Date of Award
8-16-2024
Document Type
Open Access Thesis
Department
Biomedical Science
First Advisor
Norma Frizzell
Abstract
The immunometabolite itaconate accumulates during lipopolysaccharide (LPS) stimulation of macrophages and microglia. Itaconate non-enzymatically reacts with cysteine residues to generate 2,3-dicarboxypropylcysteine (2,3-DCP), referred to as protein dicarboxypropylation. The tricarboxylic acid (TCA) cycle metabolite fumarate non-enzymatically reacts with the amino acid cysteine to form S-(2-succino)-cysteine (2SC), resulting in irreversible protein succination. Since fumarate and itaconate levels dynamically change in activated immune cells, the levels of both 2SC and 2,3-DCP reflect the abundance of these metabolites and their capacity to modify protein structure and function. I generated esters of 2SC and 2,3-DCP from protein hydrolysates and used stable isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine the abundance of these in LPS-stimulated Highly Aggressively Proliferating Immortalized (HAPI) microglia. Itaconate derived 2,3-DCP, but not fumarate derived 2SC, increased in LPS-treated HAPI microglia. Stoichiometric measurements demonstrated that 2,3-DCP increased from 1.57 to 9.07% of total cysteines upon LPS stimulation. TCA and urea cycle metabolites were derivatized using 3-nitrophenylhydrazine (3-NPH) and quantified using LC-MS/MS. Itaconate significantly increased upon LPS stimulation, while fumarate levels remained relatively constant. Over a time course of LPS stimulation, itaconate levels peaked at 6 hours before rapidly declining. Adjusting the extracellular pH from 7.4 to 7.0 over a 24-hour period did not alter fumarate or 2SC levels. HAPI microglia were also treated with 20 M Na+/H+ exchanger (NHE) inhibitor cariporide and 30 M Na+/HCO3- cotransporter (NBC) inhibitor S0859 to determine if increased intracellular pH altered succination and dicarboxypropylation. The results show the drugs have no significant effect on intracellular or extracellular 2SC or 2,3-DCP levels. The neurotoxic metabolite quinolinic acid (Quin), produced by activated microglia upon LPS was also quantified in HAPI microglia using the ethyl esterification method.
Rights
© 2024, Justin Hunter Cox
Recommended Citation
Cox, J. H.(2024). Quantification of Immunometabolite Derived Protein Modifications During Mitochondrial Metabolic Stress. (Master's thesis). Retrieved from https://scholarcommons.sc.edu/etd/7818