Redox Regulation of Ras Proteins in Dictyostelium discoideum

Authors

Boris Castillo Chabeco, Florida International UniversityFollow

Document Type

Dissertation

Degree

Doctor of Philosophy (PhD)

Major/Program

Biology

First Advisor's Name

Lou W. Kim

First Advisor's Committee Title

Committee Chair

Second Advisor's Name

Lidia Kos

Second Advisor's Committee Title

Committee Member

Third Advisor's Name

Ophelia Weeks

Third Advisor's Committee Title

Committee Member

Fourth Advisor's Name

Fernando Noriega

Fourth Advisor's Committee Title

Committee Member

Fifth Advisor's Name

Xiaotang Wang

Fifth Advisor's Committee Title

Committee Member

Keywords

Ras, RasG, protein, sodC-, redox, ROS, Dictyostelium, superoxide, NKCD, magnesium

Date of Defense

3-19-2015

Abstract

Reactive oxygen species are a normal consequence of life in an aerobic environment. However when they deviate from the narrow permissible range in cells, oxidative damage can occur. Dictyostelium discoideum is a model organism ideal for the study of cell signaling events such as those affected by oxidative stress. It was previously shown that Ras signaling in Dictyostelium is affected by genetic inactivation of the antioxidant enzyme Superoxide dismutase C (SodC) and in vitro data suggests that the NKCD motif of Ras is the redox target of superoxide.

The main objective of this project was to determine the mechanism of superoxide mediated Ras regulation in vivo. To accomplish the main objective, we cloned, and in some cases, mutated different Ras proteins and later determined their activity in wild type and sodC^- cells. RasC and RasD showed normal activation in sodC^- cells, however RasG and RasS displayed high Ras activity. These last two Ras proteins contain the NKC¹¹⁸D motif inside the nucleotide binding region. A mutation of cysteine¹¹⁸ to alanine in RasG rendered the protein less active in sodC^- than the wild type RasG protein and a mutation alanine¹¹⁸ to cysteine in RasD conferred redox sensitivity to this small GTPase. Additionally, the propensity of RasG to be targeted by superoxide was evident when the environment of wild type cells was manipulated to induce the internal generation of superoxide through changes in the extracellular ion levels mainly magnesium. Lack of magnesium ions increased the intracellular level of superoxide and severely hampered directional cell migration. Chemotaxis of cells expressing RasG was negatively impacted by the absence of magnesium ions; however rasG^- cells did not seem to be affected in their ability to perform chemotaxis. The last experiment implies that RasG is an important mediator of cell signaling during oxidative stress, responsible for preventing cells from continuing their developmental program. Our study suggests that the cysteine residue in the NKCD motif is essential for mediating the redox sensitivity of Ras proteins in Dictyostelium and that RasG is an essential mediator of the response to oxidative stress in this organism.

Identifier

FI15032137

Recommended Citation

Castillo Chabeco, Boris, "Redox Regulation of Ras Proteins in Dictyostelium discoideum" (2015). FIU Electronic Theses and Dissertations. 1864.
https://digitalcommons.fiu.edu/etd/1864