Designing selective inhibitors for calcium-dependent protein kinases in apicomplexans

Hui, Raymond; Bakkouri, M. El; Sibley, L. David

doi:10.1016/j.tips.2015.04.011

Cited by 43 publications

(42 citation statements)

References 96 publications

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“…Phylogenetic analysis based on the kinase domains suggested that they are close to CDPK4 and CDPK7, and they were named CDPK4B (TgME49_240390) and CDPK7A (TgME49_237860), respectively (see Fig. S1 in the supplemental material), as previously discussed (15). Analyses of domain structures of all CDPKs in T. gondii were performed using an online software PROSITE (http: //prosite.expasy.org/), which suggested that these CDPKs can be classed into two groups based on their domain structure.…”

Section: Sequence and Expression Analyses Of Noncanonical Cdpks In Tmentioning

confidence: 54%

“…CDPKs likely arose by fusion of a calmodulin-like domain containing four EF hands, a structural feature first described in parvalbumin and that is responsible for calcium binding, followed by their diversification in plants and protists (10). The fact that CDPKs are not found in animal cells, combined with the findings that some of them are essential, has made them attractive targets for development of inhibitors (15).…”

Section: -Isoleucine [I]-lysine [K]-lysine [K]) Kinases In Plasmodiummentioning

confidence: 99%

“…A gene ontology (GO) term search of ToxoDB using the terms "calcium binding" and "protein kinase activity" combined with a text word search for "calcium dependent protein kinase" identified 14 protein-coding genes, including previously named CDPKs (15) and two new proteins annotated as calcium-dependent protein kinase (TgME49_240390) and protein kinase domain-containing protein (TgME49_237860). Domain predictions were performed using Prosite (http://prosite.expasy.org/).…”

Section: Domain Prediction For Cdpksmentioning

confidence: 99%

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Analysis of Noncanonical Calcium-Dependent Protein Kinases in Toxoplasma gondii by Targeted Gene Deletion Using CRISPR/Cas9

2016

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Calcium-dependent protein kinases (CDPKs) are expanded in apicomplexan parasites, especially in Toxoplasma gondii where 14 separate genes encoding these enzymes are found. Although previous studies have shown that several CDPKs play a role in controlling invasion, egress, and cell division in T. gondii, the roles of most of these genes are unexplored. Here we developed a more efficient method for gene disruption using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPRassociated protein 9) that was modified to completely delete large, multiexonic genes from the genome and to allow serial replacement by recycling of the selectable marker using Cre-loxP. Using this system, we generated a total of 24 mutants in type 1 and 2 genetic backgrounds to ascertain the functions of noncanonical CDPKs. Remarkably, although we were able to confirm the essentiality of CDPK1 and CDPK7, the majority of CDPKs had no discernible phenotype for growth in vitro or infection in the mouse model. The exception to this was CDPK6, loss of which leads to reduced plaquing, fitness defect in a competition assay, and reduced tissue cyst formation in chronically infected mice. Our findings highlight the utility of CRISPR/Cas9 for rapid serial gene deletion and also suggest that additional models are needed to reveal the functions of many genes in T. gondii.

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Section: Sequence and Expression Analyses Of Noncanonical Cdpks In Tmentioning

confidence: 54%

Section: -Isoleucine [I]-lysine [K]-lysine [K]) Kinases In Plasmodiummentioning

confidence: 99%

Section: Domain Prediction For Cdpksmentioning

confidence: 99%

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Analysis of Noncanonical Calcium-Dependent Protein Kinases in Toxoplasma gondii by Targeted Gene Deletion Using CRISPR/Cas9

2016

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“…Support for this model is provided by inhibition of PKG with Compound 1 (15,17), and selective inhibition of CDPK1 with pyrazolopyrimidine inhibitors (8,47). Both classes of compounds offer promise for development of alternative therapeutics, given the essential nature of these kinases and the potent and specific inhibition offered by these respective chemical scaffolds (48,49). Evidence that PKG may enhance Ca 2ϩ release via increased production of IP 3 links these two pathways (19).…”

Section: Discussionmentioning

confidence: 99%

Serum Albumin Stimulates Protein Kinase G-dependent Microneme Secretion in Toxoplasma gondii

Brown

Lourido

Sibley

2016

Journal of Biological Chemistry

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103

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Microneme secretion is essential for motility, invasion, and egress in apicomplexan parasites. Although previous studies indicate that Ca 2؉ and cGMP control microneme secretion, little is known about how these pathways are naturally activated. Here we have developed genetically encoded indicators for Ca 2؉ and microneme secretion to better define the signaling pathways that regulate these processes in Toxoplasma gondii. We found that microneme secretion was triggered in vitro by exposure to a single host protein, serum albumin. The natural agonist serum albumin induced microneme secretion in a protein kinase G-dependent manner that correlated with increased cGMP levels. Surprisingly, serum albumin acted independently of elevated Ca 2؉ and yet it was augmented by artificial agonists that raise Ca 2؉ , such as ethanol. Furthermore, although ethanol elevated intracellular Ca 2؉ , it alone was unable to trigger secretion without the presence of serum or serum albumin. This dichotomy was recapitulated by zaprinast, a phosphodiesterase inhibitor that elevated cGMP and separately increased Ca 2؉ in a protein kinase G-independent manner leading to microneme secretion. Taken together, these findings reveal that microneme secretion is centrally controlled by protein kinase G and that this pathway is further augmented by elevation of intracellular Ca 2؉ .Toxoplasma gondii is an important opportunistic pathogen and model organism for studying the biology of members of the phylum Apicomplexa (1). Micronemes are specialized secretory vesicles present in all motile stages of apicomplexan parasites (reviewed in Ref. 2). The majority of internal microneme (MIC) 3 proteins (i.e. cargo) consist of adhesive proteins that translocate to the surface of the parasite following the regulated fusion of the organelle with the apical plasma membrane.

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“…3 and SI Appendix, Tables S32 and S33). The kinase domain from all known Ca 2+ sensor kinases belongs to the CAMK group of kinases, whereas this uncharacterized protein belongs to the tyrosine kinase-like (TKL) class of kinases (72,73). The genes encoding homologous proteins are present on many other red algal genomes, including Chondrus, Cyanidioschyzon, and Galdieria, but appear to be absent from all other characterized eukaryotic genomes.…”

Section: Significancementioning

confidence: 99%

Insights into the red algae and eukaryotic evolution from the genome of Porphyra umbilicalis (Bangiophyceae, Rhodophyta)

Brawley¹,

Blouin²,

Ficko-Blean³

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

238

222

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Porphyra umbilicalis (laver) belongs to an ancient group of red algae (Bangiophyceae), is harvested for human food, and thrives in the harsh conditions of the upper intertidal zone. Here we present the 87.7-Mbp haploid Porphyra genome (65.8% G + C content, 13,125 gene loci) and elucidate traits that inform our understanding of the biology of red algae as one of the few multicellular eukaryotic lineages. Novel features of the Porphyra genome shared by other red algae relate to the cytoskeleton, calcium signaling, the cell cycle, and stress-tolerance mechanisms including photoprotection. Cytoskeletal motor proteins in Porphyra are restricted to a small set of kinesins that appear to be the only universal cytoskeletal motors within the red algae. Dynein motors are absent, and most red algae, including Porphyra, lack myosin. This surprisingly minimal cytoskeleton offers a potential explanation for why red algal cells and multicellular structures are more limited in size than in most multicellular lineages. Additional discoveries further relating to the stress tolerance of bangiophytes include ancestral enzymes for sulfation of the hydrophilic galactan-rich cell wall, evidence for mannan synthesis that originated before the divergence of green and red algae, and a high capacity for nutrient uptake. Our analyses provide a comprehensive understanding of the red algae, which are both commercially important and have played a major role in the evolution of other algal groups through secondary endosymbioses.cytoskeleton | calcium-signaling | carbohydrate-active enzymes | stress tolerance | vitamin B 12T he red algae are one of the founding groups of photosynthetic eukaryotes (Archaeplastida) and among the few multicellular lineages within Eukarya. A red algal plastid, acquired through secondary endosymbiosis, supports carbon fixation, fatty acid synthesis, and other metabolic needs in many other algal groups in ways that are consequential. For example, diatoms and haptophytes have strong biogeochemical effects; apicomplexans cause human disease (e.g., malaria); and dinoflagellates include both coral symbionts and toxin-producing "red tides" (1). The evolutionary processes that produced the Archaeplastida and secondary algal lineages remain under investigation (2-5), but it is clear that both nuclear and plastid genes from the ancestral red algae have contributed dramatically to broader eukaryotic evolution and diversity. Consequently, the imprint of red algal metabolism on the Earth's climate system, aquatic foodwebs, and

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Designing selective inhibitors for calcium-dependent protein kinases in apicomplexans

Cited by 43 publications

References 96 publications

Analysis of Noncanonical Calcium-Dependent Protein Kinases in Toxoplasma gondii by Targeted Gene Deletion Using CRISPR/Cas9

Analysis of Noncanonical Calcium-Dependent Protein Kinases in Toxoplasma gondii by Targeted Gene Deletion Using CRISPR/Cas9

Serum Albumin Stimulates Protein Kinase G-dependent Microneme Secretion in Toxoplasma gondii

Insights into the red algae and eukaryotic evolution from the genome of Porphyra umbilicalis (Bangiophyceae, Rhodophyta)

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