Quantification of the Immunometabolite Protein Modifications S-2-Succinocysteine and 2,3- Dicarboxypropylcysteine

Am J Physiol Endocrinol Metab. 2024 Feb 7. doi: 10.1152/ajpendo.00354.2023. Online ahead of print.ABSTRACTThe tricarboxylic acid (TCA) cycle metabolite fumarate non-enzymatically reacts with the amino acid cysteine to form S-(2-succino)cysteine (2SC), referred to as protein succination. 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. 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 thiols. We generated ethyl esters of 2SC and 2,3-DCP from protein hydrolysates and used stable isotope dilution mass spectrometry to determine the abundance of these in LPS-stimulated Highly Aggressively Proliferating Immortalized (HAPI) microglia). To quantify the stoichiometry of succination and dicarboxypropylation, reduced cysteines were alkylated with iodoacetic acid to form S-carboxymethylcysteine (CMC) which was then esterified. 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. This methodology to simultaneously distinguish and quantify both 2SC and 2,3-DCP w...
Source: American Journal of Physiology. Endocrinology and Metabolism - Category: Physiology Authors: Source Type: research