Glutathionylation of Actin in the Dictyostelium Cytoskeleton
Location
CSU Ballroom
Start Date
20-4-2015 10:00 AM
End Date
20-4-2015 11:30 AM
Student's Major
Chemistry and Geology
Student's College
Science, Engineering and Technology
Mentor's Name
Rebecca Moen
Mentor's Email Address
rebecca.moen@mnsu.edu
Mentor's Department
Chemistry and Geology
Mentor's College
Science, Engineering and Technology
Description
Glutathione (GSH) is the most abundant antioxidant in living organisms. It is a small tripeptide composed of three amino acid residues; glutamate, cysteine and glycine. GSH can be covalently linked to proteins at cysteine (Cys) amino acid residues, a process called glutathionylation. Glutathionylation has been shown to protect Cys from irreversible oxidation. However, it has more recently been shown to have detrimental effects on protein function. The cytoskeleton can be considered the “skeleton” of every cell and gives shape and support. The purpose of this research is to investigate targets of glutathionylation in the cytoskeleton. My working hypothesis is that actin, the major protein component of the cytoskeleton, will be modified by GSH increasing the molecular weight of actin by 305 Da per GSH attached. Dictyostelium discoideum (Dicty) was chosen as the model organism to study cytoskeletal proteins and their interactions. Dicty AX2 cells were cultured in HL5 axenic media until reaching a cell density of 2 x107 cells/mL and were treated with varying concentrations of GSH for one hour. The Dicty cells were lysed and the cytoskeleton was isolated. The cytoskeleton proteins were separated by gradient PAGE and transferred to a nitrocellulose membrane. Western blotting was employed using primary antibodies against either actin or glutathione. The anti-glutathione antibody recognized glutathionylated proteins. Western blots were compared to identify actin glutathionylation. Amount of actin glutathionylated was quantified by comparison to an untreated actin control. My research project provided a better understanding of the targets of glutathionylation within the cytoskeleton.
Glutathionylation of Actin in the Dictyostelium Cytoskeleton
CSU Ballroom
Glutathione (GSH) is the most abundant antioxidant in living organisms. It is a small tripeptide composed of three amino acid residues; glutamate, cysteine and glycine. GSH can be covalently linked to proteins at cysteine (Cys) amino acid residues, a process called glutathionylation. Glutathionylation has been shown to protect Cys from irreversible oxidation. However, it has more recently been shown to have detrimental effects on protein function. The cytoskeleton can be considered the “skeleton” of every cell and gives shape and support. The purpose of this research is to investigate targets of glutathionylation in the cytoskeleton. My working hypothesis is that actin, the major protein component of the cytoskeleton, will be modified by GSH increasing the molecular weight of actin by 305 Da per GSH attached. Dictyostelium discoideum (Dicty) was chosen as the model organism to study cytoskeletal proteins and their interactions. Dicty AX2 cells were cultured in HL5 axenic media until reaching a cell density of 2 x107 cells/mL and were treated with varying concentrations of GSH for one hour. The Dicty cells were lysed and the cytoskeleton was isolated. The cytoskeleton proteins were separated by gradient PAGE and transferred to a nitrocellulose membrane. Western blotting was employed using primary antibodies against either actin or glutathione. The anti-glutathione antibody recognized glutathionylated proteins. Western blots were compared to identify actin glutathionylation. Amount of actin glutathionylated was quantified by comparison to an untreated actin control. My research project provided a better understanding of the targets of glutathionylation within the cytoskeleton.
Recommended Citation
Grosberg, Benjamin. "Glutathionylation of Actin in the Dictyostelium Cytoskeleton." Undergraduate Research Symposium, Mankato, MN, April 20, 2015.
https://cornerstone.lib.mnsu.edu/urs/2015/poster_session_A/37
