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Calcium-dependent protein kinase 1 is an essential regulator of exocytosis in Toxoplasma.

Lourido S, Shuman J, Zhang C, Shokat KM, Hui R, Sibley LD - Nature (2010)

Bottom Line: Conditional suppression of TgCDPK1 revealed that it controls calcium-dependent secretion of specialized organelles called micronemes, resulting in a block of essential phenotypes including parasite motility, host-cell invasion, and egress.TgCDPK1 is conserved among apicomplexans and belongs to a family of kinases shared with plants and ciliates, suggesting that related CDPKs may have a function in calcium-regulated secretion in other organisms.Because this kinase family is absent from mammalian hosts, it represents a validated target that may be exploitable for chemotherapy against T. gondii and related apicomplexans.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology, Washington University School of Medicine, 660 S. Euclid Avenue, St Louis, Missouri 63110, USA.

ABSTRACT
Calcium-regulated exocytosis is a ubiquitous process in eukaryotes, whereby secretory vesicles fuse with the plasma membrane and release their contents in response to an intracellular calcium surge. This process regulates various cellular functions such as plasma membrane repair in plants and animals, the discharge of defensive spikes in Paramecium, and the secretion of insulin from pancreatic cells, immune modulators from lymphocytes, and chemical transmitters from neurons. In animal cells, serine/threonine kinases including cAMP-dependent protein kinase, protein kinase C and calmodulin kinases have been implicated in calcium-signal transduction leading to regulated secretion. Although plants and protozoa also regulate secretion by means of intracellular calcium, the method by which these signals are relayed has not been explained. Here we show that the Toxoplasma gondii calcium-dependent protein kinase 1 (TgCDPK1) is an essential regulator of calcium-dependent exocytosis in this opportunistic human pathogen. Conditional suppression of TgCDPK1 revealed that it controls calcium-dependent secretion of specialized organelles called micronemes, resulting in a block of essential phenotypes including parasite motility, host-cell invasion, and egress. These phenotypes were recapitulated by using a chemical biology approach in which pyrazolopyrimidine-derived compounds specifically inhibited TgCDPK1 and disrupted the parasite's life cycle at stages dependent on microneme secretion. Inhibition was specific to TgCDPK1, because expression of a resistant mutant kinase reversed sensitivity to the inhibitor. TgCDPK1 is conserved among apicomplexans and belongs to a family of kinases shared with plants and ciliates, suggesting that related CDPKs may have a function in calcium-regulated secretion in other organisms. Because this kinase family is absent from mammalian hosts, it represents a validated target that may be exploitable for chemotherapy against T. gondii and related apicomplexans.

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PP1 derivatives specifically inhibit TgCDPK1 and block micronem-mediated functionsa, Alignment of the kinase sub-domain V highlighting the gatekeeper residue. b, Structures of 3-MB-PP1 and 3-BrB-PP1. c, Effect of 5μM 3-MB-PP1 on host cell invasion. Student’s t test; *P < 0.05, mean ± s.e.m., N = 3 experiments. d, Effect of 5μM 3-MB-PP1 on MIC2 secretion. Student’s t test; *P < 0.05, mean ± s.e.m., N = 3 experiments. e-f, Effect of PP1 derivatives on host lysis by T. gondii +/− 3-MB-PP1 (e) and 3-Br- PP1 (f). Mean ± s.e.m., N = 3 experiments.
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Figure 4: PP1 derivatives specifically inhibit TgCDPK1 and block micronem-mediated functionsa, Alignment of the kinase sub-domain V highlighting the gatekeeper residue. b, Structures of 3-MB-PP1 and 3-BrB-PP1. c, Effect of 5μM 3-MB-PP1 on host cell invasion. Student’s t test; *P < 0.05, mean ± s.e.m., N = 3 experiments. d, Effect of 5μM 3-MB-PP1 on MIC2 secretion. Student’s t test; *P < 0.05, mean ± s.e.m., N = 3 experiments. e-f, Effect of PP1 derivatives on host lysis by T. gondii +/− 3-MB-PP1 (e) and 3-Br- PP1 (f). Mean ± s.e.m., N = 3 experiments.

Mentions: Having established the crucial role of TgCDPK1, we took advantage of the atypical nucleotide-binding pocket of TgCDPK112 to develop a chemical biology approach to further evaluate the essential nature of this kinase. It has been previously reported that the amino acid residue at the “gatekeeper” position within the nucleotide binding pocket radically affects inhibition by pyrazolopyrimidine (PP1) derivatives, which have limited activity against most S/T protein kinases22. Insensitivity is conferred by bulky gatekeeper residues in nearly all kinases of both animal and parasite cells; however, kinases can be rendered fully sensitive by mutation to a small gatekeeper22 (Supplementary Table 2). Fortuitously, TgCDPK1 displays a glycine at this position, which is unique among canonical CDPKs (Fig. 4a) and all other protein kinases in T. gondii (L. Peixoto and D. Roos, unpublished data). This finding predicted that wild type TgCDPK1 would be naturally sensitive to PP1-based inhibitors and, consistent with this a pilot screen of selected derivatives inhibited parasite lytic growth in vitro (Supplementary Fig. 2). Furthermore, purified TgCDPK1 enzyme was extremely sensitive to the compound 3-MP-PP1 (Fig. 4b), while mutation of the glycine gatekeeper to methionine shifted the sensitivity by more than 4 logs (Supplementary Table 2). Complementation of the TgCDPK1 cKO with either wild type or gatekeeper-mutant kinase alleles (cKO/WT and cKO/G128M, respectively) restored plaque formation, demonstrating that the mutation had no major deleterious effect (Supplementary Fig. 3). When treated with PP1 analogs, both WT and cKO/WT parasites were strongly inhibited in host cell attachment and invasion (Fig. 4c), consistent with a recent report that appeared online during the revision of the present work 23. In contrast to this recent report, which failed to provide quantitative analysis of secretion 23, we observed that microneme secretion by extracellular parasites and ionophore-induced egress (Supplementary Fig. 4) were also strongly inhibited by PP1 analogs (Fig. 4d). The reversal of these phenotypes in the G128M mutant confirms that the primary in vivo target of PP1 derivatives is TgCDPK1. Consistent with these effects, 3-MB-PP1 and the related compound 3-BrB-PP1, blocked the ability of the parasite to lyse host cell monolayers (Fig. 4e,f), demonstrating the essential role of TgCDPK1 during in vitro infection. Chemical genetic studies indicate that TgCDPK1 acts independently of the previously characterized cGMP-dependent kinase (PKG), the primary target of trisubstituted pyrole and imidazopyridine kinase inhibitors that also block microneme secretion in T. gondii24,25. Correspondingly, PKG is predicted to be insensitive to PP1 derivatives (Fig. 4a)22. Collectively these findings indicate that both kinases are essential for efficient microneme secretion, possibly reflecting a hierarchical control of this important cellular pathway.


Calcium-dependent protein kinase 1 is an essential regulator of exocytosis in Toxoplasma.

Lourido S, Shuman J, Zhang C, Shokat KM, Hui R, Sibley LD - Nature (2010)

PP1 derivatives specifically inhibit TgCDPK1 and block micronem-mediated functionsa, Alignment of the kinase sub-domain V highlighting the gatekeeper residue. b, Structures of 3-MB-PP1 and 3-BrB-PP1. c, Effect of 5μM 3-MB-PP1 on host cell invasion. Student’s t test; *P < 0.05, mean ± s.e.m., N = 3 experiments. d, Effect of 5μM 3-MB-PP1 on MIC2 secretion. Student’s t test; *P < 0.05, mean ± s.e.m., N = 3 experiments. e-f, Effect of PP1 derivatives on host lysis by T. gondii +/− 3-MB-PP1 (e) and 3-Br- PP1 (f). Mean ± s.e.m., N = 3 experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2874977&req=5

Figure 4: PP1 derivatives specifically inhibit TgCDPK1 and block micronem-mediated functionsa, Alignment of the kinase sub-domain V highlighting the gatekeeper residue. b, Structures of 3-MB-PP1 and 3-BrB-PP1. c, Effect of 5μM 3-MB-PP1 on host cell invasion. Student’s t test; *P < 0.05, mean ± s.e.m., N = 3 experiments. d, Effect of 5μM 3-MB-PP1 on MIC2 secretion. Student’s t test; *P < 0.05, mean ± s.e.m., N = 3 experiments. e-f, Effect of PP1 derivatives on host lysis by T. gondii +/− 3-MB-PP1 (e) and 3-Br- PP1 (f). Mean ± s.e.m., N = 3 experiments.
Mentions: Having established the crucial role of TgCDPK1, we took advantage of the atypical nucleotide-binding pocket of TgCDPK112 to develop a chemical biology approach to further evaluate the essential nature of this kinase. It has been previously reported that the amino acid residue at the “gatekeeper” position within the nucleotide binding pocket radically affects inhibition by pyrazolopyrimidine (PP1) derivatives, which have limited activity against most S/T protein kinases22. Insensitivity is conferred by bulky gatekeeper residues in nearly all kinases of both animal and parasite cells; however, kinases can be rendered fully sensitive by mutation to a small gatekeeper22 (Supplementary Table 2). Fortuitously, TgCDPK1 displays a glycine at this position, which is unique among canonical CDPKs (Fig. 4a) and all other protein kinases in T. gondii (L. Peixoto and D. Roos, unpublished data). This finding predicted that wild type TgCDPK1 would be naturally sensitive to PP1-based inhibitors and, consistent with this a pilot screen of selected derivatives inhibited parasite lytic growth in vitro (Supplementary Fig. 2). Furthermore, purified TgCDPK1 enzyme was extremely sensitive to the compound 3-MP-PP1 (Fig. 4b), while mutation of the glycine gatekeeper to methionine shifted the sensitivity by more than 4 logs (Supplementary Table 2). Complementation of the TgCDPK1 cKO with either wild type or gatekeeper-mutant kinase alleles (cKO/WT and cKO/G128M, respectively) restored plaque formation, demonstrating that the mutation had no major deleterious effect (Supplementary Fig. 3). When treated with PP1 analogs, both WT and cKO/WT parasites were strongly inhibited in host cell attachment and invasion (Fig. 4c), consistent with a recent report that appeared online during the revision of the present work 23. In contrast to this recent report, which failed to provide quantitative analysis of secretion 23, we observed that microneme secretion by extracellular parasites and ionophore-induced egress (Supplementary Fig. 4) were also strongly inhibited by PP1 analogs (Fig. 4d). The reversal of these phenotypes in the G128M mutant confirms that the primary in vivo target of PP1 derivatives is TgCDPK1. Consistent with these effects, 3-MB-PP1 and the related compound 3-BrB-PP1, blocked the ability of the parasite to lyse host cell monolayers (Fig. 4e,f), demonstrating the essential role of TgCDPK1 during in vitro infection. Chemical genetic studies indicate that TgCDPK1 acts independently of the previously characterized cGMP-dependent kinase (PKG), the primary target of trisubstituted pyrole and imidazopyridine kinase inhibitors that also block microneme secretion in T. gondii24,25. Correspondingly, PKG is predicted to be insensitive to PP1 derivatives (Fig. 4a)22. Collectively these findings indicate that both kinases are essential for efficient microneme secretion, possibly reflecting a hierarchical control of this important cellular pathway.

Bottom Line: Conditional suppression of TgCDPK1 revealed that it controls calcium-dependent secretion of specialized organelles called micronemes, resulting in a block of essential phenotypes including parasite motility, host-cell invasion, and egress.TgCDPK1 is conserved among apicomplexans and belongs to a family of kinases shared with plants and ciliates, suggesting that related CDPKs may have a function in calcium-regulated secretion in other organisms.Because this kinase family is absent from mammalian hosts, it represents a validated target that may be exploitable for chemotherapy against T. gondii and related apicomplexans.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology, Washington University School of Medicine, 660 S. Euclid Avenue, St Louis, Missouri 63110, USA.

ABSTRACT
Calcium-regulated exocytosis is a ubiquitous process in eukaryotes, whereby secretory vesicles fuse with the plasma membrane and release their contents in response to an intracellular calcium surge. This process regulates various cellular functions such as plasma membrane repair in plants and animals, the discharge of defensive spikes in Paramecium, and the secretion of insulin from pancreatic cells, immune modulators from lymphocytes, and chemical transmitters from neurons. In animal cells, serine/threonine kinases including cAMP-dependent protein kinase, protein kinase C and calmodulin kinases have been implicated in calcium-signal transduction leading to regulated secretion. Although plants and protozoa also regulate secretion by means of intracellular calcium, the method by which these signals are relayed has not been explained. Here we show that the Toxoplasma gondii calcium-dependent protein kinase 1 (TgCDPK1) is an essential regulator of calcium-dependent exocytosis in this opportunistic human pathogen. Conditional suppression of TgCDPK1 revealed that it controls calcium-dependent secretion of specialized organelles called micronemes, resulting in a block of essential phenotypes including parasite motility, host-cell invasion, and egress. These phenotypes were recapitulated by using a chemical biology approach in which pyrazolopyrimidine-derived compounds specifically inhibited TgCDPK1 and disrupted the parasite's life cycle at stages dependent on microneme secretion. Inhibition was specific to TgCDPK1, because expression of a resistant mutant kinase reversed sensitivity to the inhibitor. TgCDPK1 is conserved among apicomplexans and belongs to a family of kinases shared with plants and ciliates, suggesting that related CDPKs may have a function in calcium-regulated secretion in other organisms. Because this kinase family is absent from mammalian hosts, it represents a validated target that may be exploitable for chemotherapy against T. gondii and related apicomplexans.

Show MeSH
Related in: MedlinePlus