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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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| Format: | Dissertation |
| Sprache: | Englisch |
| Veröffentlicht: |
Philipps-Universität Marburg
2017
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| Zusammenfassung: | 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. |
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| Umfang: | 167 Seiten |
| DOI: | 10.17192/z2017.0659 |