Small Molecule Screen for Candidate Antimalarials Targeting Plasmodium Kinesin-5

Liu, Liqiong; Richard, Jessica; Kim, Sun‐Young; Wojcik, Edward

doi:10.1074/jbc.m114.551408

Cited by 21 publications

(22 citation statements)

References 67 publications

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“…Although the exact molecular basis of these functions remains controversial, Kinesin-8s modulate MT dynamics and may either stabilise, destabilise, slow growth, or actively depolymerize MTs in spindle function [18,19,47]. Phylogenetic analysis showed that while there are 9 kinesin genes in Plasmodium, a number of kinesin classes, typically involved in long-distance intra-cellular transport in metazoa, such as kinesin-1 and kinesin-3, are absent [3,37]. proteins will be the topic of future studies.…”

Section: Discussionmentioning

confidence: 99%

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Plasmodium Kinesin-8X associates with mitotic spindles and is essential for oocyst development during parasite proliferation and transmission

Zeeshan

Shilliday

Liu

et al. 2019

Preprint

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Kinesin-8 proteins are microtubule motors that are often involved in regulation of mitotic spindle length and chromosome alignment. They move towards the ends of spindle microtubules and regulate the dynamics of these ends due, at least in some species, to their microtubule depolymerization activity. Plasmodium spp. exhibit an atypical endomitotic cell division in which chromosome condensation and spindle dynamics are not well understood in the different proliferative stages. Genome-wide homology analysis of Plasmodium spp. revealed the presence of two Kinesin-8 motor proteins (Kinesin-8X and Kinesin-8B). Here we have studied the biochemical properties of Kinesin-8X and its role in parasite proliferation. In vitro, Kinesin-8X showed motile and depolymerization activities like other Kinesin-8 motors. To understand its role in cell division, we have used protein tagging and live cell imaging to define the location of Plasmodium Kinesin-8X during all proliferative stages of the P berghei life cycle. Furthermore, we have used gene targeting to analyse the function of Kinesin-8X. The results reveal a spatio-temporal involvement of Kinesin-8X in spindle dynamics and its association with both mitotic and meiotic spindles and the putative microtubule organising centre (MTOC). Deletion of the Kinesin-8X gene showed that this protein is required for endomitotic division during oocyst development and is therefore necessary for parasite replication within the mosquito gut, and for transmission to the vertebrate host. Consistently, transcriptome analysis of Δkinesin-8X parasites reveals modulated expression of genes involved mainly in microtubulebased processes, chromosome organisation and the regulation of gene expression supporting a role in cell division. 4 Author SummaryKinesins are microtubule-based motors that play key roles in intracellular transport, cell division and motility. Members of the Kinesin-8 family contribute to chromosome alignment during cell division in many eukaryotes. However, the roles of kinesins in the atypical cell division in Plasmodium, the causative agent of malaria, is not known.In contrast to many other eukaryotes, Plasmodium proliferates by endomitosis, in which genome replication and division occur within a nucleus bounded by a persistent nuclear envelope. We show that the Plasmodium genome encodes only nine kinesins and we further investigate the role of Kinesin-8X throughout the Plasmodium life cycle using biochemical and gene targeting approaches. We show that Plasmodium Kinesin-8X has microtubule-based motility and depolymerization activity. We also show that Kinesin-8X is probably localized on putative MTOCs and spindles during cell division in most of the stages of P. berghei life cycle. By gene deletion we demonstrate that Kinesin-8X is essential for normal oocyst development and sporozoite formation. Genome-wide RNA analysis of Δkinesin-8X parasites reveals modulated expression of genes involved in microtubule-based processes. Overall, the data suggest that Kinesin-8X is a molecular ...

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Section: Discussionmentioning

confidence: 99%

“…Phylogenetic analyses have identified 9 kinesin genes in the Plasmodium genome [2,3,37] though not much is known in other members of the group Apicomplexa. Here 7 we have analysed a high-resolution representation of the phylogenetic distribution of kinesins in all known Apicomplexa including Plasmodium.…”

Section: Introductionmentioning

confidence: 99%

Plasmodium Kinesin-8X associates with mitotic spindles and is essential for oocyst development during parasite proliferation and transmission

Zeeshan

Shilliday

Liu

et al. 2019

Preprint

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“…Steady state kinetics of Eg5 were monitored using a NADH coupled enzymatic assay ( 46 , 71 ) in a SpectraMax M2e spectrophotometer (Molecular Devices) in 96-well plate format ( 47 ). In measurements of basal ATP hydrolysis rates in the presence of saturating 1 m m MgATP, motor protein concentration was as follows: L263F and T100C/L263F, 0.625 μ m ; T100C and Y82A, 1.5 μ m ; WT, Q78A, M115A, L263A, M115A/L263A, M115I/L263F, P137A, V298C, and P137A/V298C, 2.5 μ m ; and R138A, M115I, Q78N, Y82F, R138L, Q78A/R138A, and Y82F/T100C, 5 μ m .…”

Section: Methodsmentioning

confidence: 99%

Allostery Wiring Map for Kinesin Energy Transduction and Its Evolution

Richard

Kim

Nguyen

et al. 2016

Journal of Biological Chemistry

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How signals between the kinesin active and cytoskeletal binding sites are transmitted is an open question and an allosteric question. By extracting correlated evolutionary changes within 700+ sequences, we built a model of residues that are energetically coupled and that define molecular routes for signal transmission. Typically, these coupled residues are located at multiple distal sites and thus are predicted to form a complex, non-linear network that wires together different functional sites in the protein. Of note, our model connected the site for ATP hydrolysis with sites that ultimately utilize its free energy, such as the microtubule-binding site, drug-binding loop 5, and necklinker. To confirm the calculated energetic connectivity between non-adjacent residues, double-mutant cycle analysis was conducted with 22 kinesin mutants. There was a direct correlation between thermodynamic coupling in experiment and evolutionarily derived energetic coupling. We conclude that energy transduction is coordinated by multiple distal sites in the protein rather than only being relayed through adjacent residues. Moreover, this allosteric map forecasts how energetic orchestration gives rise to different nanomotor behaviors within the superfamily.

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“…The large family of kinesins includes molecular motors that are essential for several processes during cell division [28,29], and in Plasmodium berghei there are nine kinesin genes, including two kinesin-8 genes that are important in cell division and male gamete formation [23,30]. There is a single Plasmodium kinesin-5, and since this protein plays an important role during cell proliferation in many eukaryotes, and is considered as a strong target for the development of therapeutics against many diseases including malaria [31], we decided to study its role during cell division in the malaria parasite.…”

Section: Introductionmentioning

confidence: 99%

Plasmodium bergheikinesin-5 associates with the spindle apparatus during cell division and is important for efficient production of infectious sporozoites

Zeeshan

Brady

Stanway

et al. 2020

Preprint

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AbstractKinesin-5 motors play essential roles in spindle apparatus assembly during cell division, by generating forces to establish and maintain the spindle bipolarity essential for proper chromosome segregation. Kinesin-5 is largely conserved structurally and functionally in model eukaryotes, but its role is unknown in the Plasmodium parasite, an evolutionarily divergent organism with several atypical features of both mitotic and meiotic cell division. We have investigated the function and subcellular location of kinesin-5 during cell division throughout the Plasmodium berghei life cycle. Deletion of kinesin-5 had little visible effect at any proliferative stage. The only significant effect was a decrease in the number of motile sporozoites in mosquito salivary glands, which were able to infect a new vertebrate host. Live-cell imaging showed kinesin-5-GFP located on the spindle and at spindle poles during both atypical mitosis and meiosis. Fixed-cell immunofluorescence assays revealed kinesin-5 co-localized with α-tubulin and centrin-2 and exhibited a partial overlap with kinetochore marker NDC80 during early blood stage schizogony. Dual-colour live-cell imaging during male gametogony showed that kinesin-5 is closely associated with NDC80, but not with kinesin-8B, a marker of the basal body and axonemes of the forming flagella. Treatment of gametocytes with microtubule-specific inhibitors confirmed kinesin-5 association with nuclear spindles and not cytoplasmic axonemal microtubules. Altogether, our results demonstrate that kinesin-5 is associated with the spindle apparatus and expressed in proliferating parasite stages, but it is not essential for parasite survival.ImportanceKinesin-5 is a motor protein with important roles during cell proliferation and therefore considered a strong target for therapeutics development against many diseases. The role of kinesin-5 in Plasmodium is unknown, but in this organism there are atypical aspects of cell division that differ from those of model eukaryotes. For example, classical regulators such as polo-like kinase, classical cyclins and cyclin dependent kinases are absent from Plasmodium. Here, we show that in Plasmodium, unlike in most model eukaryotes, kinesin-5 is dispensable, suggesting that the parasite uses somewhat different machinery for cell proliferation. The study is important because it shows that kinesin-5 is not essential universally for cell division and suggests that there may be other non-canonical mechanisms and regulators. The general concept that kinesin-5 is essential for spindle pole formation and chromosome segregation during cell division is not true for Plasmodium, and this is of interest to a broad readership.

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Small Molecule Screen for Candidate Antimalarials Targeting Plasmodium Kinesin-5

Cited by 21 publications

References 67 publications

Plasmodium Kinesin-8X associates with mitotic spindles and is essential for oocyst development during parasite proliferation and transmission

Plasmodium Kinesin-8X associates with mitotic spindles and is essential for oocyst development during parasite proliferation and transmission

Allostery Wiring Map for Kinesin Energy Transduction and Its Evolution

Plasmodium bergheikinesin-5 associates with the spindle apparatus during cell division and is important for efficient production of infectious sporozoites

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