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Helicobacter pylori protein JHP0290 exhibits proliferative and anti-apoptotic effects in gastric epithelial cells.

Tavares R, Pathak SK - PLoS ONE (2015)

Bottom Line: JHP0290 existed in monomeric and dimeric forms in H. pylori cell extracts and culture broth.Recombinant purified JHP0290 (rJHP0290) also showed monomeric and dimeric forms, whereas the rJHP0290 C162A mutant exhibited only a monomeric form.CPT-induced caspase 3 activation was significantly reduced in the presence of rJHP0290.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden.

ABSTRACT
The influence of Helicobacter pylori infection on gastric epithelial cell proliferation, apoptosis and signaling pathways contributes to the development of infection-associated diseases. Here we report that JHP0290, which is a poorly functionally characterized protein from H. pylori, regulates multiple responses in human gastric epithelial cells. The differential expression and release of JHP0290 homologues was observed among H. pylori strains. JHP0290 existed in monomeric and dimeric forms in H. pylori cell extracts and culture broth. Recombinant purified JHP0290 (rJHP0290) also showed monomeric and dimeric forms, whereas the rJHP0290 C162A mutant exhibited only a monomeric form. The dimeric form of the protein was found to bind more efficiently to gastric epithelial cells than the monomeric form. The exposure of gastric epithelial cells to rJHP0290 induced proliferation in a dose-dependent manner. Faster progression into the cell cycle was observed in rJHP0290-challenged gastric epithelial cells. Furthermore, we detected an anti-apoptotic effect of rJHP0290 in gastric epithelial cells when the cells were treated with rJHP0290 in combination with Camptothecin (CPT), which is an inducer of apoptosis. CPT-induced caspase 3 activation was significantly reduced in the presence of rJHP0290. In addition, the activation of ERK MAPK and the transcription factor NFκB was observed in rJHP0290-challenged gastric epithelial cells lines. Our results suggest that JHP0290 may affect H. pylori-induced gastric diseases via the regulation of gastric epithelial cell proliferation and anti-apoptotic pathways.

No MeSH data available.


Related in: MedlinePlus

Binding of Monomeric and dimeric forms of rJHP0290 to AGS and MKN45 cells.(A) rJHP0290 Wt and rJHP0290 C162A in presence (+) or absence (-) of DTT in the SDS-PAGE sample buffer were immunoblotted with anti-JHP0290 antibody. Blot shown is the representative of results obtained in four independent experiments. (B) AGS and MKN45 cells were treated either with protein storage buffer (Ctrl) or with rJHP0290 Wt (2 μg/ml) or rJHP0290 C162A (2 μg/ml) for 15 min. Cells were washed, stained with anti-JHP0290 antibody and Alexa Fluor 488 conjugated secondary antibody followed by flow cytometry analysis. Result shown is the representative of results obtained in three independent experiments. (C) Flow cytometry results of the binding assay are depicted as the Mean Fluorescence intensity (MFI). Statistically significant differences are indicated by * (p < 0.05).
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pone.0124407.g002: Binding of Monomeric and dimeric forms of rJHP0290 to AGS and MKN45 cells.(A) rJHP0290 Wt and rJHP0290 C162A in presence (+) or absence (-) of DTT in the SDS-PAGE sample buffer were immunoblotted with anti-JHP0290 antibody. Blot shown is the representative of results obtained in four independent experiments. (B) AGS and MKN45 cells were treated either with protein storage buffer (Ctrl) or with rJHP0290 Wt (2 μg/ml) or rJHP0290 C162A (2 μg/ml) for 15 min. Cells were washed, stained with anti-JHP0290 antibody and Alexa Fluor 488 conjugated secondary antibody followed by flow cytometry analysis. Result shown is the representative of results obtained in three independent experiments. (C) Flow cytometry results of the binding assay are depicted as the Mean Fluorescence intensity (MFI). Statistically significant differences are indicated by * (p < 0.05).

Mentions: SDS-PAGE analysis of rJHP0290 revealed a single band of approximately 20 kDa in size (S2A Fig). However, under non-reducing conditions, another band of approximately 40 kDa in size was observed, suggesting that rJHP0290 was able to form a homodimer (S2A Fig). Immunoblotting with an anti-JHP0290 antibody further confirmed that the 20 kDa and 40 kDa bands were two forms of the same protein (Fig 2A). Because JHP0290 has a conserved cysteine at position 162 (Fig 1C), we assumed that the cysteine contributes to homodimer formation by rJHP0290 via disulfide bonds. To investigate the importance of the cysteine at position 162, we generated a mutant of rJHP0290 in which the Cysteine residue was mutated (rJHP0290 C162A). As expected, the mutant exhibited only a 20 kDa form under non-reducing conditions (Fig 2A and S2A Fig). Both the monomeric and dimeric forms of the JHP0290 protein were detected in culture broth and cellular extracts from H. pylori J99, indicating that both forms occurred naturally in the bacterium (S2B Fig). JHP0290 existed primarily in a monomeric form both when purified (rJHP0290) and in bacterial lysates or culture supernatants.


Helicobacter pylori protein JHP0290 exhibits proliferative and anti-apoptotic effects in gastric epithelial cells.

Tavares R, Pathak SK - PLoS ONE (2015)

Binding of Monomeric and dimeric forms of rJHP0290 to AGS and MKN45 cells.(A) rJHP0290 Wt and rJHP0290 C162A in presence (+) or absence (-) of DTT in the SDS-PAGE sample buffer were immunoblotted with anti-JHP0290 antibody. Blot shown is the representative of results obtained in four independent experiments. (B) AGS and MKN45 cells were treated either with protein storage buffer (Ctrl) or with rJHP0290 Wt (2 μg/ml) or rJHP0290 C162A (2 μg/ml) for 15 min. Cells were washed, stained with anti-JHP0290 antibody and Alexa Fluor 488 conjugated secondary antibody followed by flow cytometry analysis. Result shown is the representative of results obtained in three independent experiments. (C) Flow cytometry results of the binding assay are depicted as the Mean Fluorescence intensity (MFI). Statistically significant differences are indicated by * (p < 0.05).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4400171&req=5

pone.0124407.g002: Binding of Monomeric and dimeric forms of rJHP0290 to AGS and MKN45 cells.(A) rJHP0290 Wt and rJHP0290 C162A in presence (+) or absence (-) of DTT in the SDS-PAGE sample buffer were immunoblotted with anti-JHP0290 antibody. Blot shown is the representative of results obtained in four independent experiments. (B) AGS and MKN45 cells were treated either with protein storage buffer (Ctrl) or with rJHP0290 Wt (2 μg/ml) or rJHP0290 C162A (2 μg/ml) for 15 min. Cells were washed, stained with anti-JHP0290 antibody and Alexa Fluor 488 conjugated secondary antibody followed by flow cytometry analysis. Result shown is the representative of results obtained in three independent experiments. (C) Flow cytometry results of the binding assay are depicted as the Mean Fluorescence intensity (MFI). Statistically significant differences are indicated by * (p < 0.05).
Mentions: SDS-PAGE analysis of rJHP0290 revealed a single band of approximately 20 kDa in size (S2A Fig). However, under non-reducing conditions, another band of approximately 40 kDa in size was observed, suggesting that rJHP0290 was able to form a homodimer (S2A Fig). Immunoblotting with an anti-JHP0290 antibody further confirmed that the 20 kDa and 40 kDa bands were two forms of the same protein (Fig 2A). Because JHP0290 has a conserved cysteine at position 162 (Fig 1C), we assumed that the cysteine contributes to homodimer formation by rJHP0290 via disulfide bonds. To investigate the importance of the cysteine at position 162, we generated a mutant of rJHP0290 in which the Cysteine residue was mutated (rJHP0290 C162A). As expected, the mutant exhibited only a 20 kDa form under non-reducing conditions (Fig 2A and S2A Fig). Both the monomeric and dimeric forms of the JHP0290 protein were detected in culture broth and cellular extracts from H. pylori J99, indicating that both forms occurred naturally in the bacterium (S2B Fig). JHP0290 existed primarily in a monomeric form both when purified (rJHP0290) and in bacterial lysates or culture supernatants.

Bottom Line: JHP0290 existed in monomeric and dimeric forms in H. pylori cell extracts and culture broth.Recombinant purified JHP0290 (rJHP0290) also showed monomeric and dimeric forms, whereas the rJHP0290 C162A mutant exhibited only a monomeric form.CPT-induced caspase 3 activation was significantly reduced in the presence of rJHP0290.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden.

ABSTRACT
The influence of Helicobacter pylori infection on gastric epithelial cell proliferation, apoptosis and signaling pathways contributes to the development of infection-associated diseases. Here we report that JHP0290, which is a poorly functionally characterized protein from H. pylori, regulates multiple responses in human gastric epithelial cells. The differential expression and release of JHP0290 homologues was observed among H. pylori strains. JHP0290 existed in monomeric and dimeric forms in H. pylori cell extracts and culture broth. Recombinant purified JHP0290 (rJHP0290) also showed monomeric and dimeric forms, whereas the rJHP0290 C162A mutant exhibited only a monomeric form. The dimeric form of the protein was found to bind more efficiently to gastric epithelial cells than the monomeric form. The exposure of gastric epithelial cells to rJHP0290 induced proliferation in a dose-dependent manner. Faster progression into the cell cycle was observed in rJHP0290-challenged gastric epithelial cells. Furthermore, we detected an anti-apoptotic effect of rJHP0290 in gastric epithelial cells when the cells were treated with rJHP0290 in combination with Camptothecin (CPT), which is an inducer of apoptosis. CPT-induced caspase 3 activation was significantly reduced in the presence of rJHP0290. In addition, the activation of ERK MAPK and the transcription factor NFκB was observed in rJHP0290-challenged gastric epithelial cells lines. Our results suggest that JHP0290 may affect H. pylori-induced gastric diseases via the regulation of gastric epithelial cell proliferation and anti-apoptotic pathways.

No MeSH data available.


Related in: MedlinePlus