Journal of Shanghai Jiao Tong University (Medical Science) >

2025 , Vol. 45 >Issue 2: 211 - 221

DOI: https://doi.org/10.3969/j.issn.1674-8115.2025.02.010

Techniques and methods

Synthesis of the serotonin derivative 5-PT and establishment of a research system for protein serotonylation

  • XIAO Shuyu ,
  • TULAMATI Aziguli ,
  • YANG Yan ,
  • ZHANG Zhigang ,
  • YANG Xiaomei ,
  • DU Chang ,
  • ZHANG Xueli
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  • State Key Laboratory of Systems Medicine for Cancer, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200240, China
ZHANG Xueli, E-mail: xlzhang@shsci.org
DU Chang, E-mail: cdu@shsci.org.

Received date: 2024-08-12

  Accepted date: 2024-10-28

  Online published: 2025-02-28

Supported by

National Natural Science Foundation of China(82350123);Innovative Research Team of High-level Local Universities in Shanghai(SHSMU-ZDCX20210802);"Two-hundred Talents" Program of Shanghai Jiao Tong University School of Medicine(20181708)

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Abstract

Objective ·To establish a research framework for serotonylation of proteins and to provide a methodological basis for the identification of serotonylated proteins. Methods ·The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases were used to analyze the expression of the transglutaminase 2 (TGM2) gene, which encodes the key enzyme for serotonylation, and the solute carrier family 6 (SLC6A4) gene, which encodes the serotonin transporter (SERT), in normal and pan-cancer tissues. 5-Propargyltryptamide (5-PT), a serotonin derivative, was synthesized stepwise from serotonin hydrochloride, and its structure was characterized by the Fourier transform infrared spectroscopy (FT-IR), proton nuclear magnetic resonance (1H-NMR), carbon nuclear magnetic resonance (13C-NMR), and time-of-flight mass spectrometry (TOF-MS). The intracellular uptake of 5-PT in the human pancreatic cancer cell line AsPC-1 and mouse immune cells, including CD4+ T cells, CD8+ T cells, and bone marrow-derived macrophages (BMDMs), was detected by using flow cytometry. Click chemistry, co-immunoprecipitation, and mass spectrometry analysis techniques were employed to identify serotonylated proteins, and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed. Results ·Bioinformatics analysis indicated that TGM2 and SLC6A4 were widely expressed in various normal tissues and across pan-cancer tissues. The flow cytometry results showed that the synthesized 5-PT can be taken up into the human pancreatic cancer cell line AsPC-1 and mouse immune cells, including CD4+ T cells, CD8+ T cells, and BMDMs, via the SERT. Mass spectrometry analysis data showed that a significant amount of serotonylated proteins were enriched in various cells treated with 5-PT. KEGG enrichment analysis revealed that these proteins were involved in important pathways related to glycolysis and amino acid synthesis. Conclusion ·By using the synthesized 5-PT, multiple serotonylated proteins are enriched in various cell types. A research system for identifying serotonylated proteins has been successfully established, providing a relatively simple and efficient method for studying protein serotonylation.

Key words： 5-hydroxytryptamine (5-HT); serotonylation; synthesis of the serotonin derivative; post-translational modification

Cite this article

XIAO Shuyu , TULAMATI Aziguli , YANG Yan , ZHANG Zhigang , YANG Xiaomei , DU Chang , ZHANG Xueli . Synthesis of the serotonin derivative 5-PT and establishment of a research system for protein serotonylation[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2025 , 45(2) : 211 -221 . DOI: 10.3969/j.issn.1674-8115.2025.02.010

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