Amanda Raffa ’21 Major: Biology
John Kalhorn ’21 Major: Biology
Faculty Mentor: Fr. Nicanor Austriaco, Biolog

Kalhorn, John

Raffa, Amanda

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Yeast Bxi1p/Ybh3p is a pH-sensitive calcium channel in Escherichia coli.

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Providence College DigitalCommons@Providence Biology Student Scholarship Biology 4-22-2020 Yeast Bxi1p/Ybh3p is a pH-sensitive calcium channel in Escherichia coli. Amanda Raffa Providence College John Kalhorn Providence College Follow this and additional works at: https://digitalcommons.providence.edu/bio_students  Part of the Biology Commons Raffa, Amanda and Kalhorn, John, "Yeast Bxi1p/Ybh3p is a pH-sensitive calcium channel in Escherichia coli." (2020). Biology Student Scholarship. 3. https://digitalcommons.providence.edu/bio_students/3 This Poster is brought to you for free and open access by the Biology at DigitalCommons@Providence. It has been accepted for inclusion in Biology Student Scholarship by an authorized administrator of DigitalCommons@Providence. For more information, please contact dps@providence.edu. Yeast Bxi1p/Ybh3p is a pH-sensitive calcium channel in Escherichia coli.John Kalhorn, Amanda Raffa, James Mullin, and Nicanor Austriaco, O.P.Department of Biology, Providence College, Providence, RI 02918ABSTRACTYeast Bax inhibitor-1 (BXI1/YBH3) encodes a protein thatbelongs to the Bax Inhibitor (TMBIM) family of proteins,which has been linked to different tumor types in humanpatients. The crystal structure of a prokaryotic member of thefamily, BsYetJ, has revealed that the Bxi1 proteins are pHsensitive calcium leaks. Our laboratory has shown that Bxi1p islocalized to the yeast ER and vacuole and our genetic studiessuggest that the protein is a channel that controls the efflux ofcalcium from the ER. We have also over expressed Bxi1p inE.coli and have used a fura-2 based calcium assay to show thatthe protein facilitates the influx of extracellular calcium intothe cell. Further studies have suggested that the influx ofcalcium can be altered by the pH of the extracellularenvironment, and that Bxi1p functions as a generalized cationchannel. We have initiated a screen to identify small moleculeblockers for the channel in the hopes of identifying drugs thatwould kill cancer cells that are addicted to Bxi1p. Preliminaryresults suggest that Gadolinium is a potential molecularinhibitor of yBxi1p. [Our laboratory is supported by grantNIGMS R15 GM110578, awarded to N. Austriaco.]CONCLUSIONS• Our data suggests that yBxi1p-GFP is a calcium leak whenoverexpressed in E. Coli.• Gadolinium inhibits the flow of calcium in E. coli.FIGURE 1: The crystal structure of the Bax Inhibitor family of proteins reveals that they are pH-sensitive calcium leaks.INTRODUCTIONThe Bax inhibitor-1 (BXI1) gene, a member of the Bcl-2 family, was firstdiscovered by its ability to inhibit Bax-induced program cell death in the buddingyeast Saccharomyces cerevisiae [1]. This gene was found to be conserved in awide range of fungi, plant, and animal species [3].This conservation as well as itslink to serveral kinds of cancers when over expressed have spoken to theimportance of the gene and has led to its continued study. It is known that BI-1plays a role in the regulation of endoplasmic reticulum (ER) stress [4].We are working to determine the function of BXI1 in calcium transport into thecell. By using E.coli transformed with YBXI1p we can assess the transport ofcalcium directly into the cell using fura-2. Our data suggests that bxi1 is a pHsensitive calcium leak, that is inhibited by acidic pH and is enhanced by slightlybasic pH. Data also suggests that the calcium leak is inhibited by the Gadoliniumcation.FIGURE 2: yBxi1p-GFP can be overexpressed in E. Coli.LITERATURE CITED1. Q. Xu and J.C. Reed, "BAX inhibitor-1, a mammalian apoptosis suppressor identified by functional screening inyeast", Mol. Cell 1 (1998) :337-3462. J Cebulski et al., "Yeast Bax inhibitor, Bxi1p, is an ER-localized protein that links the unfolded protein responseand programmed cell death in Saccharomyces cerevisiae," PLOS One 20882 (2011): 6.3. H.J. Chae et al., "Evolutionarily conserved cytoprotection provided by Bax Inhibitor-1 (BI-1) homologs fromanimals, plants, and yeast", Gene 323 (2003): 101-1114. H.J. Chae et al., "BI-1 regulates an apoptosis pathway linked to endoplasmic reticulum stress", Mol. Cell 15 (2004):355-366- Ara+ AraFIGURE 2: A) E. coli cells transformed with a plasmid expressing BXI1-GFP were incubated with arabinose to express Bxi1p-GFP. Cells incubated in arabinose showed GFP fluorescence. B) Flow cytometry readings of GFP fluorescence after 6 hours. C) Bxi1p-GFP E. coli expressing cells were cultured for six hours, with and without arabinose. They were then collected, washed, and incubated with Fura-2 for two hours. 250 µl of cells were aliquoted into a 96-well plate. A Cytation3 Cell Imaging Multi-Mode Reader was used to image the cells at 340nm and 380nm, which correspond to the activation threshold of Fura-2. After a 6 minute baseline, 5 µl of CaCl2 was added to both conditions. (n=6; **** p<0.001)FIGURE 3: yBxi1p-GFP is permeable to calcium in a pH-dependent manner in E. coli.FIGURE 4: Gadolinium inhibits yBxi1p-GFP dependentcalcium permeability in E. coli.A) A)B)B)C)FIGURE 3: Bxi1p-GFP E. coliexpressing cells were cultured for six hours, with and without arabinose. They were then collected, washed, and incubated with Fura-2 for two hours. Cells were then placed in buffers of various pH.250 µl of cells were aliquoted into a 96-well plate. A Cytation3 Cell Imaging Multi-Mode Reader was used to image the cells at 340nm and 380nm, which correspond to the activation threshold of Fura-2. After a 6 minute baseline 5 µl of CaCl2 was added to both conditions. (n=3; **** p<0.001, ***p<0.005, **p<0.001, *p<0.05)FIGURE 4: A) Bxi1p-GFP E. coli expressing cells were cultured for six hours, with and without arabinose. They were then collected, washed, and incubated with Fura-2 for two hours. Cells were then placed in buffers of various pH. 250 µl of cells were aliquoted into a 96-well plate. A Cytation3 Cell Imaging Multi-Mode Reader was used to image the cells at 340nm and 380nm, which correspond to the activation threshold of Fura-2. After a 6 minute baseline, 5 µl of GdCl3 was added to both conditions and after an additional 12 minutes, 5 µl of CaCl2 was added. B) To ensure that Gadolinium does not kill the E. coli, cells were grown on plates of different concentrations to test their viability.+ CaCl2+ GdCl3

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Yeast Bxi1p/Ybh3p is a pH-sensitive calcium channel in Escherichia coli.

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