Distinct Palmitoylation of FoxP3 Regulates the Function of Regulatory T Cells via Palmitoyltransferases
Unique Foxp3 Palmitoylation Regulates Tregs Function through Palmitoyltransferases
Regulatory T cells (Tregs) play a crucial role in maintaining immune homeostasis and preventing excessive immune responses. As important suppressors of immune reactions, Tregs help maintain immune tolerance, thereby preventing autoimmune diseases. However, in the tumor microenvironment (TME), Tregs weaken the activity of effector cells through various mechanisms, thus promoting tumor occurrence and development. Foxp3 (Forkhead box protein P3) is a key transcription factor for Tregs, and its expression is critical for Tregs development and function. It is well known that Foxp3 can be regulated through various post-translational modifications (including phosphorylation, ubiquitination, glycosylation, and acetylation), but it remains unknown whether palmitoylation, as a post-translational modification, regulates Foxp3 function under physiological conditions and in the TME.
Research Background and Objectives
Palmitoylation is a reversible lipid post-translational modification that adds a 16-carbon palmitoyl group to the cysteine residues of proteins via a thioester bond. Palmitoylation is crucial for regulating protein subcellular localization, protein-protein interactions, stability, and signal transduction activity. Recent studies have identified key roles in the palmitoylation process, particularly the family of 24 palmitoyltransferases containing the Asp-His-His-Cys (DHHC) motif, which are responsible for catalyzing the addition of palmitoyl groups to target proteins. Additionally, the discovery of various depalmitoylating enzymes (such as APT1/2, PPT1/2, and ABHD17A/B/C) has revealed the process of removing palmitoyl groups. The dynamic balance between palmitoylation and depalmitoylation is critical for regulating protein function and coordinating cellular signaling. This study aims to detect Foxp3 palmitoylation and its impact on anti-tumor immune responses.
Research Team and Publication Information
This study was jointly completed by multiple teams from the Second Affiliated Hospital of Xinxiang Medical University, the Institute of Mental Health and Neuroscience, Henan Molecular Diagnosis and Laboratory Medicine Collaborative Innovation Center, and other institutions. The main authors include Binhu Zhou, Mengjie Zhang, Haoyuan Ma, Ying Wang, and Eryan Kong. This research was published in the journal “Cellular & Molecular Immunology” on May 8, 2024.
Research Process and Experimental Methods
The study first determined the palmitoylation status of Foxp3 in vitro by overexpressing Foxp3-Myc in HEK293T cells, using ABE (Acyl-Biotin Exchange) and Acyl-RAC (Acyl-Resin-Assisted Capture) experiments. The results indicated that Foxp3 indeed undergoes palmitoylation. Next, lymph nodes from C57BL/6 mice were collected for protein extraction and ABE detection, with Western blot analysis showing that Foxp3 is also palmitoylated in lymph nodes.
To identify specific palmitoylation sites on Foxp3, the study designed and expressed Flag-tagged Foxp3 protein for mass spectrometry analysis. Results showed that Foxp3 is palmitoylated at five cysteine residues: Cys204, Cys218, Cys280, Cys281, and Cys424, with each palmitoylation event causing a 238 Da increase in protein molecular weight. In further validation experiments, various Foxp3 mutants were expressed in HEK293T cells, and palmitoylation levels were measured by Acyl-RAC. Results showed that alanine substitutions at these cysteine residues reduced Foxp3 palmitoylation levels, with the 5CA mutant (all five cysteines mutated) almost completely eliminating palmitoylation.
Next, the study analyzed the interaction between Foxp3 and various palmitoyltransferases through co-immunoprecipitation (Co-IP) experiments, confirming physical interactions between Foxp3 and DHHC2, DHHC3, DHHC7, DHHC13, DHHC17, DHHC19, and DHHC23. Quantitative real-time PCR analysis (qRT-PCR) found that among these palmitoyltransferases, DHHC2, DHHC3, and DHHC7 were abundantly expressed in Tregs from spleen and tumor tissues.
To further explore the role of these palmitoyltransferases in Foxp3 palmitoylation, the study conducted multiple experiments, including Co-IP and Acyl-RAC analyses. Results showed that overexpression of DHHC2, DHHC3, or DHHC7 alone significantly increased Foxp3 palmitoylation levels.
Research Results and Conclusions
To further investigate the function of Foxp3 palmitoylation in vivo, the study also generated DHHC2-, DHHC3-, and DHHC7-knockout mice. Flow cytometry data showed that DHHC2 knockout led to decreased Foxp3 protein expression in lymph nodes and spleen. Using CRISPR/Cas9 gene editing technology, the study further analyzed the three palmitoyltransferases, showing that knockout of DHHC3 or DHHC7 also significantly reduced Foxp3 protein expression levels in Tregs.
Finally, the study confirmed this phenomenon in Foxp3-Cre mice, demonstrating that Foxp3 palmitoylation plays an important role in the suppressive function of Tregs in the TME. By injecting YUMM3.3 melanoma cells intratumorally, the study found that mice specifically lacking DHHC2 exhibited tumor growth inhibition and showed significantly reduced Foxp3 expression in tumor-infiltrating Tregs.
Research Significance and Value
This study reveals for the first time the dynamics of Foxp3 palmitoylation and its crucial role in anti-tumor immunity. By analyzing the regulation of Foxp3 function by various palmitoyltransferases, the research provides new insights for developing immunotherapies targeting Treg regulation.
The results of this study not only deepen our understanding of Foxp3 and Tregs but also provide important references for future research on tumor immune evasion mechanisms. The methodologies used in the study (such as mass spectrometry analysis, CRISPR/Cas9 gene editing) also demonstrate a high degree of integration between scientific innovation and experimental design, and the research conclusions have broad potential for further research and clinical applications.