5-羟色胺衍生物5-PT的合成及5-羟色胺化修饰蛋白研究体系的建立

doi:10.3969/j.issn.1674-8115.2025.02.010

上海交通大学学报（医学版） ›› 2025, Vol. 45 ›› Issue (2): 211-221.doi: 10.3969/j.issn.1674-8115.2025.02.010

• 论著 · 技术与方法 • 上一篇

5-羟色胺衍生物5-PT的合成及5-羟色胺化修饰蛋白研究体系的建立

肖舒予(), 阿孜古丽·吐拉麦提, 杨岩, 张志刚, 杨小妹, 杜畅(), 张雪莉()

上海交通大学医学院附属仁济医院上海市肿瘤研究所，肿瘤系统医学全国重点实验室，上海 200240

收稿日期:2024-08-12 接受日期:2024-10-28 出版日期:2025-02-28 发布日期:2025-02-28
通讯作者: 杜畅,张雪莉 E-mail:xiao.sy@sjtu.edu.cn;cdu@shsci.org;xlzhang@shsci.org
作者简介:肖舒予（1999—），女，硕士生；电子信箱：xiao.sy@sjtu.edu.cn。
基金资助:
国家自然科学基金(82350123);上海市地方高水平高校创新团队(SHSMU-ZDCX20210802);上海交通大学医学院“双百人”项目(20181708)

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()

State Key Laboratory of Systems Medicine for Cancer, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200240, China

Received:2024-08-12 Accepted:2024-10-28 Online:2025-02-28 Published:2025-02-28
Contact: DU Chang, ZHANG Xueli E-mail:xiao.sy@sjtu.edu.cn;cdu@shsci.org;xlzhang@shsci.org
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)

摘要/Abstract

摘要：

目的·构建5-羟色胺化（serotonylation，5-HT化）修饰蛋白研究体系，为寻找和发现5-HT化修饰蛋白提供方法学基础。方法·基于癌症基因组图谱（The Cancer Genome Atlas，TCGA）数据库和基因型-组织表达（Genotype-Tissue Expression，GTEx）数据库分析编码5-HT化修饰的关键酶的基因转谷氨酰胺酶2（transglutaminase 2，TGM2）和编码5-羟色胺转运体（serotonin transporter，SERT）的基因溶质载体家族6（solute carrier family 6，SLC6A4）在正常生理组织和肿瘤组织中的表达情况。利用5-羟色胺盐酸盐分步合成5-羟色胺衍生物5-PT（5-propargyltryptamide），并利用傅里叶变换红外光谱（FT-IR）、核磁共振氢谱¹H-NMR、核磁共振碳谱¹³C-NMR和飞行时间质谱（TOF-MS）等手段进行分析和结构表征。通过流式细胞术检测5-PT在人胰腺癌细胞AsPC-1和小鼠免疫细胞［包括CD4⁺T细胞、CD8⁺T细胞以及髓系巨噬细胞（bone marrow derived macrophage，BMDM）］的胞内摄入情况。通过点击化学反应、免疫共沉淀以及质谱分析技术寻找和鉴定发生5-HT化修饰的蛋白，并进行京都基因和基因组数据库（Kyoto Encyclopedia of Genes and Genomes，KEGG）富集分析。结果·生物信息学相关分析显示TGM2和SLC6A4在生物体内正常组织和肿瘤组织广泛存在。流式细胞术结果显示合成的5-PT可通过细胞表面的SERT摄取进入人胰腺癌细胞AsPC-1和小鼠免疫细胞（包括CD4⁺T细胞、CD8⁺T细胞以及BMDM）。质谱分析数据显示，在各个细胞蛋白的5-PT处理组都富集到了丰富的5-HT化修饰蛋白。KEGG富集分析显示这些蛋白参与糖酵解和氨基酸合成相关通路。结论·利用合成的5-PT成功构建了5-HT化修饰蛋白的研究体系，在不同的细胞中富集得到多个5-HT化修饰的蛋白，为研究蛋白的5-HT化修饰及其功能提供了相对简单高效的研究手段。

关键词: 5-羟色胺, 5-羟色胺化, 5-羟色胺衍生物合成, 翻译后修饰

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

中图分类号:

R735.9

肖舒予, 阿孜古丽·吐拉麦提, 杨岩, 张志刚, 杨小妹, 杜畅, 张雪莉. 5-羟色胺衍生物5-PT的合成及5-羟色胺化修饰蛋白研究体系的建立[J]. 上海交通大学学报（医学版）, 2025, 45(2): 211-221.

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.

图/表 6

图1 TGM2 和 SLC6A4 在各种正常组织和肿瘤组织中的表达情况Note： A. The mRNA expression of TGM2 in different normal tissues based on GTEx database. B. The mRNA expression of TGM2 in pan-cancer tissues and adjacent normal tissues from the TCGA database. C. The mRNA expression of SLC6A4 in different normal tissues based on GTEx database. D. The mRNA expression of SLC6A4 in pan-cancer tissues and adjacent normal tissues from the TCGA database.

Fig 1 Expression of TGM2 and SLC6A4 in various normal tissues and pan-cancer tissues

图2 中间体 tert-butyl (2-(5-hydroxy-1H-indol-3-yl)ethyl)carbamate的结构表征Note： A. 1H-NMR spectrum of serotonin hydrochloride. B‒D. 1H-NMR spectrum (B), 13C-NMR spectrum (C), and FT-IR spectrum (D) of tert-butyl (2-(5-hydroxy-1H-indol-3-yl)ethyl)carbamate. *indicates solvent peaks; **indicates higher intensity solvent peak.

Fig 2 Structural characterization of the intermediate tert-butyl (2-(5-hydroxy-1H-indol-3-yl)ethyl)carbamate

图3 中间体 tert-butyl (2-(5-(prop-2-yn-1-yloxy)-1H-indol-3-yl)ethyl) carbamate的结构表征Note： A. 1H-NMR spectrum. B. 13C-NMR spectrum. C. FT-IR spectrum.

Fig 3 Structural characterization of the intermediate tert-butyl (2-(5-(prop-2-yn-1-yloxy)-1H-indol-3-yl)ethyl) carbamate

图4 5-PT的结构表征Note： A. 1H-NMR spectrum. B. 13C-NMR spectrum. C. FT-IR spectrum. D. TOF-MS spectrum.

Fig 4 Structural characterization of 5-PT

图5 5-PT通过SERT转运体进入细胞内的验证Note： A/B. Schematic illustration (A) and chemical reaction diagram (B) of intracellular 5-PT uptake detection. C. Representative flow cytometry analysis of 5-PT levels in AsPC-1 cells, CD4+ T cells, CD8+ T cells and CD11b+ cells (BMDM) under the indicated treatments for 48 h. D. The bar graph shows the quantification of 5-PT levels under different treatments, measured by mean fluorescence intensity (MFI).

Fig 5 Validation of 5-PT uptake into cells via SERT

图6 利用5-PT体系成功富集可发生5-HT化修饰的蛋白Note： A/B. Workflow (A) and chemical reaction diagram (B) of the enrichment of serotonylated proteins. C. Heatmap representation of label-free quantitation (LFQ) intensity data of the top 25 serotonylated proteins enriched from AsPC-1 cells, BMDMs, CD8+ T cells, and CD4+ T cells by using the 5-PT system. The heatmap colors (from red to blue) represent the fold changes in protein levels, from increased to decreased. D. KEGG enrichment analysis of the top 40 serotonylated proteins in AsPC-1 cells, BMDMs, CD8+ T cells, and CD4+ T cells by using the 5-PT system.

Fig 6 Successful enrichment of serotonylated proteins using 5-PT system

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5-羟色胺衍生物5-PT的合成及5-羟色胺化修饰蛋白研究体系的建立

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

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