Novel approaches for the discovery of dephosphorylation control in oocyte meiosis

项目摘要 卵母细胞减数分裂M期的精确调控对于成功的胚胎发育和雌性发育至关重要。 生育突出这一点，约1%的女性由于减数分裂失败而生育力低下/不育。尽管如此，精确 在卵母细胞中，控制M期的分子机制仅被部分理解。本研究将集中在 磷酸酶PP 1（蛋白磷酸酶1）。PP 1是有丝分裂M期的重要调节因子， 约占所有脱磷作用的50%。然而，PP 1在哺乳动物卵母细胞中的具体作用是 不清楚PP 1是一种全酶，由一个催化亚基（PP 1c）和一至两个与PP 1相互作用的蛋白质组成 （PIP）。在哺乳动物细胞中，有三种PP 1cs（α，β和γ）和>180个PIP。从历史上看，PP 1研究 孤立地关注PP 1c，导致人们误以为PP 1c是混杂的。然而，PIP控制 PP 1c的定位、酶活性和底物。此外，异常PP 1介导的疾病状态 功能（例如，心脏病）是由PP 1c相互作用组的变化引起的，而不是PP 1c。此外，研究 使用PP 1/PP 2A双重抑制剂、过表达和抗PP 1c抗体注射， 产生了相互矛盾的结果。重要的是，我的初步数据使用一个特定的和新颖的PIP为基础的方法， 为了抑制PP 1c，已经发现PP 1活性对于减数分裂I的完成是必不可少的。为了进一步了解卵母细胞 减数分裂，这项拟议的研究将：（1）确定PP 1在卵母细胞减数分裂中的重要作用;（2） 开发新的工具，用于在整个M期对PP 1c进行特异性和时间控制，以解决争议 以及PP 1在卵母细胞中的作用的未知数。目的1探讨PP 1c在卵母细胞减数分裂中的作用 与一种新的PIP为基础的操作方法结合诱导蛋白质降解。在《目标2》中，一部小说 发展了一种可逆的小分子介导的细胞笼化方法--生长素可控笼化（ACC）。 ACC将被用来特异性地和时间性地调节PP 1c，进一步确定PP 1c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用于研究任何感兴趣的蛋白质。最后，Aim 3将 使用假设驱动和发现驱动的方法来确定PP 1全酶功能， 在减数分裂期间形成。具体来说，这一目标将建立两个PP 1全酶的功能 (PP1c：PNUTS和PP 1c：NIPP 1），并在整个卵母细胞中绘制PP 1c：PIP相互作用组 减数分裂总的来说，这个项目将揭示一个知之甚少，但与健康相关的磷酸酶，PP 1， 阐明了其在卵母细胞中的功能以及更广泛的M期调节。最终，这项工作将开发一个 为研究界提供了非常有价值的工具，并为M期的新型治疗方法的开发提供了信息。 包括不孕症在内的疾病。

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.