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Journal ArticleDOI

Molecular dynamics simulations and biochemical characterization of Pf14-3-3 and PfCDPK1 interaction towards its role in growth of human malaria parasite

26 Jun 2020-Biochemical Journal (Portland Press)-Vol. 477, Iss: 12, pp 2153-2177
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.
Citations
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Journal ArticleDOI
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

Journal ArticleDOI
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

Journal ArticleDOI
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

Journal ArticleDOI
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

Journal ArticleDOI
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

References
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Journal ArticleDOI
TL;DR: It is suggested that isoforms of the 14-3-3 family may play an important physiological role as inhibitors of PKC activity in human platelets but are unlikely to be involved in controlling association ofPKC with the membrane.
Abstract: The 14-3-3 proteins inhibit protein kinase C (PKC) activity in vitro and contain conserved sequences that resemble the pseudosubstrate domain of PKC and the C-terminus of the annexins. In the present study we have identified the isoforms of 14-3-3 in human platelets and used synthetic peptides derived from the regions with similarity to PKC and annexins to examine the potential role of 14-3-3 in regulating platelet activity. Immunoblotting studies with isoform-specific antisera raised against the acetylated peptides corresponding to the N-termini of 14-3-3 showed that these cells express high levels of the beta, gamma and zeta 14-3-3 isoforms. In addition, low levels of the epsilon and eta 14-3-3 isoforms were detected. In washed, saponin-permeabilized platelets incubated with [gamma-32P]ATP, thrombin- and phorbol 12-myristate 13-acetate (PMA)-induced phosphorylation of several proteins (66, 45, and 20kDa) was inhibited by preincubation with AS peptide (KNVVGARRSSWRVISSIEQK) based on the pseudosubstrate-like region of the 14-3-3 family. A control peptide of similar size had no effect on PKC-mediated phosphorylation. PMA caused a rapid translocation of PKC activity from the cytosol to the particulate fraction of saponin-permeabilized platelets that was unaffected by either the AS peptide or a peptide derived from the annexin-like 14-3-3 domain (MKGDYYRYLAEVATGDD). These results suggest that isoforms of the 14-3-3 family may play an important physiological role as inhibitors of PKC activity in human platelets but are unlikely to be involved in controlling association of PKC with the membrane.

31 citations

Journal ArticleDOI
TL;DR: It is shown here that Mtb enolase is a surface exposed plasminogen binding protein which upon immunization confers significant protection against Mtb challenge.

31 citations

Journal ArticleDOI
TL;DR: The fungal phytotoxin fusicoccin (FC) is shown to be able to stabilize 14‐3‐3 interaction to several client proteins with a mode III binding motif, and is proposed as a promising tool to control cellular processes regulated by 14‐ 3‐3 proteins.
Abstract: Modulation of the interaction of regulatory 14-3-3 proteins to their physiological partners through small cell-permeant molecules is a promising strategy to control cellular processes where 14-3-3s are engaged. Here, we show that the fungal phytotoxin fusicoccin (FC), known to stabilize 14-3-3 association to the plant plasma membrane H(+) -ATPase, is able to stabilize 14-3-3 interaction to several client proteins with a mode III binding motif. Isothermal titration calorimetry analysis of the interaction between 14-3-3s and different peptides reproducing a mode III binding site demonstrated the FC ability to stimulate 14-3-3 the association. Moreover, molecular docking studies provided the structural rationale for the differential FC effect, which exclusively depends on the biochemical properties of the residue in peptide C-terminal position. Our study proposes FC as a promising tool to control cellular processes regulated by 14-3-3 proteins, opening new perspectives on its potential pharmacological applications.

30 citations

Journal ArticleDOI
TL;DR: Northern blot analyses show that the Pk14-3-3 transcript in asexual blood stages begins to be expressed in the ring-stage, predominates in young trophozoites, and thereafter declines, while protein expression in P. knowlesi closely mimics the pattern of the transcript.

28 citations

Journal ArticleDOI
TL;DR: It is demonstrated that drugs with distinct binding modes use different mechanism to interfere with DNA condensation, with EtBr intercalation challenging the distortion of the DNA helix and separation of DNA base pairs by protamine.
Abstract: DNA condensation is a ubiquitous phenomenon in biology, yet the physical basis for it has remained elusive. Here, we have explored the mechanism of DNA condensation through the protamine-DNA interaction, and by examining on it the influence of DNA binding drugs. We observed that the DNA condensation is accompanied by B to Ψ-DNA transition as a result of DNA base pair distortions due to protamine binding, bringing about the formation of toroidal structure through coil-globule transition. The binding energetics suggested that electrostatic energy, bending energy and hydration energy must play crucial roles in DNA condensation. EtBr intercalation interferes with the protamine-DNA interaction, challenging the distortion of the DNA helix and separation of DNA base pairs by protamine. Thus, EtBr, by competing directly with protamine, resists the phenomenon of DNA condensation. On the contrary, netropsin impedes the DNA condensation by an allosteric mechanism, by resisting the probable DNA major groove bending by protamine. In summary, we demonstrate that drugs with distinct binding modes use different mechanism to interfere with DNA condensation.

27 citations