Analysis of a macrophage carbamylated proteome reveals a function in post-translational modification crosstalk

Abstract

Background. Lysine carbamylation is a biomarker of rheumatoid arthritis and kidney diseases. However, its cellular function is understudied due to the lack of tools for systematic analysis of this post-translational modification (PTM). Methods. We adapted a method to analyze carbamylated peptides by co-affinity purification with acetylated peptides based on the cross-reactivity of anti-acetyllysine antibodies. We integrated this method into a mass spectrometry-based multi-PTM pipeline to simultaneously analyze carbamylated and acetylated peptides in addition to phosphopeptides were enriched by sequential immobilized-metal affinity chromatography. Results. By testing the pipeline with RAW 264.7 macrophages treated with bacterial lipopolysaccharide, 7,299, 8,923 and 47,637 acetylated, carbamylated, and phosphorylated peptides were identified, respectively. Our analysis showed that carbamylation occurs on proteins from a variety of functions on sites with similar as well as distinct motifs compared to acetylation. To investigate possible PTM crosstalk, we integrated the carbamylation data with acetylation and phosphorylation data, leading to the identification 1,183 proteins that were modified by all 3 PTMs. Among these proteins, 54 had all 3 PTMs regulated by lipopolysaccharide and were enriched in immune signaling pathways, and in particular, the ubiquitin-proteasome pathway. We found that carbamylation of linear diubiquitin blocks the activity of the anti-inflammatory deubiquitinase OTULIN. Conclusions Overall, our data show that anti-acetyllysine antibodies can be used for effective enrichment of carbamylated peptides. Moreover, carbamylation may play a role in PTM crosstalk with acetylation and phosphorylation, and that it is involved in regulating ubiquitination in vitro .

Figure 1. Quantitative multi-post-translational modification analysis of RAW 264.7 cells treated with bacterial lipopolysaccharide.

(a) Workflow of the sample preparation procedure and proteomics analysis of phosphorylated, carbamylated and acetylated peptides. (b) Number of unique carbamylated, acetylated, and phosphorylated peptides. (c) Box plot of intensities of carbamylated peptides obtained from samples prepared with urea or sodium deoxycholate (SDC) denaturing. (d) Volcano plot of carbamylated peptides intensities from samples denatured with urea vs. SDC.

Figure 2. Carbamylated proteomics analysis of macrophages treated with bacterial lipopolysaccharide (LPS).

(a) Box plot of carbamylated peptide intensities of LPS-treated vs untreated group. (b) Principal component analysis of carbamylated peptides from LPS-treated and control samples. (c) Heatmap of carbamylated peptides of LPS-treated and control samples. (d) A DAVID functional-enrichment analysis of proteins with carbamylation sites significantly regulated by the LPS treatment.

Figure 3. Motif analysis of carbamylation site.

Sequence motif logo of carbamylated lysine (a) and acetylated lysine (b). Glutamic acid (c and d), phenylalanine (e and f), or aspartic acid (g and h) at −1 position from the carbamylated or acetylated lysine was fixed in the sequence logos. Phenylamine at +1 position from the carbamylated lysine was fixed in the sequence logo (i).

Figure 4. Number of proteins modified with carbamylation, acetylation, or phosphorylation and a functional enrichment of proteins commonly modified by all three post-translational modifications.

(a) Venn diagram of carbamylated, acetylated, or phosphorylated proteins. (b) A functional-enrichment analysis of the 1183 proteins commonly modified with all three post-translational modifications.

Figure 5. Protein-protein interaction network and functional enrichment analysis of proteins with acetylation, carbamylation and phosphorylation sites regulated by bacterial lipopolysaccharide.

The network contains 54 common proteins which had altered levels of carbamylation, acetylation, and phosphorylation by the LPS treatment. The bar graph shows the direction of regulation (p < 0.05) of the three modifications after LPS treatment. The network was enriched in proteins of the innate immune system, cytokine signaling pathway in immune system, and protein post-translation modification pathway using Reactome and they are highlighted the color of pink, blue, green, respectively. The line colors represent if the interactions were experimental determined (pink) and curated in databases (purple).

Figure 6. Ubiquitination carbamylation, acetylation, and phosphorylation sites, their regulation by bacterial lipopolysaccharide, and the effect of carbamylation in deubiquitination.

(a) Carbamylated, acetylated, or phosphorylated residues on ubiquitin and their regulation by the LPS treatment. (b) Interactions between lysine residues of M1-linear diubiquitin and OTULIN. Proximal ubiquitin and distal ubiquitin are shown in dark and light green ribbon structures, respectively. OTULIN ribbon structure is shown in blue. Lysine residues of M1-linear diubiquitin and residues of OTULIN in magenta and cyan, respectively. Met-1 of the distal ubiquitin, which is linked to the proximal ubiquitin C-terminus, is highlighted as green line structure. Hydrogen bonds, electrostatic interactions, and hydrophobic interactions are shown in green, orange, and pink dashed lines, respectively. All the interactions were identified using Discovery Studio Visualizer 4.5 program. (c) SDS-PAGE of unmodified and carbamylated M1-linear diubiquitin chains incubated with OTULIN. Image is representative of 3 replicates. (d) Mass spectra of unmodified and carbamylated M1-linear diubiquitin chains.