Malaria and Plasmodium falciparum PKG Activation
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Questions and Answers

What was the approximate number of malaria deaths in 2015?

  • 438,000 (correct)
  • 300,000
  • 500,000
  • 600,000

    • Which organism is responsible for transmitting malaria?

  • Male Anopheles mosquito
  • Female Aedes mosquito
  • Female Anopheles mosquito (correct)
  • Female Culex mosquito

    • Which variant of malaria is considered the most dangerous?

  • Plasmodium ovale
  • Plasmodium malariae
  • Plasmodium vivax
  • Plasmodium falciparum (correct)

    • What role does cGMP play in Plasmodium?

    <p>Facilitates gametogenesis and exflagellation</p> Signup and view all the answers

    Which of the following is an effector of cGMP in Plasmodium?

    <p>Pf PKG</p> Signup and view all the answers

    What is the primary role of cAMP signaling in Plasmodium?

    <p>Controlling apical exocytosis and sporozoite motility</p> Signup and view all the answers

    Which stage of the malaria lifecycle is associated with schizogony?

    <p>Asexual blood stage</p> Signup and view all the answers

    The distribution of malaria is most directly related to which factor?

    <p>Climate and living area of the mosquito</p> Signup and view all the answers

    What was the activation constant (Kact) for the full length Pf PKG?

    <p>330 ± 20 nM</p> Signup and view all the answers

    Which mutation was made to disrupt cGMP binding in CNB-B?

    <p>R250K</p> Signup and view all the answers

    How did the specific activity of both mutant proteins compare to Pf PKG 158-853?

    <p>Reduced to 50%</p> Signup and view all the answers

    What effect did the mutation of CNB-D (R492K) have on Pf PKG activation?

    <p>Strongly influenced Pf PKG activation</p> Signup and view all the answers

    What range did the comparable affinities for cGMP of the mutant proteins display?

    <p>70 to 110 nM</p> Signup and view all the answers

    Which peptide was tested on Pf PKG kinase activity?

    <p>ERNKKKAIFSNDDF</p> Signup and view all the answers

    What are the residues that make up the capping triad of CNB-D?

    <p>R484, Q532, D533</p> Signup and view all the answers

    What method was used to assess Pf PKG kinase activity?

    <p>Spectrophotometric assay</p> Signup and view all the answers

    What is the primary function of the catalytic domain in the Pf PKG encoded by the falciparum genome?

    <p>Catalyzes cyclic nucleotide signaling</p> Signup and view all the answers

    How many cGMP binding domains are present in the regulatory domain of Pf PKG?

    <p>Four</p> Signup and view all the answers

    Which antimalarial drug is not mentioned in the context of treatment challenges for malaria?

    <p>Quinidine</p> Signup and view all the answers

    In what way does Pf PKG differ from its mammalian orthologue?

    <p>Pf PKG has a larger size and lacks a dimerization domain</p> Signup and view all the answers

    What characteristic of P. falciparum’s life cycle complicates malaria treatment?

    <p>It involves multiple life stages in both hosts</p> Signup and view all the answers

    Which binding domain is degenerate in the regulatory domain of Pf PKG?

    <p>CNB-C</p> Signup and view all the answers

    What aspect of drug resistance significantly challenges malaria treatment?

    <p>Development of next generation antimalarial drugs is essential</p> Signup and view all the answers

    What is the total molecular weight of Pf PKG as mentioned?

    <p>97.5 kDa</p> Signup and view all the answers

    What effect did replacing the conserved Arg (R492) in CNB-D with a Lys residue have on cGMP binding?

    <p>Decreased the binding affinity for cGMP</p> Signup and view all the answers

    Which of the following constructs showed the highest basal activity in the absence of cGMP?

    <p>Pf PKG 401-853</p> Signup and view all the answers

    What was the specific activity of Pf PKG 401-853 when activated by cGMP?

    <p>5.3 U/mg</p> Signup and view all the answers

    What method was used to determine the active protein concentration for calculating specific activities?

    <p>Inhibition by Compound 2</p> Signup and view all the answers

    Which statement about the catalytic domain Pf PKG 519-853 is correct?

    <p>It exhibited no detectable kinase activity with peptide substrates.</p> Signup and view all the answers

    Which CNB is described as a high-affinity, cGMP selective site?

    <p>CNB-D</p> Signup and view all the answers

    What percentage of activity of the full-length protein does Pf PKG 401-853 achieve when activated by cGMP?

    <p>60%</p> Signup and view all the answers

    What is the result of the expression of Pf PKG 158-853 and 275-853 in E. coli TP2000?

    <p>Low basal activity with activation by cGMP</p> Signup and view all the answers

    What was a major consequence of TP2000 resulting in low yields?

    <p>It limited the analysis of Pf PKG regulation.</p> Signup and view all the answers

    What is indicated about the regulatory domain of Pf PKG in comparison to hPKG?

    <p>It shows significant differences that suggest it could be a drug target.</p> Signup and view all the answers

    At what concentrations of cGMP is the full-length Pf PKG protein activated?

    <p>315 nM</p> Signup and view all the answers

    What was the result of the interaction analysis of FITC-labeled IS peptide with Pf PKG?

    <p>The interaction was not demonstrated.</p> Signup and view all the answers

    Which part of the Pf PKG regulatory domain is crucial for its activation?

    <p>cGMP binding.</p> Signup and view all the answers

    What characteristic of CNB-C of Pf PKG was established in previous studies?

    <p>It binds neither cGMP nor cAMP.</p> Signup and view all the answers

    What was inferred about Pf PKG 32-853 mutant protein's activity?

    <p>It can be activated by cGMP despite lacking the IS.</p> Signup and view all the answers

    What does the study suggest about the potential of Pf PKG as a drug target?

    <p>It is a unique Plasmodium kinase with significant differences from hPKG.</p> Signup and view all the answers

    What is the effect of mutating the residues R209K and R333K in bovine PKA on cAMP affinity?

    <p>It reduces cAMP affinity only by a factor of 10.</p> Signup and view all the answers

    How does the specific activity of Pf PKG cGMP deficient construct compare to Pf PKG 401-853?

    <p>It is only slightly decreased.</p> Signup and view all the answers

    What is the main control mechanism for the catalytic activity of Pf PKG?

    <p>Interactions involving the αC-helix.</p> Signup and view all the answers

    What happens when cGMP binds to Pf PKG?

    <p>The αC-helix moves toward the PBC.</p> Signup and view all the answers

    What is indicated by the interaction between Y480 and R528 in Pf PKG?

    <p>It stabilizes the αC-helix in the active state.</p> Signup and view all the answers

    Which CNB disruption resulted in a significant decrease (>70%) in activity?

    <p>CNB-D</p> Signup and view all the answers

    What could be targeted for potential allosteric inhibitors in Pf PKG?

    <p>The interaction between Y480 and R528.</p> Signup and view all the answers

    What effect did the addition of CNB-B and -C have on the activation constants?

    <p>They had no change on the activation constants.</p> Signup and view all the answers

    What is the primary role of the N-terminal autoinhibitory sequence (IS) in Pf PKG?

    <p>To inhibit catalytic activity</p> Signup and view all the answers

    Which cyclic nucleotide binding domain (CNB) is associated with high-affinity cGMP binding but is mentioned as degenerate?

    <p>CNB-C</p> Signup and view all the answers

    Which construct demonstrates the highest basal activity in the absence of cGMP?

    <p>Pf PKG 401-853</p> Signup and view all the answers

    What type of cooperativity is observed with CNB-A and CNB-B in Pf PKG?

    <p>Positive cooperativity</p> Signup and view all the answers

    What was the method used to determine the effect of cGMP on Pf PKG kinase activity?

    <p>Microfluidic mobility-shift assay (MSA)</p> Signup and view all the answers

    What is described as the activation constant (Kact) in relation to Pf PKG?

    <p>The concentration where half-maximal kinase activity is observed</p> Signup and view all the answers

    Which residue in CNB-A is highlighted as crucial for cGMP binding?

    <p>R132</p> Signup and view all the answers

    How does the specific activity of a cGMP deficient construct compare to Pf PKG 401-853?

    <p>It is significantly lower in the presence of cGMP</p> Signup and view all the answers

    What was the activation constant (Kact) for the Pf PKG 32-853 mutant protein when activated by cGMP?

    <p>315 nM</p> Signup and view all the answers

    Which part of Pf PKG is necessary for its activation by cGMP?

    <p>Regulatory domain</p> Signup and view all the answers

    What significant property of CNB-C in Pf PKG was established in earlier studies?

    <p>It does not bind either cGMP or cAMP.</p> Signup and view all the answers

    What mechanism is indicated for the autoinhibition of Pf PKG?

    <p>Interaction of CNB-D without cGMP present.</p> Signup and view all the answers

    What was the observed effect of the R492K mutation on the cGMP binding affinity of the Pf PKG?

    <p>It decreased cGMP binding affinity significantly.</p> Signup and view all the answers

    Which Pf PKG construct expressed in E. coli showed low basal activity in the absence of cGMP?

    <p>Pf PKG 401-853</p> Signup and view all the answers

    What outcome was observed regarding the interaction of a FITC-labeled IS peptide with Pf PKG?

    <p>No interaction was demonstrated.</p> Signup and view all the answers

    What conclusion can be drawn regarding the regulatory domain of Pf PKG compared to hPKG?

    <p>Pf PKG's regulatory domain differs significantly.</p> Signup and view all the answers

    What effect does the addition of cGMP have on the Pf PKG protein's activity?

    <p>Activates the catalytic domain.</p> Signup and view all the answers

    What is the role of the conserved Arg (R492) in CNB-D of Pf PKG?

    <p>It is critical for cGMP interaction.</p> Signup and view all the answers

    Which construct yielded no detectable kinase activity with PKStide or Kemptide?

    <p>Pf PKG 519-853</p> Signup and view all the answers

    What was indicated about the specific activity of Pf PKG 401-853 when activated by cGMP?

    <p>It showed enhanced activity.</p> Signup and view all the answers

    Which cGMP binding domain is classified as low affinity and non-selective for cAMP and cGMP?

    <p>CNB-B</p> Signup and view all the answers

    What percentage of activity does Pf PKG 401-853 achieve compared to the full-length protein when activated?

    <p>60%</p> Signup and view all the answers

    What was noted about the specific activities determined by the Bradford assay?

    <p>50-80% of protein concentration was catalytically active.</p> Signup and view all the answers

    What was the EC50 value for the cGMP binding of recombinant mutant protein Pf PKG 401-853 R492K?

    <blockquote> <p>100,000 nM</p> </blockquote> Signup and view all the answers

    Which mutant proteins showed increased Kact values for cGMP?

    <p>Pf PKG 401-853 R492K</p> Signup and view all the answers

    What change was observed in the Hill slopes when comparing Pf PKG 401-853 and full-length Pf PKG?

    <p>Increased from 0.7 to 1.6</p> Signup and view all the answers

    What was the specific activity of Pf PKG 401-853 when activated by cGMP compared to its mutants?

    <p>Increased significantly</p> Signup and view all the answers

    Which method was used to determine the EC50 values for cGMP binding for the different constructs?

    <p>Solution competition assay</p> Signup and view all the answers

    What does the term 'Kact' refer to in the context of cGMP activation?

    <p>An indicator of catalytic efficiency</p> Signup and view all the answers

    What is indicated by the specific activities of the cGMP deficient constructs compared to Pf PKG 401-853?

    <p>They show lower activities than Pf PKG 401-853</p> Signup and view all the answers

    Which construct displayed the highest basal activity in the absence of cGMP?

    <p>Pf PKG 401-853FP</p> Signup and view all the answers

    What impact does the binding of cGMP to CNB-D have on the Pf PKG protein?

    <p>It induces a conformational change and stabilizes the active state.</p> Signup and view all the answers

    Which amino acid is involved in stabilizing the αC-helix when cGMP is not bound?

    <p>Arginine (R528)</p> Signup and view all the answers

    What was the purpose of introducing the mutations Y480F, R528K, and D597N?

    <p>To investigate the activation mechanism of Pf PKG.</p> Signup and view all the answers

    At what temperature and OD600 nm was expression of Pf PKG proteins induced in E. coli TP2000?

    <p>37 °C, OD600 nm of 0.6−0.8</p> Signup and view all the answers

    What is the purpose of using SDS-PAGE in protein analysis?

    <p>To verify the purity of protein samples.</p> Signup and view all the answers

    What role does the CNB-D play in the activation of Pf PKG?

    <p>It facilitates the binding of cGMP and induces conformational changes.</p> Signup and view all the answers

    What component was included in the elution buffer for His-tagged proteins?

    <p>250 mM imidazole</p> Signup and view all the answers

    What was the purpose of using a French press in the procedure?

    <p>To lyse the cells and release the protein content.</p> Signup and view all the answers

    What solvent was used to equilibrate the HiLoad 16/60 Superdex 75 gel filtration column?

    <p>20 mM MOPS (pH 7.0)</p> Signup and view all the answers

    How does the introduction of a Lys at position 528 (R528K) affect Pf PKG?

    <p>It disrupts the interaction with D597, affecting activation.</p> Signup and view all the answers

    Which method was used to calculate Kact values?

    <p>Sigmoidal dose-response curves</p> Signup and view all the answers

    What is indicated by the interaction between residues Y480 and R528 in Pf PKG?

    <p>It contributes to stabilizing the active form upon cGMP binding.</p> Signup and view all the answers

    What role does 2-mercaptoethanol serve in the buffers used?

    <p>Stabilizes protein structure.</p> Signup and view all the answers

    What components were included in the reaction buffer for the fluorescence polarization assay?

    <p>1 mM ATP, 220 μM NADH</p> Signup and view all the answers

    What was suggested by the 3-fold lower Kact value for the His-tagged protein constructs?

    <p>The αC-helix cannot be stabilized longer in the inactive state.</p> Signup and view all the answers

    What does the Bradford assay measure?

    <p>The concentration of proteins in samples.</p> Signup and view all the answers

    Study Notes

    Plasmodium falciparum PKG Activation

    • PfPKG, a cGMP-dependent protein kinase, is crucial for malaria parasite proliferation (sexual and asexual)
    • PfPKG differs significantly from human PKG (hPKG) in domain organization
    • CNB-D (Cyclic Nucleotide Binding Domain D) plays a critical role in PfPKG activation and regulation unlike hPKG which is regulated by a pseudosubstrate autoinhibitory sequence (IS)
    • cGMP binding to CNB-D induces a conformational change, specifically involving the aC-helix
    • The inactive state is characterized by an interaction between Asp597 (catalytic domain) and Arg528 (aC-helix)
    • Arg528 also stabilizes cGMP binding by interacting with Tyr480 (phosphate binding cassette) in the active state
    • This unique activation mechanism of PfPKG, distinct from hPKG, suggests it as a potential drug target for malaria.

    Malaria Disease Overview

    • Malaria is a major global infectious disease caused by Plasmodium parasites
    • Transmitted by female Anopheles mosquitoes
    • 214 million cases per year, with 438,000 deaths in 2015
    • Drug resistance is a significant challenge in malaria treatment.

    cGMP Signaling in Malaria

    • cGMP signaling plays a crucial role in the asexual blood stage, exflagellation, gametogenesis, and schizogony in liver stages of malaria.
    • cAMP signaling controls events like apical exocytosis, sporozoite motility, anion transport, and RBC and liver cell invasion.
    • Plasmodium falciparum has a single PKG isoform, larger than mammalian orthologs, lacking a dimerization domain
    • The multiple CNBs (cyclic nucleotide binding domains) in the regulatory domain of PfPKG differ significantly from mammalian PKGs.

    PfPKG Structure and Function

    • PfPKG consists of an N-terminal regulatory domain and a C-terminal catalytic domain.
    • The regulatory domains contain 4 CNBs, with CNB-C being degenerate (not functional)
    • PfPKG is larger than its mammalian orthologue with about 97.5 kDa
    • A recent study identified 107 phosphorylation sites on 69 proteins as cellular targets of PfPKG. These are involved in cell signalling, gene regulation, proteolysis, and transport.

    Mechanism of PfPKG activation

    • The activation of PfPKG does not depend on the N-terminal IS motif interacting with the catalytic domain as in hPKG.
    • Deleting specific CNBs (such as CNB-D) in PfPKG creates constructs that still show activation by cGMP
    • The isolated CNB-D has high-affinity cGMP binding.
    • The amino acid arginine within the phosphate binding cassette (PBC), specifically Arg492 in CNB-D, is critical for cGMP binding and subsequent activation. Mutations affecting this residue show significant reduction in activity.
    • The C-helix of CNB-D plays a crucial role in PfPKG activation; its interaction with the catalytic domain is crucial for regulated activity. Mutations in the relevant amino acids (R528) significantly reduce activation.

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    cGMP Binding Domain D Mediates a Unique Activation Mechanism PDF

    Description

    This quiz explores the role of PfPKG, a crucial protein kinase in the proliferation of malaria parasites. It discusses its unique activation mechanism in contrast to human PKG, highlighting its potential as a drug target. Gain insights into the molecular dynamics of malaria and the distinctive aspects of Plasmodium falciparum biology.

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