Event Title

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.

This document is currently not available here.

Share

COinS
 
Apr 20th, 10:00 AM Apr 20th, 11:30 AM

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.
http://cornerstone.lib.mnsu.edu/urs/2015/poster_session_A/37