Non-enzymatic post-translational modification of lysine clusters in C2 domains

Presenter(s)

Cisloynny Beauchamp-Perez

Abstract or Description

C2 domains are membrane-binding motifs found in a wide range of proteins involved in signal transduction and membrane trafficking, including key proteins in neurotransmitter and hormone secretion. A large subset of C2 domains bind membranes containing the signaling lipid, phosphatidylinositol-(4,5)- bisphosphate (PIP2), via a conserved cluster of lysine residues. Because lysine residues can be non- enzymatically modified by reactive compounds such as lipid aldehydes formed during oxidative stress, we are investigating the susceptibility of C2 domain lysine clusters to modification by carbonyl-containing compounds. Previous research has shown that synaptotagmin-like protein 4 (Slp-4), a C2 domain protein, becomes carbonylated in alcoholic liver disease. Our in vitro results using the purified protein domain indicate that the lysine cluster is among several sites that react with the lipid aldehyde 4-hydroxynonenone, a major byproduct of cellular oxidative stress. Furthermore, when expressed in E. coli, some of the expressed Slp-4 C2A domain becomes phosphogluconoylated at the lysine cluster, a nonenzymatic modification that is normally found only at the low-pKa amino termini of His-tagged proteins. Using mass spectrometry, Western blotting, and cation exchange chromatography, we are now seeking to if the most reactive residue is indeed contained in the lysine cluster. Further investigation will include the study of other highly reactive metabolites and their reactivity towards C2 domain containing proteins. Nonenzymatic damage to secretory proteins as a result of oxidative stress represents an underexplored possible mechanism for deterioration of secretory pathways in diseases of exocytotic cells.