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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要受精过程中两个配子（例如精子和卵子）之间的膜融合是真核生物的关键步骤生活。在所有生物体中，融合反应分两个步骤进行：膜粘附和双层合并。值得注意的是，对于任何一种生物体来说，我们还没有配子和融合体的粘附蛋白蛋白质。最近，我们实验室和其他人证明，古代雄性配子特异性蛋白HAP2是对于包括致病性疟疾生物在内的多种真核生物的受精至关重要疟原虫、绿藻、纤毛虫、高等植物和许多后生动物。我们的合作研究还表明疟疾 HAP2 的一个关键功能基序可以作为阻断传播的疟疾疫苗的目标。最近的工作表明，HAP2 在结构上与病毒 II 类融合蛋白（例如登革热和寨卡）。有包膜病毒上的 II 类融合蛋白由内体的酸性环境触发，经历从同二聚体或异二聚体到驱动双层的同三聚体的构象重组感染期间的合并和病毒进入。然而，HAP2 存在于细胞表面并可能受到调节之所以不同，是因为它在不同的环境中发挥作用。在这里，我建议在双纤毛绿化中使用施肥藻类作为模型系统来研究调节真核 II 类融合蛋白的机制。对于我们现在在任何系统中首次鉴定出配子和融合蛋白上的粘附受体。正配子上的粘附蛋白 FUS1 和粘附蛋白 MAR1（我们刚刚鉴定）和负配子上的融合原HAP2。我认为这是一个重大进步，我还确定 MAR1 是双功能。除了与 FUS1 结合外，MAR1 还在生化和功能上与 HAP2 相关在负配子上。此外，我们已经确定 FUS1 和 MAR1 依赖的配子粘附是 HAP2 从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 HAP2 在静息状态下的融合前构象配子，识别 MAR1 和 HAP2 的域，这些域是它们在静息配子中相互作用的基础，识别 FUS1 和 MAR1 中的结构域对于配子相互作用期间的结合很重要，并决定变化 MAR1 和 HAP2 在 FUS1-MAR1 结合过程中发生，激活 HAP2 进行融合。长期来看该提案的目的是增强我们对膜融合机制的基本理解受精时，同时为开发针对传播的疫苗提供新策略致病原虫。