Integrating 'omics', evolution, and biochemistry to understand fertilization

项目摘要 自20世纪80年代以来，不孕症的发病率增加了4%，其中高达20%的病例没有已知的原因。 其中一个流行的假设是同源卵子和精子蛋白质之间的不相容性;然而， 在任何生物体中，已经鉴定出几对相互作用的生殖蛋白。最好的模型之一， 研究受精核心机制的是海洋腹足动物鲍鱼，那里的精子高度富集， 只有几种蛋白质。第一对相互作用的生殖蛋白是鲍鱼精子溶素和 其卵被受体VERL。作为鲍鱼卵壳的主要成分，VERL是一种巨大的纤维状结构， 由~22个ZP-N重复组成的糖蛋白，可能形成分子间氢键，以产生高度的 稳定和保护性的卵膜。溶素通过竞争这些氢在蛋壳上形成一个洞 结合，允许精子通过并与卵母细胞融合。这些ZP-N重复序列也是 哺乳动物的卵被，以及多条证据支持人类蛋白质ZP 2作为功能类似物 的VERL。然而，三个主要的悬而未决的问题是（1）除了溶素和VERL，精子和卵子 蛋白质相互作用，（2）这些蛋白质中的哪些位点对于介导这种相互作用是重要的，以及（3）如何 这些位点的突变可能会影响蛋白质结构和生育能力。为了解决这些基本问题， 繁殖鲍鱼（K99）和人类（R 00），提出了两个具体的目标，利用国家的- 本发明涉及基因组、蛋白质组和结构方法。在目标1中，对于K99阶段，我将结合联合收割机长读 测序技术，分子进化测试和定量蛋白质组学，以确定潜在的对 鲍鱼中相互作用的生殖蛋白;对于R 00阶段，我将扩展这些定量蛋白质组学 方法，以确定潜在的新途径，有助于男性不育症。在目标2中，对于K99阶段， VERL重复序列1溶液结构及其与溶素的相互作用将通过NMR测定， 突变扫描用于鉴定干扰溶素-VERL相互作用的突变。在R 00阶段，这些 技术将应用于人类ZP 2，我将识别干扰精子结合的突变， 描述其结构效应。拟议的研究是创新的，因为它结合使用基因组， 蛋白质组学和结构技术来表征相互作用的分子机制， 快速进化的生殖蛋白预计结果将揭示核心过程， 介导卵子与精子的相互作用，并为了解更多 复杂的哺乳动物系统

Project Summary The incidence of infertility has increased 4% since the 1980s, with up to 20% cases having no known cause. One of the prevailing hypotheses is incompatibility between cognate egg and sperm proteins; however, very few pairs of interacting reproductive proteins have been identified in any organism. One of the best models for studying core mechanisms of fertilization is the marine gastropod abalone, where sperm are highly enriched for only a few proteins. The first identified pair of interacting reproductive proteins were abalone sperm lysin and its egg coat receptor VERL. As a major component of the abalone egg coat, VERL is a giant, fibrous glycoprotein composed of ~22 ZP-N repeats that likely form intermolecular hydrogen bonds to create the highly stabilized and protective egg coat. Lysin creates a hole in the egg coat by competing for these hydrogen bonds, allowing sperm to pass and fuse with the oocyte. These ZP-N repeats are also a principal component of mammalian egg coats, and multiple lines of evidence support the human protein ZP2 as the functional analog of VERL. However, three major outstanding questions are (1) besides lysin and VERL, which sperm and egg proteins are interacting, (2) what sites in these proteins are important for mediating this interaction, and (3) how might mutations in these sites impact protein structure and fertility. To address these fundamental questions of reproduction in both abalone (K99) and humans (R00), two specific aims are proposed that utilize state-of-the- art genomic, proteomic, and structural methods. In aim 1, for the K99 phase, I will combine long read sequencing technology, tests for molecular evolution, and quantitative proteomics to identify potential pairs of interacting reproductive proteins in abalone; for the R00 phase, I will extend these quantitative proteomic methods to identify potentially novel pathways that contribute to male infertility. In aim 2, for the K99 phase, the solution structure of VERL repeat 1 and its interactions with lysin will be determined by NMR, with deep mutational scanning used to identify mutations that interfere lysin-VERL interactions. In the R00 phase, these technologies will be applied to human ZP2 and I will identify mutations that interfere with sperm binding and characterize their structural effects. The proposed research is innovative for its combined use of genomic, proteomic, and structural techniques to characterize the molecular mechanisms underlying the interactions of rapidly evolving reproductive proteins. The results are expected to shed insight into the core processes that mediate egg-sperm interactions, and provide foundational information towards understanding the more complex mammalian system.