A molecular, cell & structural biology approach toward characterising alveolins, unique cytoskeletal proteins crucial for malaria parasite development

战略研究重点：Alveolins是一种中间丝蛋白，仅存在于顶复门寄生虫（例如疟疾、弓形虫病、隐孢子虫病的病原体）、甲藻和纤毛虫中。肺泡蛋白的定义是在其功能结构域中具有12个氨基酸的串联重复结构，其类似于其他原生动物细胞骨架蛋白如关节蛋白。在疟疾寄生虫中，肺泡蛋白在整个生命周期中对细胞发育至关重要，因此形成了有吸引力的疾病控制目标。本项目旨在确定肺泡蛋白的核心结构和原子结构，以及它们组装成疟原虫皮层细胞骨架的潜在机制。在后生动物中，基于中间丝（IF）的细胞骨架网络在细胞结构和可塑性中起主要作用，并作为机械应力吸收器。疟疾寄生虫表达一个独特的IF蛋白家族，称为肺泡蛋白。在同一细胞中表达的不同肺泡蛋白在功能上是独立的，并且累积地有助于寄生虫的拉伸强度、细胞形状、运动性和感染性。目前的模型是，每个肺泡蛋白组装成不同的细丝，然后相互连接成一个功能网络，类似于其他IF系统。肺泡蛋白在疟疾寄生虫发育中的基本性质、它们在整个生命周期中的表达以及它们在脊椎动物中的缺失使它们成为疟疾治疗、预防和传播控制的有吸引力的药物靶标。此外，这些药物可能对广泛的其他顶复门寄生虫以及相关的病原性原生动物有活性。在这种情况下，更好地了解肺泡蛋白的核心架构及其组装机制至关重要。该跨学科项目旨在通过结合分子、细胞和结构生物学方法来实现这一目标，具体目标如下：（1）确定核心肺泡蛋白结构域的原子结构。由不同数目的典型alveolin串联重复序列组成的合成alveolin基因将被重组表达（例如在大肠杆菌中）。coli），然后进行纯化、结晶和X射线晶体学。(2)阐明体外组装成纤维的机制。将使用电子显微镜确定重组肺泡蛋白（自动）组装成细丝的条件。这种体外组装系统将被开发为高通量筛选组装抑制剂的平台。(3)研究活疟原虫中组装成皮层细胞骨架的肺泡蛋白的结构要求。将使用伯氏疟原虫小鼠疟疾模型，使用表达肺泡蛋白修饰形式的转基因寄生虫系进行结构-功能分析，以确定功能结构域以及蛋白质相互作用和翻译后修饰的关键位点。

Strategic Research Priority: Bioscience for healthAlveolins are intermediate filament proteins that are exclusively found in apicomplexan parasites (causative agents of for example malaria, toxoplasmosis, cryptosporidiosis), dinoflagellate algea and ciliates. Alveolins are defined by possessing 12 amino-acid tandem repeat structures in their functional domains, which resemble those of other protozoan cytoskeletal proteins like articulins. In malaria parasites, alveolins are essential for cell development across the life cycle and as such form attractive targets for disease control. This project aims to determine the core architecture and atomic structure of the alveolins, as well as the underlying mechanisms for their assembly into the cortical cytoskeleton of malaria parasites.In metazoans, intermediate filament (IF)-based cytoskeletal networks have primary roles in cell architecture and plasticity, and as mechanical stress absorbers. Malaria parasites express a unique family of IF proteins named alveolins. Different alveolins expressed in the same cell are functionally independent and contribute cumulatively to the parasites' tensile strength, cell shape, motility and infectivity. The current model is that alveolins each assemble into distinct filaments before being interconnected into a functional network, similar to other IF systems. The essential nature of the alveolins in malaria parasite development, their expression throughout the life cycle, and their absence in vertebrates makes them attractive drug targets for malaria treatment, prophylaxis and transmission control. Moreover, such drugs may be active against a broad range of other apicomplexan parasites, as well as against related pathogenic protozoans. In this context a better understanding of the core architecture of the alveolins and their assembly mechanisms is vital. This interdisciplinary project aims to achieve this by combining molecular, cell and structural biological approaches through the following specific objectives: (1) Determine the atomic structure of the core alveolin domain. Synthetic alveolin genes composed of different numbers of canonical alveolin tandem repeats will be recombinantly expressed (e.g. in E. coli), followed by purification, crystallization and X-ray crystallography. (2) Elucidate the mechanisms of assembly into filaments in vitro. Conditions for (auto)-assembly of recombinant alveolins into filaments will be determined using electron microscopy. This in vitro assembly system will then be developed as a platform for high-throughput screening for inhibitors of assembly.(3) Study the structural requirements of the alveolins for assembly into the cortical cytoskeleton in live malaria parasites. Structure-function analyses using GM parasite lines expressing modified forms of the alveolins will be conducted to determine functional domains as well as critical sites of protein interaction and post-translational modification, using the Plasmodium berghei mouse malaria model.