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Titel: A destabilisation domain approach to define the in vivo functional importance of PfHsp70-1 and PfHsp40 in the intraerythrocytic life cycle of Plasmodium falciparum
Autor: Mandal, Pradipta
Weitere Beteiligte: Przyborski, Jude (Prof. Dr.)
Veröffentlicht: 2017
URI: https://archiv.ub.uni-marburg.de/diss/z2017/0659
DOI: https://doi.org/10.17192/z2017.0659
URN: urn:nbn:de:hebis:04-z2017-06590
DDC: Biowissenschaften, Biologie
Titel(trans.): Destabilisierungsdomänen Ansatz zur Definition der in vivo funktionalen Bedeutung von PfHsp70-1 und PfHsp40 für den intraerythrozytischen Lebenszyklus von P. falciparum.
Publikationsdatum: 2019-10-16
Lizenz: https://rightsstatements.org/vocab/InC-NC/1.0/

Dokument

Schlagwörter:
PfHsp40, PfHsp70-1, PFB0595w

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.

Zusammenfassung:
Der apikomplexe Parasit Plasmodium falciparum ist in der Lage, Erythrozyten zu invadieren und verursacht die virulenteste Form der Malaria. Der Lebenszyklus von P. falciparum umfasst den Übergang vom poikilothermen Moskito Vektor zum warmblütigen, menschlichen Wirt und zurück. Ein solcher Wechsel bedeutet gleichzeitig einen radikalen Wechsel der zellulären Umgebung, in welcher der Parasit sich befindet und letzten Endes physiologischen Stress. Die unterschiedlichen Stressfaktoren durch die Umgebung stellen zusätzlich zu den Fieberepisoden eine Herausforderung bezüglich der Proteostase dar, was zur evolutionären Selektion eines Netzwerks unterschiedlicher molekularer Chaperone geführt hat. Tatsächlich sind einige dieser molekularen Chaperone für das Überleben von Plasmodium unabdinglich. Aufgrund der immer weiter fortschreitenden Ausbildung von Resistenzen gegenüber den verfügbaren Medikamenten kam den Hitzeschock Proteinen (Hsp) große Beachtung bezüglich der möglichen Verwendung als Angriffspunkte für neue Antimalaria Therapien zu. Plasmodium kodiert für ein Hsp90 Homolog sowie ein konstitutiv exprimiertes, hitzeinduziertes zytosolisches Hsp70 namens PfHsp70-1. Grundsätzlich interagiert Hsp70 mit dem Co-Chaperon Hsp40 und initiiert die durch die Interaktion mit Hsp90 vervollständigte Proteinfaltungsmaschinerie, welche die Proteostase in der Zelle aufrechterhält. PfHsp90 hat sich als essentiell für die intraerythrozytäre Entwicklung von P. falciparum erwiesen. Obwohl bezüglich der Biologie von PfHsp70-1 einige in vitro Studien durchgeführt wurden, existiert wenig Information bezüglich der essentiellen Funktion der Interaktion von PfHsp70- 1 und PfHsp40 in vivo. Im Zuge der hier vorliegenden Arbeit sollte in vivo die biologische Bedeutung von PfHsp70- 1 und einem seiner vorhergesagten Co-Chaperonen, PfHsp40, untersucht werden. Dazu sollte mit Hilfe von Überexpression der dominant negativen Allele, welche an die kürzlich charakterisierte Destabilisierungsdomäne (dd) gebunden wurden, der intrazelluläre Proteinlevel reguliert werden. Dabei wurde eine dominant negative Mutante von PfHsp70-1 exprimiert, welche eine Punktmutation (E187K) trägt, die die für die normale Funktion des Proteins wichtige Beweglichkeit der Domänen stark beeinflusst. PfHsp40 wurde im konservierten HPD Motiv (D34N) mutiert, welches wichtig für die Ausbildung der Interaktion mit PfHsp70-1 ist. Eine ausreichende Überexpression der episomalen dominant negativen Versionen, welche notwendig ist, um die Funktion der endogenen Proteine kompetitiv auszuschalten, war nicht möglich. Hierbei lagen die zellulären Level der endogenen Proteine um viele Stufen höher als die der episomal exprimierten dominant negativen Allele. In der Literatur wurde der Destabilisierungsansatz als erfolgreich bei der Untersuchung vieler Plasmodienproteine beschrieben. Im Gegensatz dazu konnte im Zuge dieser Arbeit bei nahezu keinem der ausgewählten Chaperone der Proteinlevel durch die Liganden FKBP oder DHFR (eine aus E. coli genutzte Destabilisierungsdomäne) kontrolliert werden. Obwohl der Level von Wildtyp PfHsp70-1 über diese Strategie reguliert werden konnte, war die dominant negative Version des Proteins mit einer mutierten Aminosäure gegenüber dem dd-Tag und Zugabe des Liganden resistent. Gleichzeitig war es möglich, die als Kontrolle fungierenden Proteine effektiv durch die stabilisierenden Liganden zu regulieren. Kürzlich konnte die erfolgreiche Nutzung des Destabilisierungsdomänen Ansatzes für konditionelle Knock-Outs verschiedener Gene gezeigt werden. Die in der vorliegenden Arbeit dargestellten Ergebnisse weisen im Gegensatz dazu aber auch auf die möglichen Nachteile dieser Technik hin. Wir vermuten dabei, dass der Erfolg dieses Ansatzes stark vom jeweiligen Protein abhängig ist. Die Wahl dieses Ansatzes bei der vorliegenden Arbeit basierte auf verschiedenen Veröffentlichungen, welche auf eine erfolgreiche mögliche Nutzung hindeuteten. Die Tatsache jedoch, dass es nicht möglich war, diese Strategie erfolgreich zu implementieren, verlangt ein vorsichtiges Vorgehen bei der Wahl zukünftiger Ansätze zur Untersuchung der Funktion essentieller Gene.

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