Chemical proteomic profiling of protein dopaminylation in colorectal cancer cells

Abstract

Histone dopaminylation is a newly identified epigenetic mark that plays a role in the regulation of gene transcription, where an isopeptide bond is formed between the fifth amino acid residue of H3 (i.e., glutamine) and dopamine. In our previous studies, we discovered that the dynamics of this post-translational modification (including installation, removal, and replacement) were regulated by a single enzyme, transglutaminase 2 (TGM2), through reversible transamination. Recently, we developed a chemical probe to specifically label and enrich histone dopaminylation via bioorthogonal chemistry. Given this powerful tool, we found that histone H3 glutamine 5 dopaminylation (H3Q5dop) was highly enriched in colorectal tumors, which could be attributed to the high expression level of TGM2 in colon cancer cells. Due to the enzyme promiscuity of TGM2, non-histone proteins have also been identified as targets of dopaminylation on glutamine residues, however, the dopaminylated proteome in cancer cells still remains elusive. Here, we utilized our chemical probe to enrich dopaminylated proteins from colorectal cancer cells in a bioorthogonal manner and performed the chemical proteomics analysis. Therefore, 425 dopaminylated proteins were identified, many of which are involved in nucleic acid metabolism and transcription pathways. More importantly, a number of modification sites of these dopaminylated proteins were identified, attributed to the successful application of our chemical probe. Overall, these findings shed light on the significant association between cellular protein dopaminylation and cancer development, further suggesting that to block the installation of protein dopaminylation may become a promising anti-cancer strategy.

pH-Controlled chemoselective rapid azo-coupling reaction (CRACR) enables global profiling of serotonylation proteome in cancer cells

“…Notably, there are a number of overlapping proteins between the serotonylation and dopaminylation proteome ( Fig. S3 ), 22 which further confirms the activation-substitution catalytic mechanism of TGM2 and the microenvironment-driven nature of monoaminylation. 17 Finally, we identified a variety of modification sites on the key serotonylated proteins ( Fig.…”

“…Identification of serotonylation sites. Unlike protein dopaminylation that was found on both glutamine and cystine, 22 serotonylation can only occur on glutamine residues via transamination. 33 The C4 position of 5-hydroxyindole is electron-rich and can be selectively modified by our aryldiazonium-biotin probe via pH-controlled CRACR.…”

“…This step was performed according to our previous protocols. 22 Briefly, the protein lanes on the gel corresponding to the experimental and control samples were excised by using a surgical blade. Each gel strip was then placed into a 1.5 mL centrifuge tube and sectioned into small fragments (0.5 X 0.5 mm).…”

“…The colorectal cancer cell lines were cultured based on the previously reported protocol with slight modifications. 22 Briefly, HCT 116 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% sodium pyruvate under 5% CO2 at 37 o C. Upon reaching 80% confluence, 10 mL of medium containing 50 µL of 100 mM serotonin hydrochloride was added to achieve a final concentration of 0.5 mM. The medium was gently mixed, and cells were collected through centrifugation after 12 hours of culture.…”

“…These steps were performed according to our previous protocols. 22 Briefly, the processed streptavidin beads were mixed with 20 μL of 2X loading buffer (Thermo Fisher Scientific) and heated at 98°C for 10 minutes. The samples were then centrifuged at 16000X g for 10 minutes, and the resulting supernatant was loaded onto the SDS-gel for SDS-PAGE analysis.…”