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1.
DNA Cell Biol ; 2024 Jun 17.
Article in English | MEDLINE | ID: mdl-38885136

ABSTRACT

Plasmodium parasites, the causative agents of malaria, rely on sophisticated cellular mechanisms to survive and proliferate within their hosts. Plasmodium complex life cycle requires posttranslational modifications (PTMs) to control cellular activities. Neddylation is a type of PTM in which NEDD8 is covalently attached to target proteins and plays an important role in cell cycle control and metabolism. Covalent attachment to its substrates requires the Nedd8-activating enzyme, E1; the NEDD8-conjugating enzyme, E2; and the ligase, E3. In Plasmodium, protein neddylation is essential for parasite development during the stage I-II transition from zygote to ookinete differentiation and malaria transmission. Here, we discuss the current understanding of protein neddylation in Plasmodium, which is involved in malaria transmission.

2.
mBio ; 15(4): e0023224, 2024 Apr 10.
Article in English | MEDLINE | ID: mdl-38411954

ABSTRACT

Neddylation is a type of posttranslational modification known to regulate a wide range of cellular processes by covalently conjugating the ubiquitin-like protein Nedd8 to target proteins at lysine residues. However, the role of neddylation in malaria parasites has not been determined. Here, for the first time, we showed that neddylation plays an essential role in malaria transmission in Plasmodium berghei. We found that disruption of Nedd8 did not affect blood-stage propagation, gametocyte development, gamete formation, or zygote formation while abolishing the formation of ookinetes and further transmission of the parasites in mosquitoes. These phenotypic defects in Nedd8 knockout parasites were complemented by reintroducing the gene that restored mosquito transmission to wild-type levels. Our data establish the role of P. berghei Nedd8 in malaria parasite transmission.IMPORTANCENeddylation is a process by which Nedd8 is covalently attached to target proteins through three-step enzymatic cascades. The attachment of Nedd8 residues results in a range of diverse functions, such as cell cycle regulation, metabolism, immunity, and tumorigenesis. The potential neddylation substrates are cullin (CUL) family members, which are implicated in controlling the cell cycle. Cullin neddylation leads to the activation of cullin-RING ubiquitin ligases, which regulate a myriad of biological processes through target-specific ubiquitylation. Neddylation possibly regulates meiosis in zygotes, which subsequently develop into ookinetes. Our findings point to an essential function of this neddylation pathway and highlight its possible importance in designing novel intervention strategies.


Subject(s)
Plasmodium berghei , Ubiquitins , Animals , Ubiquitins/genetics , Ubiquitins/metabolism , Plasmodium berghei/genetics , Plasmodium berghei/metabolism , Cullin Proteins/metabolism , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/metabolism
3.
Microbiol Res ; 260: 127051, 2022 Jul.
Article in English | MEDLINE | ID: mdl-35490588

ABSTRACT

Protein kinases uniquely expressed in Plasmodium represent attractive drug targets. Previous studies have reported that Plasmodium falciparum Protein kinase 9 (Pk9) phosphorylates regulatory serine 106 of the ubiquitin-conjugating enzyme (Ubc13) thereby negatively regulating its activity. We investigated the effect of Pk9 depletion and Ubc13 mutation at S106 on the progression of rodent malaria model P. berghei life cycle. Our studies demonstrate that while phosphorylation of the regulatory serine 106 of Ubc13 is essential in blood stages, the lack of Pk9 expression neither altered Ubc13 phosphorylation nor parasite viability at all life cycle stages, though Ubc13 and Pk9 showed co-localization in the cytosol of erythrocytic and liver stages. Further, phosphorylation of Ubc13 in the absence of Pk9 reiterated the redundancy of its regulation in P. berghei. These results highlight the indispensable role of Ubc13 in P. berghei life cycle and redundancy in its phosphorylation by protein kinase and reiterate the need to validate novel gene function through genetic approaches for drug development strategies.


Subject(s)
Plasmodium berghei , Protein Kinases , Animals , Life Cycle Stages , Plasmodium berghei/genetics , Plasmodium falciparum/genetics , Plasmodium falciparum/metabolism , Protein Kinases/metabolism , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Serine
4.
ACS Infect Dis ; 8(6): 1116-1123, 2022 06 10.
Article in English | MEDLINE | ID: mdl-35594144

ABSTRACT

C-Mannosylation of the thrombospondin type I repeat (TSR) domains is one of the most important factors involved in their function. It occurs on the first tryptophan of the WXXWXXC conserved motif where the tryptophan is usually surrounded by arginine or lysine forming the ligand-binding stretch of this sticky domain. It is found in its canonical or modified forms in many Plasmodium proteins. TSR containing proteins such as thrombospondin-like anonymous protein (TRAP), circumsporozoite protein (CSP), CSP and TRAP related protein (CTRP), and secreted protein with altered thrombospondin repeat (SPATR) have all been shown to be important for various parasite processes and life cycle stages. Here, we show that C-mannosylation catalyzing enzyme C-mannosyltransferase (CmanT) plays an essential role in malaria transmission in Plasmodium berghei. Disruption of the CmanT does not affect asexual blood stage propagation or gametocyte development but abolishes the formation of oocysts in mosquitoes. CmanT knockout (CmanT-) parasites showed normal ookinete formation; however, these ookinetes failed in their ability to glide. CmanT- was complemented by reintroducing the gene, restoring mosquito transmission to wild-type level. We also investigated the effect of C-mannosylation on the folding and heparin-binding capacity of the Plasmodium falciparum TRAP TSR domain in silico, which suggested that this phenotype should be due to its involvement in the global stabilization of TSR residue side chain interactions.


Subject(s)
Culicidae , Malaria , Animals , Malaria/parasitology , Mannosyltransferases/genetics , Plasmodium berghei/genetics , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Thrombospondins/genetics , Thrombospondins/metabolism , Tryptophan
5.
Mol Microbiol ; 113(2): 478-491, 2020 02.
Article in English | MEDLINE | ID: mdl-31755154

ABSTRACT

Upon entering its mammalian host, the malaria parasite productively invades two distinct cell types, that is, hepatocytes and erythrocytes during which several adhesins/invasins are thought to be involved. Many surface-located proteins containing thrombospondin Type I repeat (TSR) which help establish host-parasite molecular crosstalk have been shown to be essential for mammalian infection. Previous reports indicated that antibodies produced against Plasmodium falciparum secreted protein with altered thrombospondin repeat (SPATR) block hepatocyte invasion by sporozoites but no genetic evidence of its contribution to invasion has been reported. After failing to generate Spatr knockout in Plasmodium berghei blood stages, a conditional mutagenesis system was employed. Here, we show that SPATR plays an essential role during parasite's blood stages. Mutant salivary gland sporozoites exhibit normal motility, hepatocyte invasion, liver stage development and rupture of the parasitophorous vacuole membrane resulting in merosome formation. But these mutant hepatic merozoites failed to establish a blood stage infection in vivo. We provide direct evidence that SPATR is not required for hepatocyte invasion but plays an essential role during the blood stages of P. berghei.


Subject(s)
Plasmodium berghei , Protozoan Proteins/metabolism , Sporozoites/metabolism , Thrombospondins/metabolism , Animals , Erythrocytes/parasitology , Gene Knockout Techniques , Hepatocytes/parasitology , Host-Parasite Interactions , Malaria/parasitology , Membrane Proteins/genetics , Membrane Proteins/metabolism , Merozoites/metabolism , Phylogeny , Plasmodium berghei/genetics , Plasmodium berghei/metabolism , Protozoan Proteins/genetics , Thrombospondins/genetics
6.
Int J Biol Macromol ; 126: 673-684, 2019 Apr 01.
Article in English | MEDLINE | ID: mdl-30599160

ABSTRACT

The inner membrane complex (IMC) is a defining feature of apicomplexans comprising of lipid and protein components involved in gliding motility and host cell invasion. Motility of Plasmodium parasites is accomplished by an actin and myosin based glideosome machinery situated between the parasite plasma membrane (PPM) and IMC. Here, we have studied in vivo expression and localization of a Plasmodium falciparum (Pf) IMC protein 'PfIMC1l' and characterized it functionally by using biochemical assays. We have identified cytoskeletal protein 'actin' and motor protein 'myosin' as novel binding partners of PfIMC1l, alongside its interaction with the lipids 'cholesterol' and 'phosphatidyl-inositol 4, 5 bisphosphate' (PIP2). While actin and myosin compete for interaction with PfIMC1l, actin and either of the lipids (cholesterol or PIP2) simultaneously bind PfIMC1l. Interestingly, PfIMC1l showed enhanced binding with actin in the presence of calcium ions, and displayed direct binding with calcium. Based on our in silico analysis and experimental data showing PfIMC1l-actin/myosin and PfIMC1l-lipid interactions, we propose that this protein may anchor the IMC membrane with the parasite gliding apparatus. Considering its binding with key proteins involved in motility viz. myosin and actin (with calcium dependence), we suggest that PfIMC1l may have a role in the locomotion of Plasmodium.


Subject(s)
Actins/metabolism , Cytoskeleton/metabolism , Membrane Lipids/metabolism , Myosins/metabolism , Plasmodium falciparum/metabolism , Protozoan Proteins/metabolism , Binding, Competitive , Calcium/metabolism , Cholesterol/metabolism , Immune Sera/metabolism , Ions , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Models, Biological , Phosphatidylinositol 4,5-Diphosphate/metabolism , Protein Binding , Protein Domains , Protozoan Proteins/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Solutions
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