Membrane proteins driving a cell-cell fusion reaction during fertilization

受精过程中驱动细胞-细胞融合反应的膜蛋白

• 批准号: 10598164
• 负责人: Jennifer Fricke Pinello
• 金额: $ 12.5万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-01 至 2024-08-19
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10598164
• 关键词: Adhesions; Algae; Angiosperms; Animals; Arabidopsis; Arthropods; Automobile Driving; Binding; Biochemical; Biological Models; Cell fusion; Cell membrane; Cell surface; Cells; Chimeric Proteins; Chlamydomonas; Clinical; Cryoelectron Microscopy; Cryptosporidium; Dengue; Development; Dissection; Disulfides; Eimeria; Endosomes; Environment; Eukaryota; Event; Family; Fertilization; Genes; Germ Cells; Green Algae; Hantavirus; Health; Homo; Human; Huntingtin-Associated protein 1; Infection; Laboratories; Life; Lipid Bilayers; Malaria; Mammals; Membrane; Membrane Fusion; Membrane Proteins; Molecular; Molecular Conformation; Morphology; Mus; Organism; Orthologous Gene; PDAP2 Gene; Parasites; Partner in relationship; Pathogenicity; Plants; Plasmodium; Property; Protein Family; Proteins; Protozoa; Reaction; Regulation; Rest; Sexual Reproduction; Site; Specific qualifier value; Sperm-Ovum Interactions; Structure; System; Tetrahymena; Time; Toxoplasma; Vascular Plant; Vertebrates; Viral; Virus; Work; ZIKA; adhesion receptor; dimer; disulfide bond; egg; in vivo; malaria transmission-blocking vaccine; male; mutant; pathogen; receptor; sperm cell; transmission blocking; transmission process; vaccine development; zygote

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.

项目摘要 受精过程中两个配子(如精子和卵子)之间的膜融合是真核生物中至关重要的一步。 生活。在所有生物体中，融合反应分两步进行，即膜粘连和双层合并。 值得注意的是，对于任何一个生物体来说，我们还没有同时拥有配子和融合的黏附蛋白 蛋白。最近，我们的实验室和其他实验室发现，古老的雄性配子特异蛋白HAP2是 在广泛的真核生物分类群中受精是必不可少的，包括致病的疟疾有机体 疟原虫、绿藻、纤毛虫、高等植物和许多后生动物。我们的合作研究也表明 疟原虫HAP2的一个关键功能基序可以作为阻断传播的疟疾疫苗的靶标。 最近的研究表明，HAP2在结构上与病毒II类融合蛋白(如登革热和 寨卡病毒)。被囊膜病毒上的II类融合蛋白是由内体的酸性环境触发的 经历从同源或异源二聚体到驱动双分子层的同源三聚体的构象重组 在感染期间合并和病毒进入。然而，HAP2存在于细胞表面，并可能受到调控 不同的是因为它在不同的环境中发挥作用。在这里，我建议在双纤毛绿色中使用受精 藻类作为模型系统来研究调控真核细胞II类融合蛋白的机制。对于 在任何系统中，我们现在第一次确定了配子和融合蛋白上的黏附受体。 正配子上的黏附蛋白FUS1和黏附蛋白MaR1(我们刚刚鉴定过)和 FusoGen HAP2在负配子上。我觉得这是一个很大的进步，我还确定了MaR1是 两种功能。除了与FUS1结合外，MaR1还与HAP2在生化和功能上有关联 在负配子上。此外，我们还确定了依赖于FUS1和MaR1的配子黏附是 HAP2从休眠配子上的预融合形式重新配置为同源三聚体所必需的 推动膜融合。在这项工作中，我打算确定HAP2在静息负数中的融合前构象 配子，识别MaR1和HAP2的结构域，这些结构域是它们在静止配子中相互作用的基础，识别 FUS1和MaR1中的结构域在配子相互作用过程中对它们的结合很重要，并决定了这些变化 MaR1和HAP2在FUS1-MaR1结合过程中经历的激活HAP2进行融合的过程。长期的 这项建议的目的是增进我们对膜融合机制的基本了解。 在受精的同时提供了新的策略来开发疫苗来靶向传播 致病原生动物。