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

Abstract

Serotonylation has been identified as a novel protein post-translational modification (PTM) for decades, where an isopeptide bond is formed between the glutamine residue and serotonin through transamination. Transglutaminase 2 (also known as TGM2 or TGase2) was proven to act as the main writer enzyme for this PTM and a number of key regulatory proteins (including small GTPases, fibronectin, fibrinogen, serotonin transporter, and histone H3) have been characterized as the substrates of serotonylation. However, due to the lack of pan-specific antibody for serotonylated glutamine, the precise enrichment and proteomic profiling of serotonylation still remain challenging. In our previous research, we developed an aryldiazonium probe to label protein serotonylation in a bioorthogonal manner. This chemical biology tool can be utilized alternatively for the antibody-free enrichment of serotonylated proteins, which depends on a pH-controlled chemoselective rapid azo-coupling reaction (CRACR). Here, we report the application of a photoactive aryldiazonium-biotin probe for the global profiling of serotonylation proteome in cancer cells. Thus, over 500 serotonylated proteins were identified from HCT 116 cells. Importantly, a number of modification sites of these serotonylated proteins were determine, attributed to the successful application of our chemical proteomic approach. Overall, these findings provided new insights into the significant association between cellular protein serotonylation and cancer development, further suggesting that to target TGM2-mediated monoaminylation may serve as a promising strategy for cancer therapeutics.

Bioorthogonal Labeling and Enrichment of Histone Monoaminylation Reveal Its Accumulation and Regulatory Function in Cancer Cell Chromatin

“…Utilizing these tools, we have discovered that histone monoaminylation is highly enriched in breast and colon tumors, which plays a role in cancer epigenetic regulations through the crosstalk with H3K4 methylation (known as a key mark in cancer epigenetics) and steric hindrance . Importantly, these powerful chemical tools can be used as sensitive pan-specific antibodies for the global profiling of monoaminylation proteome. ,, In summary, we observed for the first time that histone monoaminylations were accumulated in tumor tissues, which may serve as a biomarker for cancer screening and diagnosis in clinic. Moreover, we uncovered the essential epigenetic functions of H3Q5ser in cancer development, which included recruiting the “reader” WDR5, stabilizing the epigenetic mark H3K4me3, and directly decompacting the chromatin structures.…”

“…To make probe 1 easier to use for the biomarker detection of tissue samples, we first transferred a commercial fluorophore, 6-aminofluorescein, to a fluorescent diazonium (Compound 3 ) through a one-step diazotization reaction (Figure B). Similar to probe 1 , 3 can react with serotonylated histones in a bioorthogonal manner based on the same chemistry, pH-controlled CRACR. − In comparison with 1 , 3 is much easier to make and does not require UV-activation. Importantly, 3 can be directly used for fluorescent and in-gel imaging of serotonylated targets, which is convenient for tissue sample analyses and has potential for high-throughput screening assays.…”

“…One effective strategy is to design chemical biology probes that can specifically react with the modified residues of histones via bioorthogonal reactions. In this study, we designed and developed diazonium and bicyclononyne (BCN) probes to label and enrich histone serotonylation (H3Q5ser) and dopaminylation (H3Q5dop), respectively, via the pH-controlled chemoselective rapid azo-coupling reaction (CRACR) − and strain-promoted oxidation-controlled cyclooctyne-1,2-quinone cycloaddition (SPOCQ) . Employing these powerful chemical tools, we have successfully uncovered that histone H3Q5 monoaminylations are enriched in breast and colorectal cancer tissues, which can exhibit regulatory functions through affecting H3K4 methylation and chromatin compaction status.…”