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

点击翻译按钮获取中文摘要

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.