Date of Award

6-30-2016

Document Type

Open Access Thesis

Sub-Department

School of Medicine

First Advisor

Norma Frizzell

Abstract

We have identified the irreversible protein modification S-(2-succino)cysteine, a product of the Kreb’s cycle intermediate fumarate reacting with the thiol group of cysteine (Cys) residues, also termed succination. Succination is increased in adipose tissue of diabetic mice in vivo and in adipocytes matured in high glucose medium (25 mM) in vitro. Selenocysteine (Sec) is a less common but highly conserved amino acid that has major functions in redox chemistry. Sec possesses a lower pKa (~5.4) and is much more reactive in comparison to Cys. This project aimed to determine if succination occurred on Sec residues in two selenoproteins that play roles of maintaining intracellular redox environments and contain Sec in their active sites, thioredoxin reductase (TrxR) and glutathione peroxidase (GPx). We hypothesized that increased fumarate levels will lead to the succination of Sec and causing decreased enzymatic activity for each enzyme.

Two cellular models were used with elevated fumarate levels: (1) 3T3-L1 adipocytes matured in high glucose medium (25 mM) to mimic diabetic conditions and (2) 3T3-L1 fibroblasts where fumarase was knocked-down (Fh k/d) to mimic fumarase deficient cancers. As a positive control to demonstrate the ability to succinate TrxR in vitro, recombinant TrxR1 was incubated with the reactive fumarate ester, dimethyl fumarate (DMF). After analysis, a ~56% reduction in activity was detected due to succination of an active site residue, which was confirmed by mass spectrometric analyses.

Unexpectedly, TrxR activity was found to be significantly increased in 3T3-L1 adipocytes matured in 25 mM glucose compared to 5 mM glucose, despite no changes in protein levels. No statistically significant changes in TrxR activity were found in Fh k/d 3T3-L1 fibroblasts compared to controls. GPx activity was found to be significantly reduced in 3T3-L1 adipocytes matured in high glucose with no changes in levels of GPx protein. GPx activity was also analyzed in Fh k/d 3T3-L1 fibroblasts, however no significant changes were detected.

Overall, the results demonstrate that TrxR can be modified readily by reactive fumarate esters, leading to decreased enzymatic activity. However, endogenously produced fumarate does not appear to significantly contribute to TrxR or GPx modification in the models studied.

Rights

© 2016, Scott Lanci

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