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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: The 14‐3‐3 proteins have an overall inhibitory effect on cell cycle progression and apoptosis, whereas in signal transduction they may act as stimulatory or inhibitory factors.
Abstract: The 14-3-3 proteins constitute a family of conserved proteins present in all eukaryotic organisms so far investigated. These proteins have attracted interest because they are involved in important cellular processes such as signal transduction, cell-cycle control, apoptosis, stress response and malignant transformation and because at least 100 different binding partners for the 14-3-3 proteins have been reported. Although the exact function of 14-3-3 proteins is still unknown, they are known to (1) act as adaptor molecules stimulating protein-protein interactions, (2) regulate the subcellular localisation of proteins and (3) activate or inhibit enzymes. In this review, we discuss the role of the 14-3-3 proteins in three cellular processes: cell cycle control, signal transduction and apoptosis. These processes are regulated by the 14-3-3 proteins at multiple steps. The 14-3-3 proteins have an overall inhibitory effect on cell cycle progression and apoptosis, whereas in signal transduction they may act as stimulatory or inhibitory factors. This article contains supplementary material which may be viewed at the BioEssays website at http://www.interscience.wiley.com/jpages/0265-9247/Suppmat/23/v23_10.936.

528 citations

Journal ArticleDOI
TL;DR: This work indicates that the cellular complement of 14-3-3 proteins may integrate the specificity and strength of signalling through to different cellular responses, and linking specific 14- 3-3 isoforms to genetic disorders and cancers, and the cellular effects of 13.3-2 agonists and antagonists, indicate that this role is important.
Abstract: 14-3-3 proteins exert an extraordinarily widespread influence on cellular processes in all eukaryotes. They operate by binding to specific phosphorylated sites on diverse target proteins, thereby forcing conformational changes or influencing interactions between their targets and other molecules. In these ways, 14-3-3s ‘finish the job’ when phosphorylation alone lacks the power to drive changes in the activities of intracellular proteins. By interacting dynamically with phosphorylated proteins, 14-3-3s often trigger events that promote cell survival – in situations from preventing metabolic imbalances caused by sudden darkness in leaves to mammalian cell-survival responses to growth factors. Recent work linking specific 14-3-3 isoforms to genetic disorders and cancers, and the cellular effects of 14-3-3 agonists and antagonists, indicate that the cellular complement of 14-3-3 proteins may integrate the specificity and strength of signalling through to different cellular responses.

527 citations

Journal ArticleDOI
TL;DR: These structures reveal an evolutionarily conserved network of peptide-protein interactions within all 14-3-3 isotypes, explain both binding motifs, and identify a novel intrachain phosphorylation-mediated loop structure in one of the peptides.

518 citations

Journal ArticleDOI
TL;DR: A family of proteins known as 14-3-3 is currently receiving increased attention by investigators studying a broad range of biological systems, including plants and invertebrates, and current thinking indicates that these proteins may function as regulators in signal transduction/phosphorylation mechanisms.

481 citations

Journal ArticleDOI
02 Jul 1998-Nature
TL;DR: It is proposed that dimeric 14-3-3 is needed both to maintain Raf in an inactive state in the absence of GTP-bound Ras and to stabilize an active conformation of Raf produced during activation in vivo.
Abstract: cRaf-1 is a mitogen-activated protein kinase that is the main effector recruited by GTP-bound Ras in order to activate the MAP kinase pathway. Inactive Raf is found in the cytosol in a complex with Hsp90, Hsp50 (Cdc37) and the 14-3-3 proteins. GTP-bound Ras binds Raf and is necessary but not sufficient for the stable activation of Raf that occurs in response to serum, epidermal growth factor, platelet-derived growth factor or insulin. These agents cause a two- to threefold increase in overall phosphorylation of Raf on serine/threonine residues, and treatment of cRaf-1 with protein (serine/threonine) phosphatases can deactivate it, at least partially. The role of 14-3-3 proteins in the regulation of Raf's kinase activity is uncertain and is investigated here. Active Raf can be almost completely deactivated in vitro by displacement of 14-3-3 using synthetic phosphopeptides. Deactivation can be substantially reversed by addition of purified recombinant bacterial 14-3-3; however, Raf must have been previously activated in vivo to be reactivated by 14-3-3 in vitro. The ability of 14-3-3 to support Raf activity is dependent on phosphorylation of serine residues on Raf and on the integrity of the 14-3-3 dimer; mutant monomeric forms of 14-3-3, although able to bind Raf in vivo, do not enable Raf to be activated in vivo or restore Raf activity after displacement of 14-3-3 in vitro. The 14-3-3 protein is not required to induce dimerization of Raf. We propose that dimeric 14-3-3 is needed both to maintain Raf in an inactive state in the absence of GTP-bound Ras and to stabilize an active conformation of Raf produced during activation in vivo.

472 citations