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

整合“组学”、进化和结构生物化学来了解动物受精

基本信息

批准号：
9761558
负责人：
Damien Beau Wilburn
金额：
$ 11.83万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-08-09 至 2021-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9761558
关键词：
Address Affinity Animal Model Animals Binding Biochemical Biochemistry Biological Process Capsid Proteins Cell membrane Code Complex Egg Proteins Evolution Fertility Fertilization Fertilizers Foundations Genes Genome Genomics Germ Cells Glycoproteins Human Hydrogen Bonding Incidence Individual Infertility Knowledge Male Infertility Mammals Marines Mediating Methods Modeling Molecular Molecular Evolution Multidimensional NMR Techniques Mutagenesis Mutation Oocytes Organism Pathway interactions Phase Pheromone Process Proteins Proteome Proteomics Reproduction Reproductive Biology Research Sampling Site Sperm-Ovum Interactions Structural Biochemistry Structural Protein Structure Structure-Activity Relationship Study models System Techniques Technology Testing Training ZP2 abalone analog design egg experimental study infertility treatment innovation insight lysin men mutation screening new therapeutic target novel polymerization protein expression protein protein interaction protein structure receptor reproductive sex sperm cell sperm protein three dimensional structure transcriptomics

项目摘要

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.

项目概要自 20 世纪 80 年代以来，不孕症的发生率增加了 4%，其中高达 20% 的病例原因不明。流行的假设之一是同源卵子和精子蛋白质之间不相容。然而，非常在任何生物体中都已发现很少对相互作用的生殖蛋白。最好的模型之一研究受精的核心机制是海洋腹足动物鲍鱼，其中精子高度富集只有少数蛋白质。第一个确定的相互作用的生殖蛋白对是鲍鱼精子溶素和它的蛋壳受体 VERL。作为鲍鱼蛋壳的主要成分，VERL 是一种巨大的纤维状物质由约 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稳定且具有保护作用的蛋壳。赖氨酸通过竞争这些氢而在蛋壳上形成一个洞结合，使精子能够通过并与卵母细胞融合。这些 ZP-N 重复序列也是哺乳动物卵膜和多种证据支持人类蛋白质 ZP2 作为功能类似物维尔。然而，三大悬而未决的问题是（1）除了溶素和VERL之外，精子和卵子还蛋白质正在相互作用，(2) 这些蛋白质中的哪些位点对于介导这种相互作用很重要，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为了在鲍鱼（K99）和人类（R00）中繁殖，提出了两个利用最新技术的具体目标艺术基因组、蛋白质组和结构方法。目标1，对于K99阶段，我会结合长读测序技术、分子进化测试和定量蛋白质组学，以识别潜在的鲍鱼中相互作用的生殖蛋白；对于 R00 阶段，我将扩展这些定量蛋白质组学确定导致男性不育的潜在新途径的方法。在目标 2 中，对于 K99 阶段， VERL重复1的溶液结构及其与溶素的相互作用将通过NMR确定，具有深突变扫描用于识别干扰溶素-VERL 相互作用的突变。在R00阶段，这些技术将应用于人类 ZP2，我将识别干扰精子结合的突变描述它们的结构效应。拟议的研究因其组合使用基因组而具有创新性，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。预计结果将深入了解核心流程介导卵子与精子的相互作用，并为了解更多信息提供基础信息复杂的哺乳动物系统。