iGIST—A Kinetic Bioassay for Pertussis Toxin Based on Its Effect on Inhibitory GPCR Signaling

Paramonov, Valeriy M.; Sahlgren, Cecilia; Rivero-Müller, Adolfo; Pulliainen, Arto T.

doi:10.1021/acssensors.0c01340

Cited by 14 publications

(22 citation statements)

References 41 publications

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“…A consequence of PT-mediated Gαi-ADP-ribosylation is its inactivation, which leads to disturbed cAMP signaling, because Gαi can no longer efficiently inhibit the adenylate cyclase. The iGIST bioassay has been developed to measure the effect of PT on Gαi signaling in cells [ 37 ]. The method is based on HEK293 cells that ectopically express the Gαi-coupled GPCR somatostatin receptor 2 (SSTR2), together with a luminescent cAMP probe (iGIST sensor cells).…”

Section: Resultsmentioning

confidence: 99%

“…Chinese hamster ovary cells strain K1 (CHO-K1, DSMZ, Braunschweig, Germany) were cultivated in DMEM and HAM’s F12 (1:1) supplemented with 5% heat-inactivated fetal calf serum (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), 1 mM sodium pyruvate and penicillin–streptomycin (1:100) (Thermo Fisher Scientific, Waltham, MA, USA). HEK293 cells (HEK-Gs/SSTR2_HA), ectopically expressing Gαi-coupled somatostatin receptor 2 (SSTR2) GPCR as well as a luminescence cAMP probe (GloSensor-22F, Promega, Mannheim, Germany) [ 37 ], were cultivated in DMEM/F12 (containing glutamine and sodium pyruvate) supplemented with 10% heat-inactivated fetal calf serum (Invitrogen) and penicillin–streptomycin (1:100) (Thermo Fisher Scientific). Cells were grown under humidified conditions at 37 °C with 5% CO 2 , and trypsinized and reseeded every two to three days for at most 25 times.…”

Section: Methodsmentioning

confidence: 99%

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Human Peptides α-Defensin-1 and -5 Inhibit Pertussis Toxin

Kling

Pulliainen

Barth

et al. 2021

Toxins

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Bordetella pertussis causes the severe childhood disease whooping cough, by releasing several toxins, including pertussis toxin (PT) as a major virulence factor. PT is an AB5-type toxin, and consists of the enzymatic A-subunit PTS1 and five B-subunits, which facilitate binding to cells and transport of PTS1 into the cytosol. PTS1 ADP-ribosylates α-subunits of inhibitory G-proteins (Gαi) in the cytosol, which leads to disturbed cAMP signaling. Since PT is crucial for causing severe courses of disease, our aim is to identify new inhibitors against PT, to provide starting points for novel therapeutic approaches. Here, we investigated the effect of human antimicrobial peptides of the defensin family on PT. We demonstrated that PTS1 enzyme activity in vitro was inhibited by α-defensin-1 and -5, but not β-defensin-1. The amount of ADP-ribosylated Gαi was significantly reduced in PT-treated cells, in the presence of α-defensin-1 and -5. Moreover, both α-defensins decreased PT-mediated effects on cAMP signaling in the living cell-based interference in the Gαi-mediated signal transduction (iGIST) assay. Taken together, we identified the human peptides α-defensin-1 and -5 as inhibitors of PT activity, suggesting that these human peptides bear potential for developing novel therapeutic strategies against whooping cough.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Human Peptides α-Defensin-1 and -5 Inhibit Pertussis Toxin

Kling

Pulliainen

Barth

et al. 2021

Toxins

Self Cite

View full text Add to dashboard Cite

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“…2 b). This assay is based on HEK293 cells that express the Giα-coupled somatostatin receptor 2 (SSTR2) as well as a luminescent cAMP probe 38 . Control samples treated with forskolin to stimulate adenylate cyclase activity and octreotide to activate SSTR2 which inhibits adenylate cyclase activity show a moderate increase in luminescence signal, i.e.…”

Section: Resultsmentioning

confidence: 99%

“…The iGIST bioassay was performed as previously described 38 . In brief, HEK293 cells that have been stably transfected with Giα-coupled somatostatin receptor 2 (SSTR2) GPCR were seeded as 60,000 cells/well into 96-well plates with light-tight walls in DMEM/F-12 (1:1) medium (Gibco), supplemented with fetal calf serum (10%) and Pen-Strep (1%) 1 day prior to the experiment.…”

Section: Methodsmentioning

confidence: 99%

“…Cells were lysed by freezing in ADP-ribosylation buffer (0.1 mM Tris-HCl (pH 7.6), 20 mM DTT and 0.1 µM ATP) or plus protease inhibitor complete (Roche, Basel, Switzerland) as described earlier 17,29,70 iGIST bioassay. The iGIST bioassay was performed as previously described 38 . In brief, HEK293 cells that have been stably transfected with Giα-coupled somatostatin receptor 2 (SSTR2) GPCR were seeded as 60,000 cells/well into 96-well plates with light-tight walls in DMEM/F-12 (1:1) medium (Gibco), supplemented with fetal calf serum (10%) and Pen-Strep (1%) 1 day prior to the experiment.…”

Section: Cell Culture and Intoxication Experimentsmentioning

confidence: 99%

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Pharmacological targeting of host chaperones protects from pertussis toxin in vitro and in vivo

Ernst

Mittler

Winkelmann

et al. 2021

Sci Rep

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Whooping cough is caused by Bordetella pertussis that releases pertussis toxin (PT) which comprises enzyme A-subunit PTS1 and binding/transport B-subunit. After receptor-mediated endocytosis, PT reaches the endoplasmic reticulum from where unfolded PTS1 is transported to the cytosol. PTS1 ADP-ribosylates G-protein α-subunits resulting in increased cAMP signaling. Here, a role of target cell chaperones Hsp90, Hsp70, cyclophilins and FK506-binding proteins for cytosolic PTS1-uptake is demonstrated. PTS1 specifically and directly interacts with chaperones in vitro and in cells. Specific pharmacological chaperone inhibition protects CHO-K1, human primary airway basal cells and a fully differentiated airway epithelium from PT-intoxication by reducing intracellular PTS1-amounts without affecting cell binding or enzyme activity. PT is internalized by human airway epithelium secretory but not ciliated cells and leads to increase of apical surface liquid. Cyclophilin-inhibitors reduced leukocytosis in infant mouse model of pertussis, indicating their promising potential for developing novel therapeutic strategies against whooping cough.

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The antiarrhythmic drugs amiodarone and dronedarone inhibit intoxication of cells with pertussis toxin

Jia,

Lietz,

Barth

et al. 2024

Naunyn-Schmiedeberg's Arch Pharmacol

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Pertussis toxin (PT) is a virulent factor produced by Bordetella pertussis, the causative agent of whooping cough. PT exerts its pathogenic effects by ADP-ribosylating heterotrimeric G proteins, disrupting cellular signaling pathways. Here, we investigate the potential of two antiarrhythmic drugs, amiodarone and dronedarone, in mitigating PT-induced cellular intoxication. After binding to cells, PT is endocytosed, transported from the Golgi to the endoplasmic reticulum where the enzyme subunit PTS1 is released from the transport subunit of PT. PTS1 is translocated into the cytosol where it ADP-ribosylates inhibitory α-subunit of G-protein coupled receptors (Gαi). We showed that amiodarone and dronedarone protected CHO cells and human A549 cells from PT-intoxication by analyzing the ADP-ribosylation status of Gαi. Amiodarone had no effect on PT binding to cells or in vitro enzyme activity of PTS1 but reduced the signal of PTS1 in the cell suggesting that amiodarone interferes with intracellular transport of PTS1. Moreover, dronedarone mitigated the PT-mediated effect on cAMP signaling in a cell-based bioassay. Taken together, our findings underscore the inhibitory effects of amiodarone and dronedarone on PT-induced cellular intoxication, providing valuable insights into drug repurposing for infectious disease management.

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iGIST—A Kinetic Bioassay for Pertussis Toxin Based on Its Effect on Inhibitory GPCR Signaling

Cited by 14 publications

References 41 publications

Human Peptides α-Defensin-1 and -5 Inhibit Pertussis Toxin

Human Peptides α-Defensin-1 and -5 Inhibit Pertussis Toxin

Pharmacological targeting of host chaperones protects from pertussis toxin in vitro and in vivo

The antiarrhythmic drugs amiodarone and dronedarone inhibit intoxication of cells with pertussis toxin

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