚少的 Bcl-2 蛋白家族成员，具有独特的功能，它与 强烈反对 IP3R。 Bcl-2 蛋白家族控制着内在的细胞凋亡途径，并且在很大程度上发挥着重要作用。 从基础细胞生物学的角度以及作为抗癌药物的目标进行了研究。博克是最 与已充分表征的促凋亡蛋白 Bax 和 Bak 相似，但其细胞作用仍然神秘且 极具争议性；然而，共识是 Bok 定位于 ER 膜并在 凋亡信号传导。在过去的三年里，我的实验室已经开始表征新颖的 Bok-IP3R 相互作用 及其意义，但仍有许多工作要做。现在，我计划通过三个具体目标来测试 假设 Bok 与 IP3R 结合调节 IP3R 功能，并且是 Bok 发挥作用所必需的 细胞凋亡，很大程度上依赖于新开发的细胞系，其中 Bok 已使用 CRISPR/Cas9 删除 - 为基础的方法。 目标 1. 解析 Bok 在 IP3R1 上的结合位点并开发结合缺陷型 IP3R1 抑制 Bok-IP3R1 相互作用的构建体和肽。获得的结果将提供关于如何 Bok 与 IP3R 相互作用，并将生成可用于探索 Bok-IP3R 相互作用的重要性的工具。 目标 2. 定义 Bok 与 IP3R 的结合是否调节 IP3R 功能。获得的结果应表征 IP3R 调节的新模式，并深入了解 Bok 影响细胞凋亡的机制。 目标 3. 定义 Bok 在细胞凋亡信号传导中的作用以及 Bok-IP3R 相互作用对此作用的重要性。 获得的结果将与其他特定目标的数据相结合，生成一个模型，说明 Bok 如何 影响细胞凋亡，并将确定 Bok-IP3R 界面是否是药物靶点。 总而言之，该提案代表了在描述该计划的性质和意义方面所做的首次努力。 Bok-IP3R 相互作用无疑将阐明 IP3R 功能和 Bok 的细胞作用。工作 将增进我们对细胞信号传导和细胞凋亡的理解，并可能确定新的治疗靶点。

Inositol 1,4,5-trisphosphate receptors (IP3Rs) play a pivotal role in vertebrate intracellular signaling. They are a family of three very similar proteins that form tetrameric Ca2+ channels in endoplasmic reticulum (ER) membranes. For the last two decades I have been studying the molecular biology of IP3Rs and recently discovered that Bok, a very poorly understood Bcl-2 protein family member with unique features, binds very strongly to IP3Rs. The Bcl-2 protein family controls the intrinsic apoptosis pathway and is intensively investigated, both from a basic cell biology perspective, and as a target for anti-cancer drugs. Bok is most similar to the well-characterized pro-apoptotic proteins Bax and Bak, but its cellular role remains enigmatic and highly controversial; consensus is, however, that Bok is localized to the ER membrane and plays a role in apoptotic signaling. In the last 3 years, my laboratory has begun to characterize the novel Bok-IP3R interaction and its significance, but there is still much work to do. Now, through three Specific Aims, I plan to test the hypotheses that Bok binding to IP3Rs regulates IP3R function and is required for the role that Bok plays in apoptosis, leaning heavily on newly-developed cell lines in which Bok has been deleted using CRISPR/Cas9- based methods. Aim 1. Resolution of the binding site for Bok on IP3R1 and the development of binding-deficient IP3R1 constructs and peptides that inhibit the Bok-IP3R1 interaction. Results obtained will provide insight into how Bok interacts with IP3Rs and will generate tools useful for probing the significance of the Bok-IP3R interaction. Aim 2. Define whether Bok binding to IP3Rs regulates IP3R function. Results obtained should characterize a new mode of IP3R regulation and provide insight into the mechanism by which Bok influences apoptosis. Aim 3. Define the role of Bok in apoptotic signaling and the importance of the Bok-IP3R interaction to this role. Results obtained will be integrated with the data from the other Specific Aims to generate a model for how Bok influences apoptosis and will establish whether the Bok-IP3R interface is a drug target. In summary, this proposal represents the first effort at characterizing the nature and significance of the Bok-IP3R interaction and will undoubtedly shed light on IP3R function and the cellular role of Bok. The work will both advance our understanding of cell signaling and apoptosis, and may identify a new therapeutic target.