Novel approaches for the discovery of dephosphorylation control in oocyte meiosis

项目总结 卵母细胞减数分裂过程中M期的精确调控是胚胎发育和雌性发育成功的关键 生育能力。突显这一点的是，由于减数分裂失败，约1%的女性处于亚生育/不育状态。尽管如此，准确的 在卵母细胞中，控制M期的分子机制只有一部分被了解。这项研究将集中在 磷酸酶PP1(蛋白磷酸酶1)。PP1是有丝分裂M期的重要调节因子 负责约50%的脱磷作用。然而，PP1在哺乳动物卵母细胞中的具体作用是 不清楚。PP1是由一个催化亚基(PP1c)和一到两个与PP1相互作用的蛋白质组成的全酶 (点数)。在哺乳动物细胞中，有三个PP1cs(α，β和γ)和180PIPs。从历史上看，PP1研究已经 孤立地关注PP1c，导致人们错误地认为PP1c是混杂的。然而，PIP控制着 PP1c的定位、酶活性和底物。此外，异常的PP1介导的疾病状态 功能(例如，心脏病)是由PP1c相互作用组的变化引起的，而不是PP1c。此外，研究 在卵母细胞中，使用双重PP1/PP2A抑制剂，过表达和抗PP1c抗体注射，有 产生了相互矛盾的结果。重要的是，我的初步数据使用了一种特定和新颖的基于PIP的方法 抑制PP1c已发现PP1活性是完成减数分裂I所必需的。进一步了解卵母细胞 开发新的工具，在整个M阶段对PP1c进行特定和时间的控制，以解决争议 以及PP1在卵母细胞中的作用尚不清楚。目标1将确定PP1c在卵母细胞减数分裂中的功能 一种新的基于PIP的操作方法与可诱导的蛋白质降解相结合。在《目标2》中，一部小说 可逆性小分子介导的笼化方法将被开发--生长素可控笼化(ACC)。 ACC将被用来特异性和时间性地调节PP1c，进一步确定PP1c在卵母细胞中的作用 减数分裂。值得注意的是，这种新的最先进的工具可以用来研究任何感兴趣的蛋白质。最后，《目标3》将 使用假设驱动和发现驱动的方法来确定PP1全酶功能和 在减数分裂过程中形成。具体地说，这个目标将确定两个PP1全酶的功能 (PP1c：PNUTS和PP1c：NIPP1)，并定位整个卵母细胞的PP1c：PIP相互作用体 减数分裂。 阐明其在卵母细胞中的功能以及更广泛的M期调控。最终，这项工作将开发出一种 为研究界提供非常有价值的工具，并为M期新疗法的开发提供信息 包括不孕不育在内的基础疾病。

PROJECT SUMMARY Precise regulation of M-Phase in oocyte meiosis is essential for successful embryo development and female fertility. Highlighting this, ~1% of women are subfertile/infertile due to meiotic failure. Despite this, the precise molecular mechanisms governing M-Phase is only partly understood in oocytes. This research will focus on the phosphatase PP1 (Protein Phosphatase 1). PP1 is an important regulator of mitotic M-Phase and is responsible for ~50% of all dephosphorylations. However, the specific roles of PP1 in mammalian oocytes is unclear. PP1 is a holoenzyme consisting of a catalytic subunit (PP1c) and one to two PP1-interacting proteins (PIPs). In mammalian cells, there are three PP1cs (α, β, and γ) and >180 PIPs. Historically, PP1 research has focused on PP1c in isolation, leading to the misconception that PP1c is promiscuous. However, PIPs control localization, enzyme activity, and substrates of PP1c. Additionally, disease states mediated by aberrant PP1 function (e.g., heart disease) result from changes in the PP1c interactome, and not PP1c. Furthermore, studies of PP1c in oocytes, using dual PP1/PP2A inhibitors, overexpression, and anti-PP1c antibody injections, have yielded conflicting outcomes. Importantly, my preliminary data using a specific and novel PIP-based approach to inhibit PP1c has found PP1 activity is essential for meiosis I completion. To gain further insights into oocyte meiosis, this proposed research will: (1) determine the essential roles of PP1 in oocyte meiosis and; (2) develop novel tools for the specific and temporal control of PP1c throughout M-Phase to resolve controversies and unknowns about the roles of PP1 in oocytes. Aim 1 will determine the function of PP1c in oocyte meiosis with a new PIP-based manipulation approach combined with inducible protein degradation. In Aim 2, a novel method for reversible small molecule-mediated caging will be developed -- auxin-controllable caging (ACC). ACC will be used to specifically and temporally regulate PP1c, further defining the roles of PP1c in oocyte meiosis. Of note, this new state-of-the-art tool can be used to study any protein-of-interest. Finally, Aim 3 will use both hypothesis-driven and discovery-driven approaches to determine PP1 holoenzyme function and formation during meiosis. Specifically, this aim will establish the function of two PP1 holoenzymes (PP1c:PNUTS and PP1c:NIPP1) during oocyte meiosis, and map the PP1c:PIP interactome throughout oocyte meiosis. Overall, this project will shed light on a poorly understood, but health-relevant phosphatase, PP1, elucidating its functions in oocytes and more broadly M-Phase regulation. Ultimately, this work will develop a highly valuable tool for the research community, and inform the development of novel treatments for M-Phase based diseases including infertility.