Protein abundance and folding rather than the redox state of Kelch13 determine the artemisinin susceptibility of Plasmodium falciparum

Decreased susceptibilities of the human malaria parasite Plasmodium falciparum towards the endoperoxide antimalarial artemisinin are linked to mutations of residue C580 of PfKelch13, a homologue of the redox sensor Keap1 and other vertebrate BTB-Kelch proteins. Here, we addressed whether mutations a...

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Autores principales:	Robin Schumann, Eileen Bischoff, Severina Klaus, Sophie Möhring, Julia Flock, Sandro Keller, Kim Remans, Markus Ganter, Marcel Deponte
Formato:	article
Lenguaje:	EN
Publicado:	Elsevier 2021
Materias:	Malaria Artemisinin Keap1 Kelch13 Protein folding Medicine (General) R5-920 Biology (General) QH301-705.5
Acceso en línea:	https://doaj.org/article/d643779b262a4fcd8ac0dc6cb6438b67
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spelling	oai:doaj.org-article:d643779b262a4fcd8ac0dc6cb6438b672021-11-12T04:33:04ZProtein abundance and folding rather than the redox state of Kelch13 determine the artemisinin susceptibility of Plasmodium falciparum2213-231710.1016/j.redox.2021.102177https://doaj.org/article/d643779b262a4fcd8ac0dc6cb6438b672021-12-01T00:00:00Zhttp://www.sciencedirect.com/science/article/pii/S2213231721003372https://doaj.org/toc/2213-2317Decreased susceptibilities of the human malaria parasite Plasmodium falciparum towards the endoperoxide antimalarial artemisinin are linked to mutations of residue C580 of PfKelch13, a homologue of the redox sensor Keap1 and other vertebrate BTB-Kelch proteins. Here, we addressed whether mutations alter the artemisinin susceptibility by modifying the redox properties of PfKelch13 or by compromising its native fold or abundance. Using selection-linked integration and the glmS ribozyme, efficient down-regulation of PfKelch13 resulted in ring-stage survival rates around 40%. While the loss of the thiol group of C469 or of the potential disulfide bond between residues C580 and C532 had no effect on the artemisinin susceptibility, the thiol group of C473 could not be replaced. Furthermore, we detected two different forms of PfKelch13 with distinct electrophoretic mobilities around 85 and 95 kDa, suggesting an unidentified post-translational modification. We also established a protocol for the production of recombinant PfKelch13 and produced an antibody against the protein. Recombinant PfKelch13 adopted alternative oligomeric states and only two of its seven cysteine residues, C469 and C473, reacted with Ellman’s reagent. While common field mutations resulted in misfolded and completely insoluble recombinant PfKelch13, cysteine-to-serine replacements had no effect on the solubility except for residue C473. In summary, in contrast to residues C469, C532, and C580, the surface-exposed thiol group of residue C473 appears to be essential. However, not the redox properties but impaired folding of PfKelch13, resulting in a decreased PfKelch13 abundance, alters the artemisinin susceptibility and is the central parameter for mutant selection.Robin SchumannEileen BischoffSeverina KlausSophie MöhringJulia FlockSandro KellerKim RemansMarkus GanterMarcel DeponteElsevierarticleMalariaArtemisininKeap1Kelch13Protein foldingMedicine (General)R5-920Biology (General)QH301-705.5ENRedox Biology, Vol 48, Iss , Pp 102177- (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Malaria Artemisinin Keap1 Kelch13 Protein folding Medicine (General) R5-920 Biology (General) QH301-705.5
spellingShingle	Malaria Artemisinin Keap1 Kelch13 Protein folding Medicine (General) R5-920 Biology (General) QH301-705.5 Robin Schumann Eileen Bischoff Severina Klaus Sophie Möhring Julia Flock Sandro Keller Kim Remans Markus Ganter Marcel Deponte Protein abundance and folding rather than the redox state of Kelch13 determine the artemisinin susceptibility of Plasmodium falciparum
description	Decreased susceptibilities of the human malaria parasite Plasmodium falciparum towards the endoperoxide antimalarial artemisinin are linked to mutations of residue C580 of PfKelch13, a homologue of the redox sensor Keap1 and other vertebrate BTB-Kelch proteins. Here, we addressed whether mutations alter the artemisinin susceptibility by modifying the redox properties of PfKelch13 or by compromising its native fold or abundance. Using selection-linked integration and the glmS ribozyme, efficient down-regulation of PfKelch13 resulted in ring-stage survival rates around 40%. While the loss of the thiol group of C469 or of the potential disulfide bond between residues C580 and C532 had no effect on the artemisinin susceptibility, the thiol group of C473 could not be replaced. Furthermore, we detected two different forms of PfKelch13 with distinct electrophoretic mobilities around 85 and 95 kDa, suggesting an unidentified post-translational modification. We also established a protocol for the production of recombinant PfKelch13 and produced an antibody against the protein. Recombinant PfKelch13 adopted alternative oligomeric states and only two of its seven cysteine residues, C469 and C473, reacted with Ellman’s reagent. While common field mutations resulted in misfolded and completely insoluble recombinant PfKelch13, cysteine-to-serine replacements had no effect on the solubility except for residue C473. In summary, in contrast to residues C469, C532, and C580, the surface-exposed thiol group of residue C473 appears to be essential. However, not the redox properties but impaired folding of PfKelch13, resulting in a decreased PfKelch13 abundance, alters the artemisinin susceptibility and is the central parameter for mutant selection.
format	article
author	Robin Schumann Eileen Bischoff Severina Klaus Sophie Möhring Julia Flock Sandro Keller Kim Remans Markus Ganter Marcel Deponte
author_facet	Robin Schumann Eileen Bischoff Severina Klaus Sophie Möhring Julia Flock Sandro Keller Kim Remans Markus Ganter Marcel Deponte
author_sort	Robin Schumann
title	Protein abundance and folding rather than the redox state of Kelch13 determine the artemisinin susceptibility of Plasmodium falciparum
title_short	Protein abundance and folding rather than the redox state of Kelch13 determine the artemisinin susceptibility of Plasmodium falciparum
title_full	Protein abundance and folding rather than the redox state of Kelch13 determine the artemisinin susceptibility of Plasmodium falciparum
title_fullStr	Protein abundance and folding rather than the redox state of Kelch13 determine the artemisinin susceptibility of Plasmodium falciparum
title_full_unstemmed	Protein abundance and folding rather than the redox state of Kelch13 determine the artemisinin susceptibility of Plasmodium falciparum
title_sort	protein abundance and folding rather than the redox state of kelch13 determine the artemisinin susceptibility of plasmodium falciparum
publisher	Elsevier
publishDate	2021
url	https://doaj.org/article/d643779b262a4fcd8ac0dc6cb6438b67
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Protein abundance and folding rather than the redox state of Kelch13 determine the artemisinin susceptibility of Plasmodium falciparum

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