Membrane proteins driving a cell-cell fusion reaction during fertilization

项目摘要 两个配子之间的膜融合（例如，精子和卵子）在受精过程中是真核生物的关键步骤， 生活在所有生物体中，融合反应分两步进行，膜粘附和双层合并。 值得注意的是，对于任何一种生物，我们还没有同时拥有配子和融合的粘附蛋白。 蛋白最近，我们的实验室和其他人已经表明，古老的雄性配子特异性蛋白HAP 2是 在包括致病性疟疾生物在内的广泛的真核生物分类群中， 疟原虫、绿色原虫、纤毛虫、高等植物和许多后生动物。我们的合作研究还表明， 疟原虫HAP 2的一个关键功能基序可以作为阻断传播的疟疾疫苗的靶点。 最近的工作表明，HAP 2在结构上与II类病毒融合蛋白（例如登革病毒和革兰氏阴性病毒）同源。 Zika）。包膜病毒上的II类融合蛋白由内体的酸性环境触发， 经历从同源或异源二聚体到同源三聚体的构象重组， 合并和病毒进入。然而，HAP 2存在于细胞表面，并且可能受到调节。 因为它在不同的环境中发挥作用。在这里，我建议在一个双纤毛绿色 作为一个模型系统，以调查机制，调节真核II类融合蛋白。为 在任何系统中，我们第一次在配子和融合蛋白上鉴定了粘附受体。 阳性配子上的粘附蛋白FUS 1和粘附蛋白MAR 1（我们刚刚鉴定出）， 负配子上的融合原HAP 2。我认为这是一个重大的进步，我还确定MAR 1是 双功能的除了与FUS 1结合外，MAR 1还与HAP 2在生物化学和功能上相关 负配子上。此外，我们已经确定，FUS 1和MAR 1依赖的配子粘附， HAP 2从其在静息配子上的融合前形式重新配置为同源三聚体所必需的， 驱动膜融合。在这项工作中，我打算确定HAP 2融合前的构象，在静息负 配子，确定MAR 1和HAP 2的结构域，这些结构域是它们在休眠配子中相互作用的基础，确定 FUS 1和MAR 1中的结构域对它们在配子相互作用期间的结合很重要，并决定了变化 MAR 1和HAP 2在FUS 1-MAR 1结合过程中所经历的活化HAP 2以进行融合。长期 这一建议的目的是加强我们对膜融合机制的基本理解 同时为开发针对传播的疫苗提供新的策略 致病原生动物的证据

Project Summary Membrane fusion between two gametes (e.g., sperm & egg) during fertilization is a crucial step in eukaryotic life. In all organisms, the fusion reaction proceeds in two steps, membrane adhesion and bilayer merger. Remarkably, for no single organism do we yet have the adhesion proteins for both gametes and the fusion protein. Recently, our laboratory and others have shown that the ancient male gamete-specific protein HAP2 is essential for fertilization across a broad range of eukaryotic taxa, including the pathogenic malaria organism Plasmodium, green alga, ciliates, higher plants, and many metazoans. Our collaborative studies have also shown that a key functional motif of Plasmodium HAP2 can be targeted for a transmission-blocking malaria vaccine. Recent work demonstrated that HAP2 is structurally homologous to viral class II fusion proteins (e.g. Dengue and Zika). Class II fusion proteins on enveloped viruses are triggered by the acidic environment of the endosome to undergo a conformational reorganization from homo- or heterodimers into homotrimers that drive bilayer merger and viral entry during infection. HAP2, however, is present at the cell surface and likely regulated differently because it functions in a variety of milieus. Here, I propose to use fertilization in a bi-ciliated green alga as a model system to investigate the mechanisms that regulate a eukaryotic class II fusion protein. For the first time in any system, we now have identified the adhesion receptors on both gametes and the fusion protein. The adhesion protein FUS1 on plus gametes and the adhesion protein MAR1 (which we have just identified) and fusogen HAP2 on minus gametes. In what I feel is a major advance, I have also determined that MAR1 is bifunctional. In addition to binding to FUS1, MAR1 is also biochemically and functionally associated with HAP2 on minus gametes. Moreover, we have determined that FUS1 and MAR1 dependent gamete adhesion is necessary for the reconfiguration of HAP2 from its prefusion form on resting gametes into homotrimers that drive membrane fusion. In this work, I intend to determine the pre-fusion conformation of HAP2 in resting minus gametes, identify the domains of MAR1 and HAP2 that underlie their interactions in resting gametes, identify the domains in FUS1 and MAR1 important for their binding during gametes interactions, and determine the changes that MAR1 and HAP2 undergo during FUS1-MAR1 binding that activate HAP2 for fusion. The long-term objectives of this proposal are to enhance our fundamental understanding of the mechanism of membrane fusion at fertilization and at the same time provide new strategies for development of vaccines to target transmission of pathogenic protozoa.