scholarly journals Plasmodium falciparum Cyclic GMP-Dependent Protein Kinase Interacts with a Subunit of the Parasite Proteasome

2018 ◽  
Vol 87 (1) ◽  
Author(s):  
K. Govindasamy ◽  
R. Khan ◽  
M. Snyder ◽  
H. J. Lou ◽  
P. Du ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Life Cycle ◽  
Protein Kinase ◽  
Cyclic Gmp ◽  
Phosphorylation Site ◽  
Complex Life Cycle ◽  
Mosquito Vector ◽  
Dependent Protein Kinase ◽  
Content Type ◽  
Dependent Protein

ABSTRACT Malaria is caused by the protozoan parasite Plasmodium, which undergoes a complex life cycle in a human host and a mosquito vector. The parasite’s cyclic GMP (cGMP)-dependent protein kinase (PKG) is essential at multiple steps of the life cycle. Phosphoproteomic studies in Plasmodium falciparum erythrocytic stages and Plasmodium berghei ookinetes have identified proteolysis as a major biological pathway dependent on PKG activity. To further understand PKG’s mechanism of action, we screened a yeast two-hybrid library for P. falciparum proteins that interact with P. falciparum PKG (PfPKG) and tested peptide libraries to identify its phosphorylation site preferences. Our data suggest that PfPKG has a distinct phosphorylation site and that PfPKG directly phosphorylates parasite RPT1, one of six AAA+ ATPases present in the 19S regulatory particle of the proteasome. PfPKG and RPT1 interact in vitro, and the interacting fragment of RPT1 carries a PfPKG consensus phosphorylation site; a peptide carrying this consensus site competes with the RPT1 fragment for binding to PfPKG and is efficiently phosphorylated by PfPKG. These data suggest that PfPKG’s phosphorylation of RPT1 could contribute to its regulation of parasite proteolysis. We demonstrate that proteolysis plays an important role in a biological process known to require Plasmodium PKG: invasion by sporozoites of hepatocytes. A small-molecule inhibitor of proteasomal activity blocks sporozoite invasion in an additive manner when combined with a Plasmodium PKG-specific inhibitor. Mining the previously described parasite PKG-dependent phosphoproteomes using the consensus phosphorylation motif identified additional proteins that are likely to be direct substrates of the enzyme.

scholarly journals The Malaria Parasite Cyclic GMP-Dependent Protein Kinase Plays a Central Role in Blood-Stage Schizogony

2009 ◽  
Vol 9 (1) ◽  
pp. 37-45 ◽  
Author(s):  
Helen M. Taylor ◽  
Louisa McRobert ◽  
Munira Grainger ◽  
Audrey Sicard ◽  
Anton R. Dluzewski ◽  
...  
Keyword(s):  
Life Cycle ◽  
Protein Kinase ◽  
Cyclic Gmp ◽  
Malaria Parasite ◽  
Kinase Inhibitor ◽  
Blood Stage ◽  
Parasite Life Cycle ◽  
Dependent Protein Kinase ◽  
Asexual Blood Stage ◽  
Dependent Protein

ABSTRACT A role for the Plasmodium falciparum cyclic GMP (cGMP)-dependent protein kinase (PfPKG) in gametogenesis in the malaria parasite was elucidated previously. In the present study we examined the role of PfPKG in the asexual blood-stage of the parasite life cycle, the stage that causes malaria pathology. A specific PKG inhibitor (compound 1, a trisubstituted pyrrole) prevented the progression of P. falciparum schizonts through to ring stages in erythrocyte invasion assays. Addition of compound 1 to ring-stage parasites allowed normal development up to 30 h postinvasion, and segmented schizonts were able to form. However, synchronized schizonts treated with compound 1 for ≥6 h became large and dysmorphic and were unable to rupture or liberate merozoites. To conclusively demonstrate that the effect of compound 1 on schizogony was due to its selective action on PfPKG, we utilized genetically manipulated P. falciparum parasites expressing a compound 1-insensitive PfPKG. The mutant parasites were able to complete schizogony in the presence of compound 1 but not in the presence of the broad-spectrum protein kinase inhibitor staurosporine. This shows that PfPKG is the primary target of compound 1 during schizogony and provides direct evidence of a role for PfPKG in this process. Discovery of essential roles for the P. falciparum PKG in both asexual and sexual development demonstrates that cGMP signaling is a key regulator of both of these crucial life cycle phases and defines this molecule as an exciting potential drug target for both therapeutic and transmission blocking action against malaria.

scholarly journals A Novel Tool for the Generation of Conditional Knockouts To Study Gene Function across the Plasmodium falciparum Life Cycle

mBio ◽  
2019 ◽  
Vol 10 (5) ◽  
Author(s):  
Marta Tibúrcio ◽  
Annie S. P. Yang ◽  
Kazuhide Yahata ◽  
Pablo Suárez-Cortés ◽  
Hugo Belda ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Life Cycle ◽  
Genetic Modification ◽  
Gene Deletion ◽  
Genetically Modify ◽  
Complex Life Cycle ◽  
Mosquito Vector ◽  
Parasite Line ◽  
Content Type ◽  
First Time

ABSTRACT Plasmodium falciparum has a complex life cycle that involves interaction with multiple tissues inside the human and mosquito hosts. Identification of essential genes at all different stages of the P. falciparum life cycle is urgently required for clinical development of tools for malaria control and eradication. However, the study of P. falciparum is limited by the inability to genetically modify the parasite throughout its life cycle with the currently available genetic tools. Here, we describe the detailed characterization of a new marker-free P. falciparum parasite line that expresses rapamycin-inducible Cre recombinase across the full life cycle. Using this parasite line, we were able to conditionally delete the essential invasion ligand AMA1 in three different developmental stages for the first time. We further confirm efficient gene deletion by targeting the nonessential kinase FIKK7.1. IMPORTANCE One of the major limitations in studying P. falciparum is that so far only asexual stages are amenable to rapid conditional genetic modification. The most promising drug targets and vaccine candidates, however, have been refractory to genetic modification because they are essential during the blood stage or for transmission in the mosquito vector. This leaves a major gap in our understanding of parasite proteins in most life cycle stages and hinders genetic validation of drug and vaccine targets. Here, we describe a method that supports conditional gene deletion across the P. falciparum life cycle for the first time. We demonstrate its potential by deleting essential and nonessential genes at different parasite stages, which opens up completely new avenues for the study of malaria and drug development. It may also allow the realization of novel vaccination strategies using attenuated parasites.

scholarly journals Discovery of isoxazolyl-based inhibitors of Plasmodium falciparum cGMP-dependent protein kinase

2020 ◽  
Vol 11 (1) ◽  
pp. 98-101 ◽  
Author(s):  
Shams Ul Mahmood ◽  
Huimin Cheng ◽  
Sreedhar R. Tummalapalli ◽  
Ramappa Chakrasali ◽  
Rammohan R. Yadav Bheemanaboina ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Life Cycle ◽  
Protein Kinase ◽  
Multiple Roles ◽  
Dependent Protein Kinase ◽  
Cgmp Dependent Protein Kinase ◽  
Dependent Protein

The cGMP-dependent protein kinase in Plasmodium falciparum (PfPKG) plays multiple roles in the life cycle of the parasite.

scholarly journals Biochemical and Antiparasitic Properties of Inhibitors of the Plasmodium falciparum Calcium-Dependent Protein Kinase PfCDPK1

2014 ◽  
Vol 58 (10) ◽  
pp. 6032-6043 ◽  
Author(s):  
Keith H. Ansell ◽  
Hayley M. Jones ◽  
David Whalley ◽  
Alisdair Hearn ◽  
Debra L. Taylor ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Protein Kinase ◽  
Parasite Growth ◽  
Biochemical Assay ◽  
Dependent Protein Kinase ◽  
Content Type ◽  
Calcium Dependent ◽  
Dependent Protein ◽  
Calcium Dependent Protein Kinase

ABSTRACTPfCDPK1 is aPlasmodium falciparumcalcium-dependent protein kinase, which has been identified as a potential target for novel antimalarial chemotherapeutics. In order to further investigate the role of PfCDPK1, we established a high-throughputin vitrobiochemical assay and used it to screen a library of over 35,000 small molecules. Five chemical series of inhibitors were initially identified from the screen, from which series 1 and 2 were selected for chemical optimization. Indicative of their mechanism of action, enzyme inhibition by these compounds was found to be sensitive to both the ATP concentration and substitution of the amino acid residue present at the “gatekeeper” position at the ATP-binding site of the enzyme. Medicinal chemistry efforts led to a series of PfCDPK1 inhibitors with 50% inhibitory concentrations (IC50s) below 10 nM against PfCDPK1 in a biochemical assay and 50% effective concentrations (EC50s) less than 100 nM for inhibition of parasite growthin vitro. Potent inhibition was combined with acceptable absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties and equipotent inhibition ofPlasmodium vivaxCDPK1. However, we were unable to correlate biochemical inhibition with parasite growth inhibition for this series overall. Inhibition ofPlasmodium bergheiCDPK1 correlated well with PfCDPK1 inhibition, enabling progression of a set of compounds toin vivoevaluation in theP. bergheirodent model for malaria. These chemical series have potential for further development as inhibitors of CDPK1.

scholarly journals Influence of Plasmodium falciparum Calcium-Dependent Protein Kinase 5 (PfCDPK5) on the Late Schizont Stage Phosphoproteome

mSphere ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Karin Blomqvist ◽  
Michaela Helmel ◽  
Chengqi Wang ◽  
Sabrina Absalon ◽  
Tetanya Labunska ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Protein Kinase ◽  
Phosphorylation Sites ◽  
Schizont Stage ◽  
Dependent Protein Kinase ◽  
Content Type ◽  
Calcium Dependent ◽  
Dependent Protein ◽  
Calcium Dependent Protein Kinase ◽  
Potential Substrate

ABSTRACT Protein kinases are important mediators of signal transduction in cellular pathways, and calcium-dependent protein kinases (CDPKs) compose a unique class of calcium-dependent kinases present in plants and apicomplexans, including Plasmodium parasites, the causative agents of malaria. During the asexual stage of infection, the human malaria parasite Plasmodium falciparum grows inside red blood cells, and P. falciparum calcium-dependent protein kinase 5 (PfCDPK5) is required for egress from the host cell. In this paper, we characterize the late-schizont-stage P. falciparum phosphoproteome by performing large-scale phosphoproteomic profiling on tightly synchronized parasites just prior to egress, identifying 2,704 phosphorylation sites on 919 proteins. Using a conditional knockdown of PfCDPK5, we identify 58 phosphorylation sites on 50 proteins with significant reduction in levels of PfCDPK5-deficient parasites. Furthermore, gene ontology analysis of the identified proteins reveals enrichment in transmembrane- and membrane-associated proteins and in proteins associated with transport activity. Among the identified proteins is PfNPT1, a member of the apicomplexan-specific novel putative transporter (NPT) family of proteins. We show that PfNPT1 is a potential substrate of PfCDPK5 and that PfNPT1 localizes to the parasite plasma membrane. Importantly, P. falciparum egress relies on many proteins unique to Apicomplexa that are therefore attractive targets for antimalarial therapeutics. IMPORTANCE The malaria parasite Plasmodium falciparum is a major cause of morbidity and mortality globally. The P. falciparum parasite proliferates inside red blood cells during the blood stage of infection, and egress from the red blood cell is critical for parasite survival. P. falciparum calcium-dependent protein kinase 5 (PfCDPK5) is essential for egress; parasites deficient in PfCDPK5 remain trapped inside their host cells. We have used a label-free quantitative mass spectrometry approach to identify the phosphoproteome of schizont-stage parasites just prior to egress and identify 50 proteins that display a significant reduction in phosphorylation in PfCDPK5-deficient parasites. We show that a member of the Apicomplexan-specific transport protein family, PfNPT1 is a potential substrate of PfCDPK5 and is localized to the parasite plasma membrane. P. falciparum egress requires several proteins not present in human cells, thus making this pathway an ideal target for new therapeutics.

scholarly journals Plasmodium falciparum Calcium Dependent Protein Kinase 4 is critical for male gametogenesis and transmission to the mosquito vector

2021 ◽  
Author(s):  
SUDHIR KUMAR ◽  
Meseret T. Haile ◽  
Michael R. Hoopmann ◽  
Linh T. Tran ◽  
Samantha A. Michaels ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Life Cycle ◽  
Mrna Translation ◽  
Complex Life Cycle ◽  
Mosquito Vector ◽  
Parasite Transmission ◽  
Calcium Dependent ◽  
Male Gametogenesis ◽  
Parasite Plasmodium ◽  
Dependent Protein

Gametocytes of the malaria parasite Plasmodium are taken up by the mosquito vector with an infectious blood meal, representing a critical stage for parasite transmission. Calcium dependent protein kinases play key roles in calcium mediated signaling across the complex life cycle of the parasite. We sought to understand their role in human parasite transmission from the host to the mosquito vector and thus investigated the role of the human infective parasite Plasmodium falciparum CDPK4 in the parasite life cycle. P. falciparum cdpk4 parasites created by targeted gene deletion showed no effect in blood stage development or gametocyte development. However, cdpk4 parasites showed a severe defect in male gametogenesis and the emergence of flagellated male gametes. To understand the molecular underpinnings of this defect, we performed mass spectrometry based phosphoproteomic analyses of wild type and Plasmodium falciparum cdpk4 late gametocyte stages, to identify key CDPK4 mediated phosphorylation events that may be important for the regulation of male gametogenesis. We further employed in vitro assays to identify these putative substrates of Plasmodium falciparum CDPK4. This indicated that CDPK4 regulates male gametogenesis by directly or indirectly controlling key essential events such as DNA replication, mRNA translation and cell motility. Taken together, our work demonstrates that PfCDPK4 is a central kinase that regulates exflagellation, and thereby is critical for parasite transmission to the mosquito vector.

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