Biology subjects

Sudhakar, R.

Publications and source records attributed to Sudhakar, R..

2 recordsLinked to original sources

bioRxiv2020

Plasmodium falciparum Atg18 localization to the food vacuole is phosphatidylinositol 3-phosphate-dependent and quinoline-sensitive

Autophagy is a lysosome-dependent degradative process involving over 35 Atg proteins. The autophagy repertoire in malaria parasites is limited and does not appear to be a major degradative process. To better understand the autophagy process, we investigated Plasmodium falciparum Atg18 (PfAtg18), a PROPPIN family protein, whose members like S. cerevisiae Atg18 (ScAtg18) and human WIPI2 are essential for autophagy. Wild type and mutant PfAtg18 were expressed in P. falciparum and assessed for localization, the effect of various inhibitors and antimalarials on PfAtg18 localization, and identification of PfAtg18-interacting proteins. PfAtg18 is expressed in asexual erythrocytic stages and localized to the food vacuole, which was also observed with other Plasmodium Atg18 proteins, indicating that food vacuole localization is a conserved feature. Interaction of PfAtg18 with the food vacuole-associated PI3P is essential for localization, as PfAtg18 mutants of PI3P-binding motifs neither bound PI3P nor localized to the food vacuole. Interestingly, ScAtg18 showed complete cytoplasmic localization despite binding with PI3P, indicating additional requirement for PfAtg18 localization. The food vacuole multi-drug resistance protein 1 (MDR1) was consistently identified in the PfAtg18 immunoprecipitate, and also interacted with PfAtg18. In contrast to PfAtg18, ScAtg18 did not interact with the MDR1, which, in addition to PI3P, could play a critical role in localization of PfAtg18. Chloroquine and amodiaquine greatly affected PfAtg18 localization, suggesting that these quinolines target PfAtg18 or the proteins that might be involved in its localization. Thus, PI3P and MDR1are critical mediators of PfAtg18 localization, and PfAtg18 may modulate MDR1 activity.

cell biology↗

bioRxiv2020

Characterization of Plasmodium falciparum NEDD8 and identification of cullins as its substrates

A variety of post-translational modifications of Plasmodium falciparum proteins, including phosphorylation and ubiquitination, are shown to have key regulatory roles. The neural precursor cell expressed developmentally downregulated protein 8 (NEDD8) is a ubiquitin-like modifier of cullin-RING E3 ubiquitin ligases, which regulate diverse cellular processes, including the cell-cycle. Although neddylation pathway is conserved in eukaryotes, it is yet to be characterized in Plasmodium and related apicomplexan parasites. Towards studying the neddylation pathway in malaria parasites, we characterized P. falciparum NEDD8 (PfNEDD8) and identified cullins as its physiological substrates. PfNEDD8 is a 76 amino acid residue protein without the C-terminal tail, indicating that it can be readily conjugated. The wild type and mutant (Gly75Gly76 mutated to Ala75Ala76) PfNEDD8 were expressed in P. falciparum. Western blot of wild type PfNEDD8-expressing parasites indicated multiple high molecular weight conjugates, which were absent in the parasites expressing the mutant, indicating conjugation of NEDD8 to proteins through Gly76. Immunoprecipitation followed by mass spectrometry of wild type PfNEDD8-expressing parasites identified several proteins, including two putative cullins. Furthermore, we expressed PfNEDD8 in mutant S. cerevisiae strains that lacked endogenous NEDD8 ({Delta}rub1) or NEDD8 conjugating E2 enzyme ({Delta}Ubc12). The western blot of complemented strains and mass spectrometry of PfNEDD8 immunoprecipitate showed conjugation of PfNEDD8 to S. cerevisiae cullin cdc53, demonstrating functional conservation and cullins as the physiological substrates of PfNEDD8. The characterization of PfNEDD8 and identification of cullins as its substrates make ground for investigation of specific roles and drug target potential of neddylation pathway in malaria parasites.

biochemistry↗