Judith L Green1, Yang Wu1, Vesela Encheva2, Edwin Lasonder3, Adchara Prommaban1,4, Simone Kunzelmann5, Evangelos Christodoulou5, Munira Grainger1, Ngoc Truongvan6, Sebastian Bothe7, Vikram Sharma3, Wei Song8, Irene Pinzuti8, Chairat Uthaipibull9, Somdet Srichairatanakool4, Veronique Birault10, Gordon Langsley11, Hermann Schindelin6, Benjamin Stieglitz8, Ambrosius P Snijders2, Anthony A Holder1. 1. Malaria Parasitology Laboratory, The Francis Crick Institute, London, United Kingdom. 2. Mass Spectrometry Proteomics, The Francis Crick Institute, London, United Kingdom. 3. School of Biomedical Science, University of Plymouth, Plymouth, United Kingdom. 4. Department of Biochemistry, Chiang Mai University, Chiang Mai, Thailand. 5. Structural Biology Science Technology Platform, The Francis Crick Institute, London, United Kingdom. 6. Rudolf Virchow Center for Experimental Biomedicine, Universität Würzburg, Würzburg, Germany. 7. Department of Chemistry and Pharmacy, University of Würzburg, Würzburg, Germany. 8. School of Biological and Chemical Sciences, Queen Mary University of London, London, United Kingdom. 9. National Center for Genetic Engineering and Biotechnology, Khlong Luang, Thailand. 10. Translation, The Francis Crick Institute, London, United Kingdom. 11. Laboratoire de Biologie Cellulaire Comparative des Apicomplexes, Institut Cochin, Université Paris Descartes, Paris, France.
Abstract
Ubiquitylation is a common post translational modification of eukaryotic proteins and in the human malaria parasite, Plasmodium falciparum (Pf) overall ubiquitylation increases in the transition from intracellular schizont to extracellular merozoite stages in the asexual blood stage cycle. Here, we identify specific ubiquitylation sites of protein substrates in three intraerythrocytic parasite stages and extracellular merozoites; a total of 1464 sites in 546 proteins were identified (data available via ProteomeXchange with identifier PXD014998). 469 ubiquitylated proteins were identified in merozoites compared with only 160 in the preceding intracellular schizont stage, suggesting a large increase in protein ubiquitylation associated with merozoite maturation. Following merozoite invasion of erythrocytes, few ubiquitylated proteins were detected in the first intracellular ring stage but as parasites matured through trophozoite to schizont stages the apparent extent of ubiquitylation increased. We identified commonly used ubiquitylation motifs and groups of ubiquitylated proteins in specific areas of cellular function, for example merozoite pellicle proteins involved in erythrocyte invasion, exported proteins, and histones. To investigate the importance of ubiquitylation we screened ubiquitin pathway inhibitors in a parasite growth assay and identified the ubiquitin activating enzyme (UBA1 or E1) inhibitor MLN7243 (TAK-243) to be particularly effective. This small molecule was shown to be a potent inhibitor of recombinant PfUBA1, and a structural homology model of MLN7243 bound to the parasite enzyme highlights avenues for the development of P. falciparum specific inhibitors. We created a genetically modified parasite with a rapamycin-inducible functional deletion of uba1; addition of either MLN7243 or rapamycin to the recombinant parasite line resulted in the same phenotype, with parasite development blocked at the schizont stage. Nuclear division and formation of intracellular structures was interrupted. These results indicate that the intracellular target of MLN7243 is UBA1, and this activity is essential for the final differentiation of schizonts to merozoites.
Ubiquitylation is a common post translational modification of eukaryotic proteins and in the humann class="Disease">malaria parasite, Plasmodium falciparum (Pf) overall ubiquitylation increases in the transition from intracellular schizont to extracellular merozoite stages in the asexual blood stage cycle. Here, we identify specific ubiquitylation sites of protein substrates in three intraerythrocytic parasite stages and extracellular merozoites; a total of 1464 sites in 546 proteins were identified (data available via ProteomeXchange with identifier PXD014998). 469 ubiquitylated proteins were identified in merozoites compared with only 160 in the preceding intracellular schizont stage, suggesting a large increase in protein ubiquitylation associated with merozoite maturation. Following merozoite invasion of erythrocytes, few ubiquitylated proteins were detected in the first intracellular ring stage but as parasites matured through trophozoite to schizont stages the apparent extent of ubiquitylation increased. We identified commonly used ubiquitylation motifs and groups of ubiquitylated proteins in specific areas of cellular function, for example merozoite pellicle proteins involved in erythrocyte invasion, exported proteins, and histones. To investigate the importance of ubiquitylation we screened ubiquitin pathway inhibitors in a parasite growth assay and identified the ubiquitin activating enzyme (UBA1 or E1) inhibitor MLN7243 (TAK-243) to be particularly effective. This small molecule was shown to be a potent inhibitor of recombinant PfUBA1, and a structural homology model of MLN7243 bound to the parasite enzyme highlights avenues for the development of P. falciparum specific inhibitors. We created a genetically modified parasite with a rapamycin-inducible functional deletion of uba1; addition of either MLN7243 or rapamycin to the recombinant parasite line resulted in the same phenotype, with parasite development blocked at the schizont stage. Nuclear division and formation of intracellular structures was interrupted. These results indicate that the intracellular target of MLN7243 is UBA1, and this activity is essential for the final differentiation of schizonts to merozoites.
Authors: Edwin Lasonder; Kunal More; Shailja Singh; Malak Haidar; Daniela Bertinetti; Eileen J Kennedy; Friedrich W Herberg; Anthony A Holder; Gordon Langsley; Chetan E Chitnis Journal: Front Microbiol Date: 2021-05-24 Impact factor: 5.640
Authors: James Abugri; Joseph Ayariga; Samuel Sunyazi Sunwiale; Cletus Adiyaga Wezena; Julien Agyemang Gyamfi; Michael Adu-Frimpong; Godfred Agongo; Julius Tieroyaare Dongdem; Daniel Abugri; Bismarck Dinko Journal: Heliyon Date: 2022-08-24