Abstract
Background
A balance between positive and negative regulation of the Toll-like receptor (TLR) signaling pathway is required to prevent harmful or inappropriate inflammatory responses. Although TLR9 has been reported to be expressed in many mammalian tissues and cells, its function and exact mechanism are not fully understood. Some protein post-translational modifications (PTMs), such as phosphorylation and ubiquitination, are involved in the development of tumors. An important PTM, methylation, which controls the TLR9 signaling pathway, still remains unclear.
Methods
Using human prostate cancer (PCa) cells PC3 and clinical PCa samples, this study employed LipidTOX staining, RNA interference, Western blot, immunoprecipitation, GST fusion protein sedimentation, and immunofluorescence techniques to investigate the molecular mechanism of TLR9 protein arginine methylation modification in PCa ferroptosis.
Results
In this study, we found that the total level of methylation was reduced in PCa, the expression of TLR9 was decreased, and the level of reactive oxygen species (ROS) was increased in PCa cells, indicating that TLR9 may be involved in the regulation of ferroptosis in PCa. In addition, protein arginine methyltransferase 5 and 9 (PRMT5 and PRMT9) are recruited to mediate methylation modification of TLR9 after stimulation of unmethylated cytosine guanine [oligodeoxynucleotide (ODN)], but PRMT9 has the strongest effect, which prompted us to focus on PRMT9. Decreased expression of PRMT9 down-regulates arginine (R) methylation levels of TLR9. Methionine 260 (G260) mediates PRMT9 to catalyze arginine methylation of TLR9 on R216. In vitro transcriptomic results showed that the expression level of acyl synthetase medium chain family 1 (ACSM1) in cancer tissues was significantly higher than that in the para-cancer group. BODIPY-C11 immunofluorescence results indicated that TLR9 methylation could regulate the growth of PC3 cells mediated by ACSM1. In addition, arginine methylation enhancement of TLR9 induced by PRMT5 or 9 increased the transcription activity of nuclear factor erythroid 2-related factor 2 (NRF2), and arginine methylation of TLR9 at sites 216 and 305 mediated the interaction between TLR9 and NRF2 and enhanced the transcription activity of NRF2. PRMT9 mutation and (R216K) mutant TLR9 decreased NRF2 transcriptional activity, and R216 mutation decreased PRMT9-mediated arginine methylation GPX4 and other related iron-death protein levels.
Conclusions
Our study reveals a key role of TLR9 protein arginine methylation in the regulation of ferroptosis, which may provide a therapeutic strategy for controlling the development and progression of PCa.
Keywords: Arginine methylation, PRMT9, Toll-like receptor 9 (TLR9), nuclear factor erythroid 2-related factor 2 (NRF2), GPX4
Highlight box.
Key findings
• The study discovers Toll-like receptor 9 (TLR9) can undergo arginine methylation modification, and reveals a key role of TLR9 protein arginine methylation in the regulation of ferroptosis.
What is known and what is new?
• TLR9 can undergo phosphorylation and ubiquitination modifications, and these modifications can regulate vital movement.
• We have identified a previously unreported novel modification of TLR9, and this modification can regulate tumor initiation and progression.
What is the implication, and what should change now?
• This suggests that post-translational modifications of proteins may serve as novel therapeutic targets for tumor regulation, calling for greater collaboration among researchers to identify and advance these mechanisms.
Introduction
Cancer cells have different metabolic needs than normal cells because they are highly proliferative and need to survive in a microenvironment with limited nutrient supply. Changes in material metabolism are one of the most common adaptive abilities shown by cancer cells (1). PCa is characterized by a high dependence on reprogramming of lipid metabolic pathways (2), which will inevitably lead to disruption of the balance of lipid metabolism, namely ferroptosis disorder (3-5). Ferroptosis is a controlled form of cell death characterized by lipid peroxidation and iron-dependent reactive oxygen species (ROS) accumulation that differs morphologically and mechanistically from apoptosis and other forms of cell death. Ferroptosis imbalances in prostate cancer (PCa) lead to disrupted cell clearance and altered cell death mechanisms, which play a key role in shaping the tumor microenvironment and influencing cancer outcomes. Among Toll-like receptors (TLRs), TLR9 is a key member, generating five isoforms in the human body by selective shearing (6,7). TLR9 binds to the non-methylated CpG motif of bacterial DNA and activates the transcription factor NF-κB via the MyD88 adaptor protein (8-10). Compared with most TLR family members localized in the plasma membrane, TLR9 is an intracellular receptor mainly localized in organelles (11). After binding to CpG DNA, TLR9 is cleaved and processed and translocated to endosomes and lysosomes, which initiate downstream signal transduction after binding to MyD88. TLR9 plays a double-edged role in many tumor cells. A recent study showed that in patients with triple-negative breast cancer, neutrophil extracellular trap (NETs) mediated TLR9/Merlin signaling pathway to resist ferroptosis and promote the progression of triple-negative breast cancer, indicating a close relationship between TLR9 and ferroptosis. TLR9 may be a key to clinical diagnosis and treatment for cancer (12).
Protein post-translational modification (PTM) processes include phosphorylation, glycosylation, lipid modification, ubiquitination, methylation, acetylation, and REDOX-related modifications (13,14). Protein methylation can occur on eight amino acid residues, such as lysine, aspartate, histidine, glutamate, cysteine, arginine, and glutamine. Protein methylation (15-17) is classified according to the atoms to which the methyl group binds, including N-methylation, O-methylation, and S-methylation. There are three types of methylation states, including monomethylation, dimethylation, and trimethylation, which occur on the N atom. K residues can undergo monomethylation, dimethylation, and trimethylation, but R residues can only undergo monomethylation and dimethylation. Two different forms of dimethylation can occur on the R-side chain, including asymmetric trimethylation and symmetric demethylation (18). Protein methylation is mainly catalyzed by two classes of protein lysine methyltransferase (PKMTs) and protein arginine methyltransferase PRMTs, and PRMTs include nine family members (PRMT1–9). Protein methylation plays an important role in the logical processes of biological variation, such as transcriptional regulation, signal transduction, DNA repair, gene activation, gene suppression and RNA regulation (19). Reduced expression of PRMT7 can inhibit ferroptosis of lung glial cells caused by inflammatory monocytes and promote monocyte exosmosis and migration in patients with chronic obstructive pulmonary disease (20). In addition, PRMT4 has been shown to increase ferroptosis and promote doxorubicin-induced cardiomyopathy by inhibiting the nuclear factor erythroid 2-related factor 2 (NRF2)/GPX4 pathway (21). Histone methyltransferase 1 (SETDB1) can reprogram lung cancer epithelial-mesenchymal transformation genes and induce ferroptosis (22). In addition, lysine-specific methyltransferase 2B (KMT2B) of histone H3 lysine 4 (H3K4) methyltransferase promotes myocardial ischemia reperfusion by accelerating Ribosomal protein kinase transcripting and activating the TNF-α/NOX2 pathway to inhibit ferroptosis (23). In cardiomyocytes, GSK-J4, a double inhibitor of histone lysine demethylase 61/6B (KDM61/6B), can reduce the production of acyl-CoA synthetase long chain family member 4 (ACSL4) and lipid peroxidation by inhibiting the demethylation of histone H3 lysine 27 (H3K27), thereby reducing ACSL4-induced hypersensitivity to iron sag (24). In this study, we found that protein arginine methylation regulates ferroptosis in PCa. After ODN treatment, G260 of PRMT9 binds to and methylates R216 of recruits and activates NRF2 to regulate GPX4-mediated ferroptosis process through methylation of R216 and R305 of TLR9, thereby affecting PCa metabolism, growth and survival. In conclusion, our study reveals the key role of TLR9 protein arginine methylation in regulating ferroptosis in PCa. This finding may provide a therapeutic strategy for controlling the development and progression of PCa. We present this article in accordance with the MDAR reporting checklist (available at https://tau.amegroups.com/article/view/10.21037/tau-2026-1-0089/rc).
Methods
Cell culture and reagents
The PC3 cells (CL-0185), HEK293T cells (CL-0005) and Raw264.7 cells (CL-0190) were purchased from ATCC. The above cells were cultured in Duchesne’s modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA), containing 10% fetal bovine serum (FBS, Gemini Bio-Products, Woodland, CA, USA) and a 1% streptomycin-penicillin mixture (Beyotime Biotechnology, Shanghai, China). The cells grew stably in a 5% CO2 cell culture at 37 ℃. ODN orders from InvivoGen (Santiago, CA, USA). Lipofectamine™ 3000 and RNAiMAX were purchased from Invitrogen (Camarillo, CA, USA). EZ Cell transfection Reagent was ordered from Life-iLab Biotechnology (Shanghai, China). 4,6-diamidino-2-phenylindole (DAPI) was ordered from Sigma-Aldrich (St. Louis, Missouri, USA). Protein G PLUS was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, California, USA). The 10× RIPA lysis buffer was purchased from Merck Millipore (Bedford, MA, USA). The plasmids include empty vector (EV), Myc-Flag-TLR9, HA-PRMT5, HA-PRMT9, HA-TLR9, His, His-TLR9, GST-PRMT9, Renilla, luciferase-NRF2 and Flag-ACSM1 homoplasms were derived from Miaoling biology (Shanghai, China). BL21(DE3) and DH-5α Competent Cells was ordered from Thermo Fisher Scientific Inc. (Shanghai, China). 4×104 cells were transferred to 96-well plates for luciferase reporting activity test, 2×105 cells were subjected to enzyme-linked immunosorbent assay (ELISA) in 24-well plates, and 8×105 cells were subjected to western blot analysis in 3.5 cm petri dishes. All antibody ratios are listed in Table S1.
TLR9 and PRMT9 mutants were constructed
TLR9 construction: TLR9 mutants (R74K, R216K, R305K, R977K) were constructed by site-directed mutation of TLR9 according to quikKIIXLsite directed mutation kit.
PRMT9 construction: G260E mutant PRMT9 was constructed by site-directed mutation.
Immunoprecipitation (IP), co-IP and western blot
Whole-cell lysates from PC3 and HEK293T cells were centrifuged and the resulting supernatant was incubated overnight at 4 ℃ with mouse anti-human TLR9, NRF2, Myc, Flag, HA or PRMT9 antibodies. The mixture was then incubated with 30 µL of Protein G-PLUS for four hours. The immune proteins were separated and washed, eluted by heat, separated by sodium dodecyl sulfate and polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to nitrocellulose membranes. The membrane was sealed using a solution of 5% skimmed milk or bovine serum albumin in phosphate-buffered saline (PBS) containing 0.2–0.4% Tween 20, and then incubated with the primary antibody overnight at 4 ℃. After washing, they were incubated with IRDye®800CW or 680RD Goat anti-mouse/Rabbit IgG (H+L) or HRP coupled friendly pure Goat anti-mouse/Rabbit IgG (H+L) secondary antibody, and Tanon 5200 multi-chemiluminescence imaging system (Tanon, Shanghai, China) or Odyssey Infrared Imaging System (LI− COR Biosciences, Lincoln, NE, USA) to detect protein bands.
RNA interference and cell transfection
Ribobio (Shanghai, China) designed and synthesized a small interfering RNA (siRNA) targeting PRMT9. The above siRNA fragments were transfected into PC3 cells using liposome RNAiMAX. The above cells were further analyzed when transfected with 72 afterbodies. Recombinant lentivirus acyl synthetase medium chain family 1 (ACSM1) was produced by Genechem (Shanghai, China). PC3 cells with logarithmic growth stage and 4×104 cells were selected for culture in a 6 cm petri dish. The cell growth density reached about 30%, and the final concentration of 1×108 cells /mL viral suspension was transfected with a new medium.
Dual-luciferase reporter assay
NRF2 and Renilla and the above plasmids were co-transfected or not co-transferred into PC3 cells. After the above cells were treated with 1× passive lysis buffer (PLB), the level of NRF2 transcriptional activity was detected by Dual-Luciferase® Reporter Assay System (Promega Biotech, Madison, WI, USA).
In vitro protein methylation assay
The purified prokaryotic protein his-TLR9 and its corresponding empty carrier were isolated by SDS-PAGE, boiled at 100 ℃ for 5 min, and analyzed by western blot with symmetric dimethylated antibody.
RNA purification and reverse transcription quantitative polymerase chain reaction (RT-qPCR)
Total RNA was isolated from PC3 cells using RNAiso Plus (Takara, Japan) and reverse transcribed using a PrimeScript™ RT kit (Takara, Japan), with amplification performed using a LightCycler 96 system (Roche, Basel, Switzerland). TB Green® Premix Ex Taq™ (Takara, Japan) was used to amplify the retroproducts of different samples. The relative mRNA level of β-actin was calculated using the 2−ΔΔC method.
Immunofluorescence
The cells (5×105) were inoculated into a special immunofluorescence petri dish. When the cell density reached 80%, it was fixed with 4% cold paraformaldehyde for 30 min, then incubated with a closed buffer for 30 min, incubated at 4 ℃ with the primary antibody overnight, and incubated with the second antibody for 60 min. After staining with DAPI for 15 min, the FluoView™ FV1000 Confocal Laser Scanning Microscope (Olympus, Tokyo, Japan) was used for image acquisition.
Measurement of ROS and ACSM1
ACSM1 in urine was determined by the Elabscience Biotechnology ELISA Kit (Wuhan, China). The level of cell ROS was determined by Elabscience Biotechnology ROS kits (Wuhan, China).
Mass spectrometry identification
HA-TLR9 and HA-PRMT9 were transferred to 293T cells, the cells were collected and IP was done. SDS-PAGE electrophoresis was performed, and then Coomassie bright blue staining and decolorization were performed to enrich the methylated TLR9 protein samples. The final mass spectrometry identification was commissioned by Nanning Bojun Biological Co., Ltd. The identification process was as follows: IP strip → proteolytic hydrolysis → LC-MS/MS analysis → database search → modification site identification results.
Statistical analysis
All experimental data were analysed using SPSS 19.0 software (SPSS Inc., Chicago, IL, USA) and are presented as the mean ± standard deviation (SD). The Student’s t-test was used to compare two groups. A significance level of P<0.05 was used. All experimental data were representative of three independent and random experiments by biological replicates.
Results
ODN induces arginine methylation of TLR9 by PRMT9
To investigate the effect of ODN on arginine methylation of TLR9, we detected its level in Raw264.7 cells treated with ODN. The results showed that ODN induced arginine methylation of TLR9 in a time-dependent manner (Figure 1A). To further investigate TLR9-specific methyltransferase, we examined the effects of type III PRMTs (PRMT5 and PRMT9) on TLR9 (Figure 1B). The results showed that TLR9 was methylated by PRMT5 and PRMT9, respectively, but PRMT9 had the most significant effect on the arginine methylation of TLR9 (Figure 1C). Subsequently, we explored the relationship between TLR9 and the two PRMT types. The results showed that TLR9 interacted with these PRMTS separately (Figure 1B). For the above reasons, we have narrowed our search to PRMT9. To further evaluate the specificity of PRMT9, we down-regulated its expression at protein and mRNA levels using three siRNA fragments (PRMT9 siRNA1, 2 and 3). The results showed that PRMT9 siRNA2 was the most efficient of the three fragments (Figure 1D,1E). We then investigated the effect of PRMT9 on the arginine methylation of TLR9. Our results showed that the level of arginine methylation of TLR9 was significantly reduced following the downregulation of PRMT9 by ODN treatment, compared to control PC3 cells (Figure 1F). Additionally, our in vitro protein methylation assay confirmed the arginine methylation of TLR9 (Figure 1G).
Figure 1.
TLR9 is methylated by PRMT9 in response to ODN. (A) Raw264.7 cells were treated with ODN (0.5 μM) for the specified time. The endogenous arginine methylation of TLR9 was detected by IP and western blot analysis. TLR9 co-transfected or non-transfected PRMT5 and 9 in HEK293T cells, respectively. Arginine methylation (B) of TLR9 and its interaction with PRMT5 and 9 (C) were detected by Co-IP and western blotting, respectively. PC3 cells were transfected instantaneously with control siRNA or PRMT9 siRNA1, 2, and 3 for 72 h. The expression of PRMT9 protein (D) and mRNA (E) was detected by Western blot and RT-qPCR. (F) Transient transfection of PC3 cells with control siRNA or PRMT9 siRNA2 followed by treatment with or without ODN. The endogenous arginine methylation of TLR9 was detected by IP and western blot analysis. (G) Arginine methylation of TLR9 was determined by in vitro protein methylation method. ***, P<0.001; ns, not significant. HA, hemagglutinin; IP, immunoprecipitation; NC, negative control; ODN, oligodeoxynucleotide; PC, prostate cancer; PRMT, protein arginine methyltransferase; RT-qPCR, reverse transcription quantitative polymerase chain reaction; siRNA, small interfering RNA; TLR, Toll-like receptor.
PRMT9 catalyzes arginine methylation of TLR9 on multiple sits by G260
In order to explore the effective part of PRMT9 in catalyzing TLR9 arginine methylation, we summarized the results of the existing literature, found the active catalytic site of PRMT9, constructed a PRMT9 mutant (G260E), which is an inactive form of PRMT9, and tested its effect on TLR9 arginine methylation. The results showed that the PRMT9 G260E mutation significantly eliminated arginine methylation of TLR9 compared to wild-type PRMT9 (Figure 2A). Subsequently, we conducted mass spectrometry analysis of TLR9 protein, and the results showed that 28 arginines could be methylated (shown in red in Figure 2B-2D). Combined with professional software, we predicted the following arginine methylation sites (Figure 2E), and then we constructed TLR9 mutants. Our results showed that when co-transfected with PRMT9, TLR9 with R216K substitution significantly reduced arginine methylation strength (Figure 2F).
Figure 2.
Co-transfection of TLR9 and PRMT9 in HEK293T cells. G260 of PRMT9 mediates arginine methylation (A) of TLR9 at multiple locations.The target band (B) was detected by gel electrophoresis and Coomassie bright blue staining after Co-IP. The TLR9 protein sequence (C) labeled as murine was identified by mass spectrometry and the result of TLR9 arginine modification (D) was identified by partial mass spectrometry. MSP was used to predict the methylation of human TLR9. According to the score, mass spectrometry analysis and sequence structure results, the following sites (E) were obtained as the last possible methylation sites. (F) WT TLR9 and its mutants (R216K, R977K) co-transfected PRMT9 in HEK293T cells. Arginine methylation of TLR9 was detected by Co-IP and western blot. EV, empty vector; HA, hemagglutinin; IP, immunoprecipitation; MSP, methylation-specific polymerase chain reaction; PRMT, protein arginine methyltransferase; TLR, Toll-like receptor; WT, wild type.
Arginine methylation regulates the growth of PCa
To explore differences in gene expression between PCa tissue and paracancer tissue, we used transcriptomic methods to measure tissue containing 20 male malignant tumors and Normal tissue adjacent to the carcinoma. This data mining revealed 12 genes of interest: TCHH, NUAK2, SPON2, ACSM1, PDXP, DESG2, ARHGDIG, GTSE1, CYP2J2, LMX1B, SNHG4 and PLA2G2A (Figure 3A). We focused on ACSM1, an enzyme encoded by this gene that catalyzes the activation of medium-chain fatty acids (MCFAs), and because their potential role in PCa is not completely unknown, we examined a series of published clinical datasets that compared with non-malignant prostate tissue. The ACSM1 gene was up-regulated in PCa, and TLR9 and PRMT9 expression were down-regulated (Figure 3B-3D). Finally, we evaluated the effect of arginine methylation of TLR9 on ROS levels in PCa cells, and the results showed that arginine methylation of TLR9 significantly increased the production of ROS (Figure 3E), and this regulation was also regulated by ACSM1. The increase in ROS and the accumulation of lipids, especially those containing polyunsaturated fatty acids, is likely to lead to lipid peroxidation, which is an important marker of ferroptosis. We confirmed this result with BODIPY-C11 staining, which showed that the TLR9 R216K mutation significantly enhanced lipid peroxidation (Figure 3F).
Figure 3.
Arginine methylation regulates the growth of prostate cancer. Box map (A) of gene differential expression in 20 male malignant tumor tissues and adjacent paracancer tissues detected by transcriptomics. Box plot for comparison of expression levels of ACSM1, TLR9 and PRMT9 in PRAD in GEPIA2 + GTEx database (B). box map (C) of ACSM1 gene expression in 20 male malignant tumor tissues and adjacent paracancer tissues detected by transcriptomics. The urine of patients with hyperplasia of prostate, prostate cancer and prostate cancer was collected for reexamination within 3 months after surgery, and the level of ACSM1 expression was detected by ELISA method (D). PC3 cells were transfected with or without TLR9, PRMT9, ACSM1 and siRNA ACSM1. ROS reagent was used to detect cell proliferation (E). The effect of TLR9 methylation on the accumulation of hydroperoxides in membrane phospholipids was assessed by BODIPY-C11 immunofluorescence (scale bar, 20 µm; F). **, P<0.01; ***, P<0.001. ACSM1, acyl synthetase medium chain family 1; ELISA, enzyme-linked immunosorbent assay; GEPIA, Gene Expression Profiling Interactive Analysis; GTEx, Genotype-Tissue Expression; PRAD, prostate adenocarcinoma; PRMT, protein arginine methyltransferase; ROS, reactive oxygen species; siRNA, small interfering RNA; TLR, Toll-like receptor.
Activation of NRF2 by TLR9 arginine methylation
It is well known that NRF2 is one of the transcription factors. In response to oxidative stress, the binding tightness of NRF2 with Kelch-like ECH-associated protein 1 (Keap1) changes, resulting in the dissociation of NRF2 and its translocation into the nucleus. To our surprise, we found that TLR9 can directly bind to NRF2 in PC3 cells, and that the TLR9 mutation in R216K, rather than R977K, hardly recruits NRF2 (Figure 4A). Additionally, we examined the impact of arginine methylation on the transcriptional activity of NRF2 in TLR9. Luciferase reporter gene analysis showed that NRF2 transcriptional activity was significantly enhanced in PC3 cells co-transfected with TLR9 and PRMT9 or PRMT5 compared with TLR9 alone (Figure 4B). R216K and R305K-mutated TLR9 significantly reduced NRF2 transcriptional activity in PC3 cells compared to full-length WT-TLR9 (Figure 4C). In contrast, PRMT9 with the G260E mutation significantly inhibited NRF2 transcriptional activity in PC3 cells (Figure 4D). Interestingly, this regulation is regulated by negative feedback from ACSM1. Also, in PC3 cells, Erastin or Liproxstatin-1 can change NRF2 transcriptional activity within TLR9 arginine methylation (Figure 4E).
Figure 4.
Activation of NRF2 by TLR9 arginine methylation. WT TLR9 and its mutants (R74K, R216K, R305K, and R977K) were transfected in PC3 cells, and the interaction between the above molecules was examined by Co-IP and western blot (A). A dual-luciferase reporter assay was used to measure NRF2 transcription activity, (B) empty vector and full-length TLR9 were co-transfected into PC3 cells with PRMT5 and 9, respectively, and the transcriptional activity of NRF2 was detected by dual luciferase reporter gene. (C) Empty vector, WT full-length TLR9, or its mutants (R216K, R305K) were transfected into PC3 cells, and NRF2 transcriptional activity was measured using a dual luciferase reporter gene. (D) Empty vector and full-length TLR9 were co-transfected into PC3 cells with WT PRMT9 and its mutant (G260E) in the presence or absence of ACSM1, respectively. (E) Empty vector, WT full-length TLR9 or its mutants R216K was transfected into PC3 cells with erastin or liproxstatin-1treatment for 12 h. The transcriptional activity of NRF2 was detected by dual luciferase reporter gene assay. Data from dual-luciferase reporter assays are shown as mean ± SD of three independent experiments. *, P<0.05; **, P<0.01; ***, P<0.001. IP, immunoprecipitation; NRF2, nuclear factor erythroid 2-related factor 2; PC, prostate cancer; PRMT, protein arginine methyltransferase; SD, standard deviation; TLR, Toll-like receptor; WT, wild type.
Arginine methylation of TLR9 enhances GPX4-mediated ferroptosis
GPX4 is a downstream gene of the transcription factor NRF2, and we further evaluated the effect of TLR9 arginine methylation on GPX4-mediated ferroptosis. Based on Western blotting and RT-qPCR data analysis, TLR9 arginine methylation significantly enhanced NOX1 and COX2 expression and reduced GPX4 production in PC3 cells (Figure 5A,5B), which further proved that TLR9 arginine methylation could regulate ferroptosis in PCa cells.
Figure 5.
Arginine methylation of TLR9 enhances GPX4-mediated ferroptosis. PC3 cells were co-transfected with WT TLR9 and its mutant (R216K) and PRMT9, followed by treatment with or without ODN, and GPX4, NOX1, and COX2 protein (A) and mRNA (B) levels were measured by immunoblotting and RT-qPCR for PRMT9 protein. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ODN, oligodeoxynucleotide; PC, prostate cancer; PRMT, protein arginine methyltransferase; RT-qPCR, reverse transcription quantitative polymerase chain reaction; TLR, Toll-like receptor; WT, wild type.
Discussion
In this study, we demonstrated that ODN can induce arginine methylation of TLR9. PRMT9 binds to TLR9 and efficiently mediates its arginine methylation by G260, indicating that PRMT9 is a methyltransferase of TLR9. Arginine methylation of TLR9 was also confirmed by our in vitro protein methylation assays. To pinpoint the precise locations of arginine methylation in TLR9, we first generated expert predictions based on our earlier research, followed by mass spectrometry identification, and finally experimentally determined that R216 could be methylated. Furthermore, TLR9 directly binds to NRF2 through the R216 and R305 methylation sites and enhances its transcriptional activity. In contrast, G260 of PRMT9 promoted TLR9-activated NRF2 transcriptional activity, and arginine methylation of TLR9 at R216 enhanced NRF2 transcriptional activity and GPX4-mediated ferroptosis. The expression of ACSM1 in cancer tissues was significantly higher than that in the paracancer normal group. Combined with the BODIPY-C11 staining, this indicated that TLR9 methylation was involved in the blocking of the transition from normal cells to cancer cells. Our dual luciferase assay demonstrated that this shift was caused by the enhanced ferroptosis resistance of cancer cells. In short, these findings suggest that arginine methylation is a key regulatory mode in the regulation of ferroptosis in PCa.
For PTMs of TLR9, phosphorylation, ubiquitination, and glycosylation of TLR9 have been reported so far. After activation, TLR9 binds to epidermal growth factor receptor (EGFR) to form a complex, which mediates TLR9 tyrosine phosphorylation modification, and then recruits MyD88, IKK and IRAK, activate the MAPK/NF-κB pathway to induce the synthesis of inflammatory factors and interferon (25). Kong et al. found that TLR9 expression was in the myocardial tissue of diabetic cardiomyopathy (DCM) mice and cardiomyocytes stimulated by high glucose. It is noteworthy that TLR9 overexpression can improve cardiac dysfunction, myocardial remodeling, oxidative stress, apoptosis, and energy metabolism in DCM. At the same time, it has no significant effect on inflammatory response and signaling pathways (26). In our study, we first clarified that arginine methylation of PRMT9 positively regulates GPX4 ferroptosis signaling through a direct interaction between TLR9 and NRF2. Given the complex classification and structure of protein methylation modifications, we only studied the symmetric dimethylation of TLR9 and its function. We also confirmed the arginine methylation of TLR9 using in vitro protein methylation assays. To our surprise, it was all methylated in the absence of GST-PRMT9, suggesting that prokaryotic proteins may be methylated by some methyltransferase in E. coli BL21. The murine methylated TLR9 protein was and its arginine methylation sites were successfully identified by mass spectrometry. It should be stated that we initially synthesized human methylated TLR9 protein and attempted to identify its arginine methylation sites using mass spectrometry, but were unable to locate them. The reason for this may be that there are not enough protein samples or that the molecular weight of the methyl group, methyl −, is too small to be detected by mass spectrometry. NRF2 is an important intracellular transcription factor in the classical ferroptosis signaling pathway. The intracellular level of NRF2 is precisely regulated by Keap1 protein. It only maintains a low physiological level to meet the needs of normal life activities of cells (27). In response to oxidative stress stimulation, the tight binding between NRF2 and Keap1 changes, leading to the dissociation of NRF2 and translocation to the nucleus. Once there, NRF2 first binds to Maf protein to form then binds to antioxidant response elements (ARE) upstream of the target gene to open the transcription process of downstream genes (28). Our results show that TLR9 binds directly to NRF2 via the R216 and R305 arginine methylation sites, respectively, suggesting that TLR9 directly interacts with NRF2 in the absence of Keap1 protein, which has not been reported previously. Of course, there is no doubt that the use of Co-IP alone to detect a direct interaction between TLR9 and NRF2 is not sufficient. The next step in our study was to confirm it using a pull-down assay. NRF2 not only maintains cellular REDOX homeostasis, but also plays an important role in cellular life activities such as ferroptosis, autophagy, apoptosis, and DNA damage repair (29,30). Kim et al. found that Plk2 acts as an antioxidant and anti-inflammatory regulator through the phosphorylation and activation of NRF2. Plk2 and NRF2 act synergistically to protect renal cells from renal toxicants and keep them alive (31). Li et al. found that NRF2 phosphorylation can promote the growth of gallbladder cancer cells (32). Kim et al. reported that NRF2 phosphorylation reduces oxidative stress and proinflammatory cytokines in patients with multiple sclerosis (MS), providing new insights into the pathogenesis of MS (33). Our study shows that ODN induces PRMT9 to methylate TLR9 at R216, directly binds to NRF2 through the R216 and R305 methylation sites, and enhances its transcriptional activity. The results of our study differ from others’ opinions. Our study unveils a novel finding: a previously unknown mechanism that regulates the NRF2 ferroptosis signaling pathway, involving PRMT9-mediated arginine methylation. Based on our results, we propose the following model: arginine methylation reduces ferroptosis activity, and upon ODN stimulation, PRMT9 is recruited to TLR9 and methylated at R216 by G260, while R216 and R305 catalyze TLR9 recruitment of NRF2. Subsequently, NRF2 is activated and transferred from the cytoplasm to the nucleus, ultimately contributing to the progression of ferroptosis.
Supplementary
The article’s supplementary files as
Acknowledgments
None.
Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.
Footnotes
Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://tau.amegroups.com/article/view/10.21037/tau-2026-1-0089/rc
Funding: This study was supported by grant from the Anhui Province clinical medicine research and transformation special project (No. 202204295107020051), Anhui Province Scientific Research Program for Universities (No. 2024AH051267), Research Program of Bengbu Medical University (Nos. 2023byzd117 and 2024byzd497), and Bengbu Municipal Health Commission (No. BBWK2024A104).
Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tau.amegroups.com/article/view/10.21037/tau-2026-1-0089/coif). The authors have no conflicts of interest to declare.
Data Sharing Statement
Available at https://tau.amegroups.com/article/view/10.21037/tau-2026-1-0089/dss
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