Fig 1: SIRT4 regulates methionine metabolism through mediating MAT2A ADP ribosylation. A Volcano plot showing differential gene expression in mice’s livers upon methionine restriction. Red circles indicates genes increased in the Ctrl set while blue ones means genes decreased in the MR set. B Western blot analysis of MAT2A mono-ADP-ribosylation (MARylation) in HEK293 and SNU449 cells in which SIRT4 was silenced (shRNA). C The MARylation of MAT2A was measured by western blot in HEK293 or SNU449 cells stably expressing HA tagged SIRT4. D Western blot analysis of MAT2A MARylation in the FLAG immunoprecipitated samples from HEK293 or SNU449 cells treated with nicotinamide (NAM) for indicated times. E Western blot analysis of MAT2A MARylation in the FLAG immunoprecipitated samples from HEK293 or SNU449 cells treated with Sirtinol for indicated times. F Relative activity of MAT2A was shown in the SNU449 cells in which either c-Myc or SIRT4 was knockdown or overexpressed or c-Myc and SIRT4 overexpressed together. G Abundances of intracellular primary methionine cycle metabolites were compared between the control cells and the SNU449 cells where either c-Myc or SIRT4 was knockdown or stably overexpressed or c-Myc and SIRT4 overexpressed together. H MARylation level of mutant FLAG-MAT2A-E111A ectopically expressed in MAT2A-knockout HEK293 or SNU449 cells was measured by using anti-ADP ribosylation antibody when SIRT4 was silenced. I MARylation level of mutant FLAG-MAT2A-E111A ectopically expressed in MAT2A-knockout HEK293 or SNU449 cells was measured by using anti-ADP ribosylation antibody when SIRT4 was overexpressed. J MAT2A dimer are shown in blue or lightblue (PDB ID: 4NDN). The active site was shown in red circle. K E111 in MAT2A is conserved. Protein sequence alignment surrounding E111 colored in light blue from indicated species. Data are means ± SEM. Group differences were analyzed by two-tailed Student’s multiple comparison test (F, G) (**p < 0.01)
Fig 2: Myc promoted SIRT4 degradation to reduce ADP ribosylation of MAT2A in the HCC. A Protein levels of c-Myc, MAT2A and MARylation level of MAT2A were determined by western blot from 85 pairs of tumour tissues. In the inserted boxplots, the circles indicate the median; the square indicate the 5th–95th percentiles. The statistical analysis was performed by the two tailed Student’s t-test (**p < 0.01). B Correlation between Myc and SIRT4 in HCC patients. 85 tissue pairs were immunohistochemically stained with indicated antibodies. The pair-wise Pearson correlation coefficient and the corresponding p-value between two genes were calculated. C The patients with high SIRT4 expression (n = 43) have longer survival compared to low SIRT4 expression (n = 42). Significance was determined using Kaplan–Meier analysis. D Schematic showing that high methionine content is sensed by mTORC1 that triggers c-Myc expression, which promotes TRIM32-mediated SIRT4 degradation. SIRT4 regulates MAT2A activity through MARylation, thereby inhibits MAT2A mediated tumour growth. SIRT4 loss resulted in the low MARylation level of MAT2A, which promotes methionine metabolism to generate SAM. In a feedback, high SAM level triggers transcription of metabolic enzymes to maintain tumour progression. Upward pointing arrows indicate an increase, and downwards pointing arrows indicate a decrease
Fig 3: Inhibition of MAT2A promotes KSHV lytic replication.A iSLK-RGB-BAC16 cells were transfected with siRNAs targeting MAT2A (siMA-1 and siMA-2) or the control siRNA (siCtrl). MAT2A mRNA and protein levels were analyzed by RT-qPCR and Western-blotting. GAPDH was used as a normalization control. B, C iSLK-RGB-BAC16 cells were transfected with siRNAs targeting MAT2A or the control siRNA for 24 h. Cells were treated with 3 mM of sodium butyrate (NaB) for 48 h, and observed with a fluorescent microscope (B) and analyzed by flow cytometry for EGFP-positive cells (C). D iSLK-RGB-BAC16 cells transfected with siRNAs targeting MAT2A or the control siRNA were treated with 3 mM of NaB for 72 h and examined for the expression of KSHV proteins RTA, ORF57, ORF-K8 and ORF65 by Western-blotting. GAPDH was used as a loading control. E, F iSLK-RGB-BAC16 cells were treated with 50 mM of cycloleucine (Cyclo) and 3 mM of NaB for 48 h and observed with a fluorescent microscope (E) and analyzed by flow cytometry for EGFP-positive cells (F). G iSLK-RGB-BAC16 cells treated with 50 mM of cycloleucine (Cyclo) and 3 mM of NaB for 72 h and examined for the expression of KSHV lytic proteins by Western-blotting. GAPDH was used as a loading control. H, I iSLK-RGB-BAC16 cells were treated with 50 mM of cycloleucine (Cyclo) and 3 mM of NaB for 24 h. Cells were then washed with PBS and cultured in fresh medium without NaB, hygromycin, puromycin and G418 for 72 h. Supernatants were collected and used for titrating infectious virions by infecting MM cells. mRFP1-positive cells were imaged (H) and quantified at 48 h post-infection (I). J iSLK-RGB-BAC16 cells were transfected with siRNAs targeting MAT2A or the control siRNA. After 24 h, cells were transfected with a plasmids expressing HA-tagged MAT2A (HA-MAT2A) or a vector control (Vector) and treated with 3 mM of NaB. Cells were lysed to collect the proteins at 72 h post-treatment. Antibodies to HA, MAT2A, METTL16 and KSHV proteins LANA, RTA, ORF57 and ORF65 were used to detect their respective proteins by Western-blotting. β-actin was used as a loading control. *, **, and *** indicate p values of <0.05, <0.01, and <0.001, respectively, ns not significant.
Fig 4: METTL16 and MAT2A regulate intracellular GSH and ROS levels.A iSLK-RGB-BAC16 cells transfected with siRNAs targeting MAT2A (siMA-1 and siMA-2) or the control siRNA (siCtrl) were treated with 3 mM of NaB for 24 h. The intracellular GSH level was analyzed by mass spectrometry. B iSLK-RGB-BAC16 cells transfected with siRNAs targeting METTL16 (siM16-1 and siM16-2) or the control siRNA (siCtrl) were treated with 3 mM of NaB for 24 h. The intracellular GSH level was analyzed by mass spectrometry. C iSLK-RGB-BAC16 cells transfected with siRNAs targeting MAT2A or the control siRNA were treated with 3 mM of NaB. The intracellular ROS level was measured and quantified by flow cytometry. D iSLK-RGB-BAC16 cells transfected with siRNAs targeting METTL16 or the control siRNA were treated with 3 mM of NaB. The intracellular ROS level was measured and quantified by flow cytometry. E ROS levels in iSLK-RGB-BAC16 cells treated with 3 mM of NaB and cycloleucine (Cyclo) at the indicated concentrations were measured by flow cytometry. F Intracellular ROS levels of iSLK-RGB-BAC16 cells treated with 3 mM of NaB and SAM at the indicated concentrations were measured by flow cytometry. *, **, and *** indicate p values of <0.05, <0.01, and <0.001, respectively, ns not significant.
Fig 5: METTL16 and MAT2A regulate intracellular ROS level to control KSHV lytic replication.A iSLK-RGB-BAC16 cells pre-treated with NAC at 10 mM for 2 h were transfected with siRNAs targeting METTL16 (siM16-1 and siM16-2) or the control siRNA (siCtrl) for 24 h in the presence of NAC. Cells were then induced with 3 mM of NaB in the presence of NAC for 48 h and the intracellular ROS level was measured and quantified by flow cytometry. B iSLK-RGB-BAC16 cells pre-treated with NAC at 10 mM for 2 h were transfected with siRNAs targeting MAT2A (siMA-1 and siMA-2) or the control siRNA (siCtrl) for 24 h in the presence of NAC. Cells were then induced with 3 mM of NaB in the presence of NAC for 48 h and the intracellular ROS level was measured and quantified by flow cytometry. C, D iSLK-RGB-BAC16 cells pre-treated with NAC at 10 mM for 2 h were transfected with siRNAs targeting METTL16 or the control siRNA for 24 h in the presence of NAC and then induced with 3 mM of NaB. Cells were observed with a fluorescent microscope (C) and analyzed by flow cytometry for EGFP-positive cells (D). E, F iSLK-RGB-BAC16 cells pre-treated with NAC at 10 mM for 2 h were transfected with siRNAs targeting MAT2A or the control siRNA for 24 h in the presence of NAC and then induced with 3 mM of NaB. Cells were observed with a fluorescent microscope (E) and analyzed by flow cytometry for EGFP-positive cells (F).
Supplier Page from Abcam for Anti-MAT2A antibody