Cellular and molecular mechanisms regulating function of a broadly conserved gamete membrane fusogen

广泛保守的配子膜融合剂功能的细胞和分子机制调节

• 批准号: 9761105
• 负责人: Jun Zhang
• 金额: $ 6.16万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-09 至 2020-12-23
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9761105
• 关键词: Adhesions; Animal Model; Antibodies; Back; Binding; Biochemical; Biological Models; CRISPR/Cas technology; Cell membrane; Cells; Chimeric Proteins; Chlamydomonas; Chordata; Culicidae; Cysteine; Dengue; Development; Dissociation; Endosomes; Ensure; Eukaryota; Event; Evolution; Gel Chromatography; Genes; Germ Cells; Green Algae; Human; Huntingtin-Associated protein 1; Hydrophobicity; Impairment; In Vitro; Laboratories; Lead; Length; Malaria; Mating Types; Membrane; Membrane Fusion; Membrane Proteins; Methods; Modeling; Molecular; Molecular Conformation; Mutation; Organism; PDAP2 Gene; Partner in relationship; Peptides; Plasmodium; Plasmodium falciparum; Proteins; Publishing; Reaction; Recombinants; Reproduction; Research; Role; Signal Pathway; Signal Transduction; Structure; System; Testing; Time; Transmembrane Domain; Vascular Plant; Viral; Viral Fusion Proteins; Virus; Zika Virus; base; cell motility; dimer; experimental study; field study; fungus; human pathogen; improved; in vivo; insight; molecular mass; monomer; mutant; pathogen; sedimentation velocity; transmission process; trimer core; virus envelope; zygote

Project Summary Our understanding of the molecular mechanisms of fusion between reproductive cells (gametes) during sexual reproduction is surprisingly limited. In studies with the unicellular green alga, Chlamydomonas and the malaria pathogen Plasmodium, the Snell laboratory has shown that the broadly conserved, gamete-specific membrane protein HAP2 (also called GCS1) is essential for bilayer merger during gamete fusion. HAP2 acts after tight adhesion of gamete plasma membranes, and is present on only one of the two gametes (e.g., mating type minus gametes in Chlamydomonas) and therefore functions unilaterally, as do viral fusion proteins during their entry into host cells. Remarkably, recent collaborative structure/function studies demonstrated that Chlamydomonas HAP2 is structurally homologous to the class II fusion proteins of many enveloped viruses, including dengue and Zika. Though recombinant forms of the ectodomain of Chlamydomonas HAP2 form trimers in vitro and the hydrophobic fusion loop is essential for function in vivo, we still know very little about the molecular mechanism of this eukaryotic class II fusion protein during gamete fusion in any HAP2 organism. Endogenous trimers that presumably form during gamete fusion have yet to be detected, and the mechanisms are unknown that restrict triggering of trimer formation until the HAP2-bearing gamete undergoes specific membrane interaction with the membrane of its partner gamete. In preliminary experiments, I have discovered a stable oligomer, which appears only after gamete fusion, and is likely to be the endogenous HAP2 trimer. I have also determined that, unlike with the viral class II proteins, low pH fails to trigger oligomer formation of HAP2. Rather, the HAP2-containing minus mating structure must bind to the adhesion protein FUS1 on the plus mating structure, ensuring that triggering takes place only at the right time and place. This research aims to define the molecular mechanisms that underlie the function of a broadly conserved eukaryotic membrane fusogen that is essential for fusion of gametes in organisms across kingdoms. The insights gained from this study will improve our understanding of fundamental, conserved mechanisms of gamete fusion, and have the potential to yield improved strategies for interfering with sexual reproduction and transmission of organism that harm humans, including the devastating malaria organism Plasmodium. Here, I will test the model that a new HAP2 oligomer detected in gametes after fusion is a trimer. I will investigate structural features of HAP2 required for oligomerization. And, I will investigate the mechanisms that underlie FUS1-dependent triggering of HAP2 structural rearrangements.

项目概要 我们对生殖细胞（配子）之间融合分子机制的理解 有性生殖期间的数量惊人地有限。在单细胞绿藻的研究中， 衣藻和疟疾病原体疟原虫，斯内尔实验室已经表明， 广泛保守的配子特异性膜蛋白 HAP2（也称为 GCS1）对于 配子融合过程中的双层合并。 HAP2在配子血浆紧密粘附后起作用 膜，并且仅存在于两个配子之一上（例如，交配型减去配子 衣藻），因此单方面发挥作用，病毒融合蛋白在进入过程中也是如此 进入宿主细胞。值得注意的是，最近的合作结构/功能研究表明 衣藻 HAP2 在结构上与许多 II 类融合蛋白同源 有包膜病毒，包括登革热和寨卡病毒。尽管胞外域的重组形式 衣藻 HAP2 在体外形成三聚体，疏水性融合环对于 体内功能，我们对这一类真核生物的分子机制仍然知之甚少 任何 HAP2 生物体配子融合过程中的 II 融合蛋白。内源性三聚体 据推测，配子融合过程中的形成尚未被发现，其机制是 未知限制三聚体形成的触发，直到携带 HAP2 的配子经历 与其伴侣配子的膜发生特异性膜相互作用。在初步 实验中，我发现了一种稳定的寡聚物，它只在配子融合后出现，并且是 可能是内源性 HAP2 三聚体。我还确定，与病毒类不同 II 蛋白，低 pH 值无法触发 HAP2 寡聚体的形成。相反，含有HAP2的 负交配结构必须与正交配结构上的粘附蛋白 FUS1 结合， 确保触发仅在正确的时间和地点发生。本研究旨在定义 广泛保守的真核生物功能的分子机制 膜融合剂，对于跨界生物体内的配子融合至关重要。这 从这项研究中获得的见解将提高我们对基本的、保守的理解 配子融合机制，并有可能产生改进的干扰策略 危害人类的有机体的有性繁殖和传播，包括 毁灭性的疟疾生物疟原虫。在这里，我将测试新的 HAP2 寡聚物的模型 融合后在配子中检测到的是三聚体。我将研究所需HAP2的结构特征 用于低聚。而且，我将研究 FUS1 依赖的机制 触发HAP2结构重排。