Molecular mechanisms of a gamete membrane fusion reaction during fertilization

项目摘要 受精过程中配子（如精子和卵子）之间的膜融合是受精的关键步骤。 真核生物的生命周期不幸的是，我们对调节这一重要的分子知之甚少。 生殖过程已知一种重要的蛋白质是HAP 2，一种保守的雄性配子， 在许多真核生物中，受精所必需特异性因子， 重要的人类和动物病原体（例如引起疟疾的疟原虫属，锥虫属，弓形虫属， 艾美耳球虫等）。最近令人兴奋的研究发现，HAP 2在结构上与II类病毒同源， 融合蛋白这些病毒（例如登革热和寨卡）II类蛋白介导病毒和寨卡病毒的合并。 在感染过程中宿主细胞膜，并依赖于寡聚体和构象的变化 以响应低pH以获得它们促融合活性。拟议的研究将测试模型，在此之前， 融合，HAP 2以类似于某些病毒II类蛋白的方式在配子膜上组织自身， 这反过来又有助于调节HAP 2在性行为中配子细胞融合中的功能。实验- 顺应性的性生活周期的单细胞微囊，衣原体reinhardtii，将被用作 模型系统遗传上易处理的披衣细胞容易形成正负配子， 存在用于定量配子中的不同步骤的实验室和完善的方法 膜融合反应衣原体也是唯一一种HAP 2蛋白结构 已经发表，其中与HAP 2相互作用的受体结合蛋白已经 已识别（MAR 1，初步数据）。此外，使用策略性设计的突变， 衣原体HAP 2，沿着生化交联方法，我们检测到寡聚 这种融合剂的形式提供了有关其在膜上的多聚体接触的诱人线索 在融合之前。这项建议的长远目标是加强我们对 受精时膜融合的机制，并确定其他治疗靶点， HAP 2可能导致更好的疫苗或预防策略，用于许多有害的寄生虫病。

Project Summary Membrane fusion between gametes (e.g. sperm & egg) during fertilization is a crucial step in eukaryotic life cycles. Unfortunately, not much is known about the molecules regulating this vital reproductive process. One protein that is known to be important is HAP2, a conserved, male-gamete- specific factor necessary for fertilization in a wide range of eukaryotic species, including many important human and animal pathogens (e.g. Plasmodium sp. causing malaria, Trypanosoma, Toxoplasma, Eimeria, etc.). Exciting recent work has discovered that HAP2 is structurally homologous to viral class II fusion proteins. These viral (e.g. Dengue and Zika) class II proteins mediate the merger of virus and host cell membranes during infection and are dependent upon oligomeric and conformational changes in response to low pH for their fusogenic activity. The proposed studies will test the model that, before fusion, HAP2 organizes itself on the gamete membrane in a similar way to certain viral class II proteins, which in turn, helps regulate HAP2's function in gamete cell fusion during sex. The experimentally- amenable sexual life cycle of the unicellular micro-alga, Chlamydomonas reinhardtii, will be used as a model system. Genetically-tractable Chlamydomonas cells readily form plus and minus gametes in the laboratory and well-established methods exist for quantifying the distinct steps in the gamete membrane fusion reaction. Chlamydomonas is also the only organism in which a HAP2 protein structure has been published and where a receptor-binding protein which interacts with HAP2 has been identified (MAR1, preliminary data). Furthermore, using strategically designed mutations of Chlamydomonas HAP2, along with biochemical crosslinking methods, we have detected oligomeric forms of this fusogen which offer tantalizing clues concerning its multimeric contacts on the membrane before fusion. The long-term objectives of this proposal are to enhance our fundamental understanding of the mechanism of membrane fusion at fertilization and to identify additional therapeutic targets on HAP2 which could lead to better vaccines or prevention strategies for many harmful parasitic diseases.