A destabilisation domain approach to define the in vivo functional importance of PfHsp70-1 and PfHsp40 in the intraerythrocytic life cycle of Plasmodium falciparum

The apicomplexan malaria parasite, Plasmodium falciparum is capable of invading red blood cells and causes the most virulent form of malaria. The life cycle of P. falciparum involves the migration from the poikilothermic mosquito vector to warm-blooded human host and vice versa. Such transition i...

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Bibliographic Details
Main Author: Mandal, Pradipta
Contributors: Przyborski, Jude (Prof. Dr.) (Thesis advisor)
Format: Dissertation
Language:English
Published: Philipps-Universität Marburg 2017
Biologie
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Summary:The apicomplexan malaria parasite, Plasmodium falciparum is capable of invading red blood cells and causes the most virulent form of malaria. The life cycle of P. falciparum involves the migration from the poikilothermic mosquito vector to warm-blooded human host and vice versa. Such transition introduces radical differences between the cellular environments where the parasite resides, imposing physiological stress. The diverse environmental insults in addition to the febrile fever episodes imparts challenge on the proteostasis, resulting in the evolutionary selection of a diverse network of molecular chaperones. In fact, some molecular chaperones are essential for the survival of Plasmodium. Due to the developing resistance of Plasmodium against currently available drugs, heat shock proteins have received extensive research attention as antimalarial targets in recent years. Plasmodium codes for one Hsp90 homologue and a constitutively expressed heat inducible cytosolic Hsp70 known as PfHsp70-1. In general, Hsp70 interacts with co-chaperone Hsp40 initiating the protein folding machinery that finally interacts with Hsp90 to maintain proteostasis in a cell. PfHsp90 has been found to be essential for the intraerythrocytic development of P. falciparum. Although there have been some in vitro studies on the biology of PfHsp70-1, the information on the in vivo essential function of PfHsp70-1 and its interaction with PfHsp40 is limited. In this study, we wanted to identify the in vivo biological importance of PfHsp70-1 and one of its predicted co-chaperones, PfHsp40 by the overexpression of the dominant negative alleles tagged to recently characterised destabilisation domain (dd) to regulate protein level. We expressed a dominant negative PfHsp70-1 possessing a point mutation (E187K), severely affecting normal domain movement important for its function. PfHsp40 was mutated in the conserved HPD motif (D34N) necessary for establishing interaction with PfHsp70-1. Unfortunately, we could not obtain sufficient overexpression of the episomal dominant negative versions to override the function of the endogenous proteins in a competitive manner. The cellular levels of endogenous proteins were higher by several folds compared to that of the episomally expressed dominant negative alleles. The destabilisation strategy has been reported to be successful for studying certain plasmodial proteins. But in contrast, during our work the level of almost none of the candidate chaperones could be controlled by either FKBP or E. coli DHFR derived destabilisation domains in a ligand dependent fashion. Although, the level of wild type PfHsp70-1 could be regulated by this strategy, the dominant negative version with only one amino acid substitution made it non responsive to dd tagging and further ligand treatment. At the same time, the level of control proteins could be efficiently regulated by stabilising ligands. Recently, success of destabilization domain strategy for conditional knockdown of several genes has been reported. But in contrast, our observations in this study unravel the possible drawbacks. We assume that the success of such an approach is greatly protein dependent. Based on the several reports initially this approach appeared to be the most beneficial system. But, the failure to successfully implement this strategy demands careful consideration in selecting an alternative future approach to study the function of essential genes in Plasmodium.
Physical Description:167 Pages
DOI:https://doi.org/10.17192/z2017.0659