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 受精期间两个配子（例如精子和鸡蛋）之间的膜融合是真核的关键步骤 生活。在所有生物体中，融合反应以两个步骤进行：膜粘附和双层合并。 值得注意的是，对于我们还没有一个生物体，我们还拥有配子和融合的粘附蛋白 蛋白质。最近，我们的实验室和其他实验室表明，古老的男配子特异性蛋白质HAP2是 在广泛的真核分类群中受精至关重要，包括致病性疟疾生物 疟原虫，绿藻，纤毛，较高的植物和许多后生动物。我们的协作研究也表明 疟原虫HAP2的关键功能基序可以针对传播疟疾疫苗。 最近的工作表明，HAP2在结构上与病毒II类融合蛋白同源（例如，登革热和 Zika）。包膜病毒上的II类融合蛋白是由内体的酸性环境触发的 从同型或异二聚体转变为驱动双层的同构二聚体的构象重组 感染期间的合并和病毒进入。但是，HAP2存在于细胞表面，并且可能调节 以不同的方式，因为它在各种环境中起作用。在这里，我提议在双纤维绿色中使用受精 藻类作为模型系统，以研究调节真核II类融合蛋白的机制。为了 在任何系统中，我们第一次都确定了配子和融合蛋白上的粘附受体。 加上配子和粘附蛋白MAR1的粘附蛋白FUS1（我们刚刚识别）和 减去配子上的Fusogen HAP2。我认为是一个重大进步，我还确定Mar1是 双功能。除了与FUS1结合外，MAR1在生化和功能上也与HAP2相关 在负配子上。此外，我们已经确定FUS1和MAR1依赖配子粘附是 HAP2从其在休息配子中重新配置的重新配置所必需的 驱动膜融合。在这项工作中，我打算确定HAP2的静止构象 配子，确定MAR1和HAP2的域，这些域是其在静止配子中的互动的基础 FUS1和MAR1中的域对于它们在配子相互作用过程中的结合很重要，并确定变化 在FUS1-MAR1结合期间，该MAR1和HAP2经历了激活HAP2以进行融合。长期 该建议的目标是增强我们对膜融合机制的基本理解 受精，同时提供了开发疫苗以靶向传播的新策略 致病原生动物。