Molecular dynamics simulations and biochemical characterization of Pf14-3-3 and PfCDPK1 interaction towards its role in growth of human malaria parasite
TL;DR: Overall, this study characterizes 14-3-3I as a scaffold protein in the malaria parasite and unveils CDPK1 as its previously unidentified target, setting a precedent for the rational design of 14- 3-3 based PPI inhibitors by utilizing 14-2-3 recognition motif peptides, as a potential antimalarial strategy.
Abstract: Scaffold proteins play pivotal role as modulators of cellular processes by operating as multipurpose conformation clamps. 14-3-3 proteins are gold-standard scaffold modules that recognize phosphoSer/Thr (pS/pT) containing conserved motifs, and confer conformational changes leading to modulation of functional parameters of their target proteins. Modulation in functional activity of kinases has been attributed to their interaction with 14-3-3 proteins. Herein, we have annotated and characterized PF3D7_0818200 as 14-3-3 isoform I in Plasmodium falciparum 3D7, and its interaction with one of the key kinases of the parasite, Calcium-Dependent Protein Kinase 1 (CDPK1) by performing various analytical biochemistry and biophysical assays. Molecular dynamics simulation studies indicated that CDPK1 polypeptide sequence (61KLGpS64) behaves as canonical Mode I-type (RXXpS/pT) consensus 14-3-3 binding motif, mediating the interaction. The 14-3-3I/CDPK1 interaction was validated in vitro with ELISA and SPR, which confirmed that the interaction is phosphorylation dependent, with binding affinity constant of 670 ± 3.6 nM. The interaction of 14-3-3I with CDPK1 was validated with well characterized optimal 14-3-3 recognition motifs: Mode I-type ARSHpSYPA and Mode II-type RLYHpSLPA, by simulation studies and ITC. This interaction was found to marginally enhance CDPK1 functional activity. Furthermore, interaction antagonizing peptidomimetics showed growth inhibitory impact on the parasite indicating crucial physiological role of 14-3-3/CDPK1 interaction. Overall, this study characterizes 14-3-3I as a scaffold protein in the malaria parasite and unveils CDPK1 as its previously unidentified target. This sets a precedent for the rational design of 14-3-3 based PPI inhibitors by utilizing 14-3-3 recognition motif peptides, as a potential antimalarial strategy.
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TL;DR: In this paper, an overview of the updated kinome of Plasmodium falciparum, the species that is the largest contributor to malaria mortality, and current knowledge pertaining to the function of parasite-encoded protein kinases during the parasite's life cycle is presented.
13 citations
TL;DR: In this paper, a set of eight proteins with a remarkable resemblance to human proteins were found to be moonlighting proteins carried by Plasmodium falciparum molecules carried by extracellular vesicles.
Abstract: Red blood cells infected with Plasmodium falciparum secrete extracellular vesicles in order to facilitate the survival and infection of human cells. Various researchers have studied the composition of these extracellular vesicles and identified the proteins contained inside. In this work, we used that information to detect potential P. falciparum molecules that could be imitating host proteins. We carried out several searches to detect sequences and structural similarities between the parasite and host. Additionally, the possibility of functional mimicry was explored in line with the potential role that each candidate can perform for the parasite inside the host. Lastly, we determined a set of eight sequences (mainly moonlighting proteins) with a remarkable resemblance to human proteins. Due to the resemblance observed, this study proposes the possibility that certain P. falciparum molecules carried by extracellular vesicles could be imitating human proteins to manipulate the host cell's physiology.
3 citations
TL;DR: In this paper , phthalimide analogs possessing the bioactive scaffolds, benzimidazole and 1,2,3-triazole, were evaluated for in vitro and in vivo anti-plasmodial activity without any apparent hemolysis, or cytotoxicity.
Abstract: Constant emergence of drug-resistant Plasmodium falciparum warrants urgent need for effective and inexpensive drugs. Herein, phthalimide (Pht) analogs possessing the bioactive scaffolds, benzimidazole and 1,2,3-triazole, were evaluated for in vitro and in vivo anti-plasmodial activity without any apparent hemolysis, or cytotoxicity. Analogs 4(a-e) inhibited the growth of 3D7 and RKL-9 strains at submicromolar concentrations. Defects were observed during parasite egress from or invasion of the red blood cells. Mitochondrial membrane depolarization was measured as one of the causes of cell death. Phts 4(a-e) in combination with artemisinin exhibited two-to three-fold increased efficacy. Biophysical and biochemical analysis suggest that Pht analogs mediate plasmodial growth inhibition by interacting with tubulin protein of the parasite. Lastly, Phts 4(a-e) significantly decreased parasitemia and extended host survival in murine model Plasmodium berghei ANKA infection. Combined, the data indicate that Pht analogs should be further explored, which could offer novel value to the antimalarial drug development pipeline.
3 citations
TL;DR: ST72 with CQ resulted in improved growth inhibitory activity than individual drugs in both in vitro and in vivo studies and did not show any significant hemolysis or cytotoxicity against human HepG2 cells suggesting a good safety profile.
Abstract: We employed a comprehensive approach of target-based virtual high-throughput screening to find potential hits from the ZINC database of natural compounds against cysteine proteases falcipain-2 and falcipain-3 (FP2 and FP3). Molecular docking studies showed the initial hits showing high binding affinity and specificity toward FP2 were selected. Furthermore, the enzyme inhibition and surface plasmon resonance assays were performed which resulted in a compound ZINC12900664 (ST72) with potent inhibitory effects on purified FP2. ST72 exhibited strong growth inhibition of chloroquine-sensitive (3D7; EC50 = 2.8 µM) and chloroquine-resistant (RKL-9; EC50 = 6.7 µM) strains of Plasmodium falciparum. Stage-specific inhibition assays revealed a delayed and growth defect during parasite growth and development in parasites treated with ST72. Furthermore, ST72 significantly reduced parasite load and increased host survival in a murine model infected with Plasmodium berghei ANKA. No Evans blue staining in ST72 treatment indicated that ST72 mediated protection of blood–brain barrier integrity in mice infected with P. berghei. ST72 did not show any significant hemolysis or cytotoxicity against human HepG2 cells suggesting a good safety profile. Importantly, ST72 with CQ resulted in improved growth inhibitory activity than individual drugs in both in vitro and in vivo studies.
2 citations
TL;DR: In this article, the role of signaling pathways in regulation of the key processes of merozoite egress and red blood cell invasion by Plasmodium falciparum and, in particular, the importance of the second messengers, cAMP and Ca2+, and cyclic nucleotide dependent kinases.
Abstract: We review the role of signaling pathways in regulation of the key processes of merozoite egress and red blood cell invasion by Plasmodium falciparum and, in particular, the importance of the second messengers, cAMP and Ca2+, and cyclic nucleotide dependent kinases. cAMP-dependent protein kinase (PKA) is comprised of cAMP-binding regulatory, and catalytic subunits. The less well conserved cAMP-binding pockets should make cAMP analogs attractive drug leads, but this approach is compromised by the poor membrane permeability of cyclic nucleotides. We discuss how the conserved nature of ATP-binding pockets makes ATP analogs inherently prone to off-target effects and how ATP analogs and genetic manipulation can be useful research tools to examine this. We suggest that targeting PKA interaction partners as well as substrates, or developing inhibitors based on PKA interaction sites or phosphorylation sites in PKA substrates, may provide viable alternative approaches for the development of anti-malarial drugs. Proximity of PKA to a substrate is necessary for substrate phosphorylation, but the P. falciparum genome encodes few recognizable A-kinase anchor proteins (AKAPs), suggesting the importance of PKA-regulatory subunit myristylation and membrane association in determining substrate preference. We also discuss how Pf14-3-3 assembles a phosphorylation-dependent signaling complex that includes PKA and calcium dependent protein kinase 1 (CDPK1) and how this complex may be critical for merozoite invasion, and a target to block parasite growth. We compare altered phosphorylation levels in intracellular and egressed merozoites to identify potential PKA substrates. Finally, as host PKA may have a critical role in supporting intracellular parasite development, we discuss its role at other stages of the life cycle, as well as in other apicomplexan infections. Throughout our review we propose possible new directions for the therapeutic exploitation of cAMP-PKA-signaling in malaria and other diseases caused by apicomplexan parasites.
2 citations
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TL;DR: The co-crystal structure of β-clamp with peptide from DNA ligase (not reported earlier in prokaryotes) is determined, revealing the region from ligase that interacts with β-Clamp.
Abstract: Helicobacter pylori, a gram-negative and microaerophilic bacterium, is the major cause of chronic gastritis, gastric ulcers and gastric cancer. Owing to its central role, DNA replication machinery has emerged as a prime target for the development of antimicrobial drugs. Here, we report 2A structure of β-clamp from H. pylori (Hpβ-clamp), which is one of the critical components of DNA polymerase III. Despite of similarity in the overall fold of eubacterial β-clamp structures, some distinct features in DNA interacting loops exists that have not been reported previously. The in silico prediction identified the potential binders of β-clamp such as alpha subunit of DNA pol III and DNA ligase with identification of β-clamp binding regions in them and validated by SPR studies. Hpβ-clamp interacts with DNA ligase in micromolar binding affinity. Moreover, we have successfully determined the co-crystal structure of β-clamp with peptide from DNA ligase (not reported earlier in prokaryotes) revealing the region from ligase that interacts with β-clamp.
15 citations
TL;DR: It is observed that AMP-activated protein kinase–mediated phosphorylation at Ser-99 promotes TET2 stability and increases global DNA 5-hydroxymethylcytosine levels and that protein phosphatase 2A can interact with Tet2 and dephosphorylate Ser- 99.
15 citations
TL;DR: It is experimentally demonstrated that the transcript for PfMA was expressed only in the late blood stages of parasite consistent with a putative role in erythrocyte invasion, and the functional role of the PfMA protein was identified as a parasite adhesin involved in direct attachment with the target ery Throcyte.
Abstract: Malaria remains a major health problem worldwide. All clinical symptoms of malaria are attributed to the asexual blood stages of the parasite life cycle. Proteins resident in apical organelles and present on the surface of P. falciparum merozoites are considered promising candidates for the development of blood stage malaria vaccines. In the present study, we have identified and characterized a microneme associated antigen, PfMA [PlasmoDB Gene ID: PF3D7_0316000, PFC0700c]. The gene was selected by applying a set of screening criteria such as transcriptional upregulation at late schizogony, inter-species conservation and the presence of signal sequence or transmembrane domains. The gene sequence of PfMA was found to be conserved amongst various Plasmodium species. We experimentally demonstrated that the transcript for PfMA was expressed only in the late blood stages of parasite consistent with a putative role in erythrocyte invasion. PfMA was localized by immunofluorescence and immuno-electron microscopy to be in the micronemes, an apical organelle of merozoites. The functional role of the PfMA protein in erythrocyte invasion was identified as a parasite adhesin involved in direct attachment with the target erythrocyte. PfMA was demonstrated to bind erythrocytes in a sialic acid independent, chymotrypsin and trypsin resistant manner and its antibodies inhibited P. falciparum erythrocyte invasion. Invasion of erythrocytes is a complex multistep process that involves a number of redundant ligand-receptor interactions many of which still remain unknown and even uncharacterized. Our work has identified and characterized a novel P. falciparum adhesin involved in erythrocyte invasion.
12 citations
TL;DR: Quinacrine, because of its ability to mimic the HIP interactions with heparin, was shown to successfully modulate the HIP‐heparin interactions, demonstrating the feasibility of inhibiting the disease relevant heparIn‐protein interactions by a small molecule, which could be an effective strategy for the development of future therapeutic agents.
Abstract: The heparin-protein interaction plays a vital role in numerous physiological and pathological processes Not only is the binding mechanism of these interactions poorly understood, studies concerning their therapeutic targeting are also limited Here, we have studied the interaction of the heparin interacting peptide (HIP) from Tat (which plays important role in HIV infections) with heparin Isothermal titration calorimetry binding exhibits distinct biphasic isotherm with two different affinities in the HIP-heparin complex formation Overall, the binding was mainly driven by the nonionic interactions with a small contribution from ionic interactions The stoichiometric analysis suggested that the minimal site for a single HIP molecule is a chain of 4 to 5 saccharide molecules, also supported by docking studies The investigation was also focused on exploiting the possibility of using a small molecule as an inhibitor of the HIP-heparin complex Quinacrine, because of its ability to mimic the HIP interactions with heparin, was shown to successfully modulate the HIP-heparin interactions This result demonstrates the feasibility of inhibiting the disease relevant heparin-protein interactions by a small molecule, which could be an effective strategy for the development of future therapeutic agents
11 citations
TL;DR: It is implicit from CD and fluorescence results that IgG does not undergo any significant conformational changes, which further suggests that proA and proG dictate the phenomenon of recognition in antibody complexes.
5 citations