Interaction of 14-3-3I and CDPK1 mediates the growth of human malaria parasite
Summary (4 min read)
Introduction
- Scaffold proteins play pivotal role as modulators of cellular processes by operating as multipurpose conformation clamps.
- Overall, this study characterizes 14-3- 3I as a scaffold protein in the malaria parasite and unveils CDPK1 as its previously unidentified target.
- Depending on the biochemical nature of their phosphorylated protein targets, physical association with 14-3-3 proteins can have different functional consequences, resulting in modulation of its enzymatic activity, subcellular sequestration, protein stability and/or alteration of protein-protein interactions (9, 19).
- Further, the authors report that 14-3-3I interacts with a highly expressed protein in the parasite, Calcium-Dependent Protein Kinase 1 (CDPK1) that plays key role in multitude of essential cellular processes, including parasite invasion and egress during intra-erythrocytic proliferative stages of the parasite.
Results
- Sequence analysis and identification of P. falciparum 14-3-3I MultAlin-based sequence alignment of 14- 3-3 isoforms from Homo sapiens and P. falciparum 3D7 demonstrated patterns of conservation and correlation in Pf14-3-3I protein sequence in light of the well-studied orthologs in humans .
- The comparable Ramachandran plot characteristics and RMSD value confirmed the reliability of the 3D-structural model of Pf14-3-3Idimer to be taken further for docking and simulation studies.
- Further, western blot analysis of native 14-3-3I protein in the parasite lysate, by using in-house generated polyclonal mice sera raised against r14-3-3I, confirmed the existence of 14-3-3I protein in the parasite .
- Updated optimal consensus 14-3-3 binding motifs constructed from amino acid sequences of these 14-3-3 binding phosphopeptides are shown .
- To probe the associated molecular interactions that regulate the binding affinity of 14-3-3Idimer with pCDPK1, the authors performed MD simulations.
14-3-3I exhibits divergent binding affinities for CDPK1 and pCDPK1 in vitro
- For ELISA based 14-3-3/CDPK1 PPI analysis, Poly-L-Lysine coated 96-welled microtitre plate was coated with rCDPK1 (or rpCDPK1), followed by addition of increasing concentrations of the prey protein, r14-3-3I.
- The interaction analysis was done by using monoclonal antibody against GST protein.
- Interaction analysis was done by injecting serial dilutions of either rCDPK1 or rpCDPK1 ranging from 100 nM to 1 μM over the r14-3-3I-immobilized sensor chip surface, followed by comparing their respective kinetics & binding affinities at RT.
- IFA was performed on synchronized P. falciparum 3D7 culture to check for colocalization of 14-3-3I and CDPK1 by probing mature stages of the parasite with anti-Pf14-3-3I mouse-serum and anti-PfCDPK1 rabbit-serum, and images were acquired using Nikon A1-R Interaction of P. falciparum 14-3-3I and CDPK1 confocal microscope using the NIS Elements software.
MD Simulation reveals stable complex formation of 14-3-3Idimer with Phosphopeptides 1, 2
- The peptide 1 was found to bind in the amphipathic groove of the receptor protein, 14-3-3Idimer.
- The observation was supported by the higher number of intermolecular Hydrogen-bonds that are formed between 14-3-3Idimer and peptide 2 as compared to peptide 1.
- This was achieved by determining kinetic parameters of the interaction, like binding affinity constant (Ka) and change in enthalpy (ΔH), under sustained salt (150 mM NaCl) and pH (7.4) conditions, at RT.
- In case of peptide 2, as further injections continued, decline in exothermic heat resulted in sigmoidal curve ending near zero baseline.
- In Figure 3B, solid line shows the best fit of non-linear experimental data, and the model reproduces experimental data fairly well.
3I protein was enthalpically favorable (ΔH = −11.34 ± 4.27 kcal/mol), whereas entropically
- Immunoblotting with HRP-conjugated anti-His antibody indicated that phosphorylation status of CDPK1 dictates its interaction with 14-3-3I, as confirmed by ELISA .
- The same blot stripped and re-probed with HRP-conjugated antiGST antibody served as experimental control to check equal coupling of r14-3-3I (or GST) with Glutathione Sepharose beads in all binding reactions.
- Towards this, mature schizonts were allowed to invade into erythrocytes in the presence of 12.5 μM concentration of the peptides.
- Untreated parasites served as control and percentage parasite growth inhibition was measured by flow cytometry, as described in experimental procedures section.
Discussion
- 14-3-3 is a novel class of dimeric, conserved scaffold proteins that recognize phosphor-serine/threonine (pS/pT) containing conserved binding motifs in a variety of signaling proteins, thus regulating their physiological functions.
- The epsilon group of 14-3-3 proteins is considered as ancestral and destined to fulfill fundamental cellular functions.
- SPR analysis indicated that 14-3-3I has approximately two fold higher affinity towards phosphorylated CDPK1, with binding affinity constants (KD) of 1.35 µM & 0.67 µM for 14-3-3I/CDPK1 and 14-3-3I/pCDPK1 interaction, respectively .
- The peptide exhibited high affinity for different isoforms of 14-3-3 by binding to the amphipathic groove of 14-3-3 via polar and hydrophobic interactions.
- In conclusion, their findings confirm the existence of Pf14-3-3I protein in the malaria parasite P. falciparum, and present insight into its sequence and structural features which may prove to be an initial lead in understanding of its function in the parasite.
Culture of P. falciparum
- Cryopreserved P. falciparum parasites (3D7 laboratory strain) were thawed and cultured according to the protocol as described by W. Trager & JB. Jensen (1976) (51).
- Briefly, parasite cultures were maintained in O + erythrocytes at 2% hematocrit level, in RPMI 1640 medium (Gibco®, USA) supplemented with 0.5% AlbuMAX TM I (Gibco®, USA), 50 mg/L hypoxanthine, 10 mg/L gentamycin (Gibco®, USA) and 2 gm/L sodium bicarbonate.
- To identify experimentally validated 14-3- 3 interacting partners from prokaryotes & eukaryotes, and collate details of phosphoSer/Thr sites on the target proteins that have been reported to bind directly to 14-3-3 proteins, literature survey followed by mining of publically available databases was done.
- 18S rRNA was also amplified from genomic DNA (gDNA) of the parasite, as positive control.
- DNA fragment encoding full-length Pf14-3-3I protein was amplified with the following primer sets: Pf14-3- 3I_Fwd, 5′- TGCGGGATCCATGGCAACATCTGAAGAAT.
Scientific ™) at 4 ºC for 15 minutes. Bacterial
- Pellet, thus obtained, was resuspended in cell lysis buffer (buffer A) containing 10 mM HEPESNaOH, pH 7.4; 1 mM Ethylene Diamine Tetraacetic Acid (EDTA); 150 mM NaCl; 25μg/ml lysozyme; 3 mM β-MercaptoEthanol (βME), Protease Inhibitor Cocktail (PIC, Roche) and 1mM PhenylMethylSulfonyl Fluoride (PMSF), followed by sonication for 15 minutes with successive pulses of 6 seconds ON and 10 seconds OFF.
- The formulation was made by thoroughly mixing equal volumes of Freund‟s complete adjuvant and saline containing r14-3-3I protein.
- Once the anti-serum was raised, native Pf14-3-3I protein was detected in schizonts lysate by immunoblotting.
- Protein purification was done in a similar manner as the purification of r14-3-3I, except for the following changes in buffers‟ compositions.
- The slides were washed and mounted with ProLong Gold antifade reagent with DAPI (4',6- diamidino-2phenylindole) and images were acquired using Nikon A1-R confocal microscope using the NIS Elements software.
Homology modeling of Pf14-3-3I and PfCDPK1
- Three-dimensional structure of a protein can provide us with precise information about its single, most stable conformation, as dictated by its sequence.
- Comparative or homology modeling, one of the most common structure prediction methods in structural genomics and proteomics, was employed to model 3D structures of 14-3- 3Idimer and CDPK1 from P. falciparum strain 3D7.
- To search for a suitable template for homology modeling, BLASTp (https://blast.ncbi.nlm.nih.gov/Blast.cgi) search was performed by using amino acid sequences of 14-3-3I and CDPK1 as query sequences, against Protein Data Bank (PDB) database (http://www.rcsb.org/) (76, 77).
- Reliability of the refined structural models of Pf14-3-3Idimer and PfCDPK1 was assessed by examining backbone dihedral angles: phi (Ø) and psi (Ψ) of the amino acid residues lying in the energetically favourable regions of Ramachandran space (83).
- This was done by using online available tool, PROCHECK v.3.5 (https://www.ebi.ac.uk/thorntonsrv/software/PROCHECK/) (84).
ELISA to confirm 14-3-3I/CDPK1 interaction
- In all experiments, minimally & maximally phosphorylated CDPK1 protein is represented as rCDPK1 and rpCDPK1, respectively.
- Surface Plasmon Resonance analysis of binding affinity between CDPK1 and immobilized 14-3-3I rCDPK1 protein was auto-phosphorylated in an in vitro kinase reaction, as mentioned above.
- Briefly, 5 μM of recombinant 14-3-3I protein was immobilized on the surface (self-assembled monolayer of 11- Mercapto-Undecanoic Acid, MUA on gold surface) of SPR sensor chip by the mechanism of covalent amine coupling.
- Data were fit to the two-state conformational change model by using AutoLab SPR Kinetic Evaluation software provided with the instrument.
- At least three independent experiments were performed.
Isothermal Titration Calorimetric analysis
- To calculate kinetic parameters such as binding affinity constant (Ka) for interaction of 14- 3-3I with consensus 14-3-3 binding peptides 1 and 2, Isothermal Titration Calorimetry (ITC) experiments were performed by using MicroCal iTC200 (Malvern Instruments Ltd, UK; at School of Physical Sciences, Jawaharlal Nehru University, New Delhi, India).
- For this purpose, Interaction of P. falciparum 14-3-3I and CDPK1 recombinant 14-3-3I protein was dialyzed extensively against HEPES-NaCl buffer (10 mM HEPES-NaOH, pH 7.4 and 150 mM NaCl) using Amicon TM Ultra-15 Centrifugal Filter Unit (10 kDa cutoff) before its subsequent use in ITC.
- Amount of heat produced per injection (corrected data) was analyzed by integration of area under individual peaks by MicroCal ORIGIN 7 software provided by the instrument manufacturer.
- Experimental data was presented as the amount of heat produced per second (μcal/sec; corrected for heat of dilution of the ligand) following each injection of the ligand into the protein solution, as a function of time .
- After progression of the parasites to ring stage, erythrocytes were washed with 1X PBS and stained with Ethidium Bromide (EtBr, 10 μM) for 20 minutes at RT, in dark.
Statistical analysis
- In the bar graphs, data is expressed as Mean ± Standard Deviation (SD) of three independent experiments, done in duplicates.
- Statistical analysis was done by using OriginPro Evaluation 2018b Graphing and Analysis software.
- Interaction of P. falciparum 14-3-3I and CDPK1 Acknowledgements: Authors acknowledge AutoLab Esprit SPR facility of Advanced Instrumentation Research Facility (AIRF), Jawaharlal Nehru University (JNU), New Delhi, India and Central Instrumentation Facility (CIF) of SCMM, JNU for flow cytometry.
- The lab facility of Shiv Nadar University is also acknowledged.
- The authors declare that they have no conflicts of interest with the contents of this article.
FOOTNOTES
- This work has been funded by DST-EMR from the Department of Science and Technology (DST), Ministry of Science and Technology, Government of India.
- E) Localization of 14-3-3. Anti-r14-3-3I mice serum was used to probe localization of the protein in mature schizonts and free merozoites by using confocal microscopy.
- The experiment was done twice in triplicates.
- RMSD: Root Mean Square Deviation; pS: Phosphorylated serine; KD: Affinity constant; DIC: Differential Interference Contrast image.
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Frequently Asked Questions (19)
Q2. What is the role of R18 in the anticancer process?
R18 efficiently blocked the binding of 14-3-3 to Raf-1 kinase, a physiological ligand of 14-3-3, thereby effectively abolishing the protective role of 14-3-3 against phosphatase-induced inactivation of Raf-1, and virulence factor exoenzyme S (ExoS) of the pathogenic bacterium Pseudomonas aeruginosa (44).
Q3. What is the effect of the structural changes on the binding of CDPK1?
the structural changes conferred in 14-3-3Idimer upon the binding of CDPK1 might assist a stable complex formation between them.
Q4. What is the role of pCDPK1 in the binding of 14-3-3Id?
The phosphorylated serine, pS64 of pCDPK1 was found to play a key role in mediating its interaction with 14-3-3Idimer, where a Hydrogen-bond was formed between pS64 of pCDPK1 and K227 of 14-3-3Idimer with a bond length of 2.92Å.
Q5. What is the peptide that inhibits the interaction between 14-3-3 and ExoS?
The peptide exhibited high affinity for different isoforms of 14-3-3 by binding to the amphipathic groove of 14-3-3 via polar and hydrophobic interactions.
Q6. How did the authors determine the binding modes of r14-3-3i?
Phosphopeptides 1 & 2 interact with 14-3-3I in vitroAfter affirming 14-3-3Idimer interactionwith phosphopeptides 1 & 2 through molecular dynamics simulation studies, the authors further sought to establish the knowledge of binding modes of complexation between r14-3-3I and the peptides, by employing ITC.
Q7. What is the effect of GST on the rpCDPK1 binding?
inhibition of binding between rCDPK1 (orInteraction of P. falciparum 14-3-3I and CDPK1rpCDPK1) and r14-3-3I was readily observed in the presence of peptides 1 & 2.
Q8. What is the role of the 14-3-3 isoforms in the development of a?
In the concurrent year, Wendy J. Fanti et al (1994) reported that 14-3-3 isoforms bind and enhance serine/threonine kinase activity of Raf-1, a key mediator of mitogenesis and differentiation, thus promoting Raf-1 dependent oocyte maturation (24).
Q9. What is the simplest way to inhibit the binding of 14-3-3 to the receptor?
Earlier reports suggest that syntheticpeptides containing phosphorylated 14-3-3 binding motifs can efficiently inhibit the association of 14- 3-3 proteins with their interacting partners, by typically binding to the conserved amphipathic groove of 14-3-3s.
Q10. What is the divergent form of Pf14-3-3I?
Residues involved in dimerization (solid circles) and phosphopeptide binding (solid squares) are conserved in all 14-3-3 Isotypes, except Pf14-3-3II which appears to be the most divergent form.
Q11. What is the role of r143-3I in the parasite?
the authors have shown the interaction of Pf14-3-3I with CDPK1, an important kinase of the parasite involved in motility and apical organelle discharge critical for invasion process.
Q12. What is the kinetics of the interaction between 14-3-3I and pCDPK?
SPR analysis indicated that 14-3-3I has approximately two fold higher affinity towards phosphorylated CDPK1, with binding affinity constants (KD) of 1.35 µM & 0.67 µM for 14-3-3I/CDPK1 and 14-3-3I/pCDPK1 interaction, respectively (Figure 2C).
Q13. What is the phosphorylation dependent interaction of 14-3-3I with CDPK1?
In a related study by their group, the phosphorylation dependent interaction of 14-3-3I with CDPK1 was validated in merozoites, wherein, by pulling down 14-3-3I, CDPK1 was detected in a Ca 2+ dependent manner (42).
Q14. What was the amount of heat produced per second?
Experimental data was presented as the amount of heat produced per second (μcal/sec; corrected for heat of dilution of the ligand) following each injection of the ligand into the protein solution, as a function of time (minutes).
Q15. What is the effect of GST on the phosphorylation of CDPK1?
Immunoblotting with HRP-conjugated anti-His antibody indicated that phosphorylation status of CDPK1 dictates its interaction with 14-3-3I, as confirmed by ELISA (Figure 4B).
Q16. What was the percentage of parasite growth inhibition measured by flow cytometry?
Untreated parasites served as control and percentage parasite growth inhibition was measured by flow cytometry, as described in experimental procedures section.
Q17. What is the kinetics of the interaction between r14-3-3I and CDPK?
14-3-3I protein was found to co-localize very nicely with CDPK1 protein towards cell periphery in mature schizonts and free merozoites.
Q18. What is the phylogenetic analysis of Pf14-3-3I?
their phylogenetic analysis shows that despite having evolved separately since the early eukaryotes, Pf14-3-3I protein has followed convergent evolution with plant nonepsilon group, as depicted in the phylogenetic model (Figure S2).
Q19. What is the peptide for preventing the interaction of Pf14-3-3I?
This study would be useful for designing target specific 14-3-3 recognition motif peptides to block interaction of Pf14-3-3I with its cognate proteins in the parasite, and develop as potential antimalarial strategy.