Biology subjects

Sijwali, P. S.

Publications and source records attributed to Sijwali, P. S..

5 recordsLinked to original sources

bioRxiv2024

The apicomplexan DedA superfamily protein VMP1 is crucial for the biogenesis and function of secretory organelles

Apicomplexan parasites, including Plasmodium falciparum and Toxoplasma gondii, contain specialized secretory organelles such as micronemes, rhoptries, and dense granules, which are essential for parasite motility, host cell invasion, development, and egress. DedA superfamily proteins are implicated in lipid mobilization, which is a key requirement for organelle biogenesis. Herein, we identified and investigated the vacuole membrane protein 1 (VMP1), a DedA superfamily member, of P. falciparum (PfVMP1) and T. gondii (TgVMP1). PfVMP1 and TgVMP1 are ER-localized lipid scramblases. TgVMP1 depletion adversely affected parasite development, motility, host cell invasion, and egress. These phenotypes were consistent with impaired rhoptry and dense granule biogenesis, and decreased secretion of micronemes, rhoptries, and dense granules in TgVMP1-depleted parasites, indicating a crucial role for TgVMP1 in the biogenesis and function of these organelles. TgVMP1 depletion impaired lipid droplet homeostasis and ER organization. Restoration of the ER-localized lipid scramblase by complementing TgVMP1-depleted parasites with PfVMP1 or a homolog as distant as human VMP1 rescued the depleted parasites, indicating their functional conservation and a crucial role for ER-resident lipid scramblase activity in the biogenesis and function of secretory organelles. The essentiality of TgVMP1 for parasite development and likely functional conservation of apicomplexan VMP1 proteins highlight their drug-target potential.

cell biology↗

bioRxiv2023

Plasmodium falciparum contains functional SCF and CRL4 ubiquitin E3 ligases, and CRL4 is critical for cell division and membrane integrity

Protein ubiquitination is essential for cellular homeostasis and regulation of several processes, including cell division and genome integrity. Ubiquitin E3 ligases determine substrate specificity for ubiquitination, and Cullin-RING ubiquitin E3 Ligases (CRLs) make the largest group among the ubiquitin E3 ligases. Although conserved and most studied in model eukaryotes, CRLs remain underappreciated in Plasmodium and related parasites. To investigate the CRLs of human malaria parasite Plasmodium falciparum, we generated parasites expressing tagged P. falciparum cullin-1 (PfCullin-1), cullin-2 (PfCullin-2), Rbx1 (PfRbx1) and Skp1 (PfSkp1). PfCullin-1 and PfCullin-2 were predominantly expressed in erythrocytic trophozoite and schizont stages, with nucleocytoplasmic localization and chromatin association, suggesting their roles in different cellular compartments and DNA-associated processes. Immunoprecipitation, in vitro protein-protein interaction and ubiquitination assay confirmed the presence of a functional SCF (PfSCF), comprising of PfCullin-1, PfRbx1, PfSkp1, PfFBXO1 and calcyclin binding protein. Immunoprecipitation, sequence analysis and ubiquitination assay indicated that PfCullin-2 forms a functional human CRL4-like complex (PfCRL4), consisting of PfRbx1, cleavage and polyadenylation specific factor subunit_A and WD40 repeat proteins. PfCullin-2 knock-down at the protein level, which would hinder PfCRL4 assembly, significantly decreased asexual and sexual erythrocytic stage development. Several pathways, including protein translation and folding, lipid biosynthesis and transport, DNA replication, and protein degradation were dysregulated upon PfCullin-2-depletion, which likely reflects association of PfCRL4 with multiple pathways. Consistent with dysregulation of multiple pathways, PfCullin-2-depleted schizonts had poorly delimited merozoites and internal membraned structures, suggesting a role of PfCRL4 in maintaining membrane integrity. PfCullin-2-depleted parasites had significantly lower number of nuclei/parasite than the normal parasites, indicating a crucial role of PfCRL4 in cell division. Taken together, we for the first time demonstrate the presence of functional CRLs in P. falciparum, with crucial roles for PfCRL4 in cell division and maintaining membrane integrity. This study will benefit investigation of similar ligases in related parasites.

microbiology↗

bioRxiv2021

Plasmodium DDI1 is an essential chromatin-associated protein with a role in DNA-protein crosslink repair

DDI1 proteins are involved in a variety of cellular processes, including proteasomal degradation of specific proteins. All DDI1 proteins contain a ubiquitin-like (UBL) domain and a retroviral aspartyl protease (RVP) domain. Some DDI1 proteins also contain a ubiquitin-associated (UBA) domain. The three domains confer distinct activities to DDI1 proteins. The presence of RVP domain makes DDI1 a potential target of HIV protease inhibitors, which also block the development of malaria parasites. Hence, we investigated the DDI1 of malaria parasites to identify its roles during parasite development and potential as a therapeutic target. DDI1 proteins of Plasmodium and other Apicomplexan parasites share the UBL-RVP domain architecture, and some also contain the UBA domain. Plasmodium DDI1 is expressed across all the major life cycle stages and is important for parasite survival, as conditional depletion of DDI1 protein in the mouse malaria parasite Plasmodium berghei and the human malaria parasite Plasmodium falciparum compromised parasite development. Infection of mice with DDI1 knock-down P. berghei was self-limiting and protected the recovered mice from subsequent infection with homologous as well as heterologous parasites, indicating potential of DDI1 knock-down parasites as a whole organism vaccine. P. falciparum DDI1 (PfDDI1) is associated with chromatin and DNA-protein crosslinks. PfDDI1-depleted parasites accumulated DNA-protein crosslinks and showed enhanced susceptibility to DNA damaging chemicals, indicating a role of PfDDI1 in removal of DNA-protein crosslinks. Knock-down of PfDDI1 increased susceptibility to the retroviral protease inhibitor lopinavir and antimalarial artemisinin, which suggests that simultaneous inhibition of DDI1 could potentiate antimalarial activity of these drugs. As DDI1 knock-down parasites confer protective immunity and it could be a target of HIV protease inhibitors, Plasmodium DDI1 is a potential therapeutic target for malaria control.

cell biology↗

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↗