Abstract
Lysine N-pyrrolation, converting lysine residues to Nϵ-pyrrole-l-lysine, is a recently discovered post-translational modification. This naturally occurring reaction confers electrochemical properties onto proteins that potentially produce an electrical mimic to DNA and result in specificity toward DNA-binding molecules such as anti-DNA autoantibodies. The discovery of this unique covalent protein modification provides a rationale for establishing the molecular mechanism and broad functional significance of the formation and regulation of Nϵ-pyrrole-l-lysine-containing proteins. In this study, we used microbeads coupled to pyrrolated or nonpyrrolated protein to screen for binding activities of human serum-resident nonimmunoglobin proteins to the pyrrolated proteins. This screen identified apolipoprotein E (apoE) as a protein that innately binds the DNA-mimicking proteins in serum. Using an array of biochemical assays, we observed that the pyrrolated proteins bind to the N-terminal domain of apoE and that oligomeric apoE binds these proteins better than does monomeric apoE. Employing surface plasmon resonance and confocal microscopy, we further observed that apoE deficiency leads to significant accumulation of pyrrolated serum albumin and is associated with an enhanced immune response. These results, along with the observation that apoE facilitates the binding of pyrrolated proteins to cells, suggest that apoE may contribute to the clearance of pyrrolated serum proteins. Our findings uncover apoE as a binding target of pyrrolated proteins, providing a key link connecting covalent protein modification, lipoprotein metabolism, and innate immunity.
Highlights
Lysine N-pyrrolation, converting lysine residues to N⑀-pyrrole-L-lysine, is a recently discovered post-translational modification
To determine the presence of serum nonimmunoglobin protein(s) that can bind pyrrolated proteins, normal human serum was incubated with beads coupled to either the native BSA or pyrrolated BSA, and the bound proteins were eluted and separated by SDS-PAGE under reducing conditions (Fig. 2A)
Nine peptides were found in the pulldown with pyrrolated BSA (pyrBSA), of which Ͼ29% could be attributed to apolipoprotein E (Table S1)
Summary
Lysine N-pyrrolation, converting lysine residues to N⑀-pyrrole-L-lysine, is a recently discovered post-translational modification. This naturally occurring reaction confers electrochemical properties onto proteins that potentially produce an electrical mimic to DNA and result in specificity toward DNAbinding molecules such as anti-DNA autoantibodies The discovery of this unique covalent protein modification provides a rationale for establishing the molecular mechanism and broad functional significance of the formation and regulation of N⑀-pyrrole-L-lysine– containing proteins. The pyrrole ring is an important structural motif found in a wide range of biologically active natural products and is highly susceptible to electrophilic attack and oxidation [13] It displays a variety of chemical properties, including – stacking and hydrogen-bonding interactions, that may contribute to the closer packing and higher density of pyrrole-containing molecules. Lysine N-pyrrolation is a physiologically relevant post-translational modification and could be an endogenous source of DNA mimic proteins These findings provide a rationale for establishing a molecular mechanism and broad functional significance of the formation and regulation of the pyrrolated proteins. We speculated the presence of an innate regulatory mechanism for the pyrrolated proteins having structural properties similar to DNA and tried to identify serum pyrrole-binding proteins
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