Authors:

Amber Johnson

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About Paper:

Hydrogen sulfide (HS) is present at high concentrations in the colon, and elevated levels are linked to diseases like colon cancer and inflammatory bowel disease. Research in our laboratory has shown that H,S disrupts colonic Wnt signaling, which is necessary for maintaining colon stem cells. Since H,S can react with disulfide bonds in proteins, we hypothesized that H2S impairs Wnt signaling by disrupting essential disulfide bonds in Wnt ligands. To determine if reduction of disulfide bonds is sufficient to disrupt Wnt signaling, we treated HEK293 cells with the membrane-impermeable reductant, tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and measured expression of the Wnt target gene Axin2, using quantitative polymerase chain reaction. We observed decreased Axin2 expression in cells exposed to TCEP, which supports disulfide bond reduction as a mechanism for Wnt signaling disruption. Next, we investigated whether H,S treatment reduces disulfides in the Wnt pathway ligands, WNT3a and RSPO, since previous data had shown that culturing HEK293 cells in H,S-exposed medium was sufficient to decrease Wnt signaling. We monitored the cysteine redox status of RSPO in medium +100 ppm H,S exposure, followed by tagging with methyl-PEG maleimide. Changes observed by western blot analysis are currently being characterized. We are also investigating the effect of HS exposure on the cysteine redox status in WNT3a by Western blot analysis and complementing the study with a luciferase-based assay that reports on Wnt activity. Our current model for the effects of H,S on Wnt signaling will be discussed.

Source:

Chicago Area Undergraduate Research Symposium

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Co-authors:

Amber Johnson