Figure 6.
Inositol maintains IMPDH2 oligomerization, directly binds to IMDPH2, and activates IMPAH2 activity, thereby promoting guanylate purine metabolism. (A) The cell lysates from PC3 cells were incubated with biotin or biotin-labeled inositol, and inositol-interacting proteins were pulled down by streptavidin beads and subjected to mass spectrometry. (B) The cell lysates from PC3 cells were incubated with 40 μM of biotin or biotin-labeled inositol, pulled down with streptavidin beads and subjected to immunoblotting with IMPDH2 antibody. Immunoblotting data were verified in at least two independent experiments. (C) GST and GST-IMPDH2 full length (FL) were expressed in Escherichia coli BL21 cells and immobilized on Glutathione-Sepharose. 2 μg of these recombinant proteins were incubated with 40 μM of biotin or biotin-labeled inositol (Biotin-Ins) for 3 h, followed by pulled down with streptavidin beads. The interaction was determined by immunoblotting with indicated antibodies. The purity of the recombinant proteins was resolved by SDS-PAGE, followed by Coomassie blue staining. (D) The illustration of the IMPDH2 full length (1–514 aa), the N-terminal (1–111 aa), CBS (111–232 aa), and C-terminal (232–514 aa). (E) GST-IMPDH2 N-terminal, GST-IMPDH2 C-terminal, and GST-CBS domain from IMPDH2 were expressed in Escherichia coli BL21 cells and immobilized on Glutathione-Sepharose. 2 μg of these recombinant proteins was incubated with 40 μM of biotin or biotin-labeled inositol (Biotin-Ins) for 3 h, followed by pulled down with streptavidin beads. The interaction was determined by immunoblotting with indicated antibodies. The purity of the recombinant proteins was resolved by SDS-PAGE, followed by Coomassie blue staining. Immunoblotting data were verified in at least two independent experiments. (F) 22RV1 cell lysates upon shLuc and shIMPA1(#1 and #2) were subjected to crosslinking by bismaleimidohexane (BHM), followed by immunoblotting with IMPDH2 antibody. The input levels of IMPA1, IMPDH2, and GAPDH were represented without crosslinking. Immunoblotting data were verified in at least two independent experiments. (G) 22RV1 cell lysates upon shLuc and shIMPA1(#2) with the treatment of vehicle and 100 μM of inositol were subjected to cross-link by bismaleimidohexane (BHM), followed by immunoblotting with IMPDH2 antibody. The input levels of IMPA1, IMPDH2, and GAPDH were represented without cross-link. Immunoblotting data were verified in at least two independent experiments. (H) Recombinant IMPDH2 protein incubated with vehicle and 100, 200 μM of inositol were subjected to cross-link by BMH, followed by immunoblotting with IMPDH2 antibody. The input levels of IMPDH2 were represented without cross-link. Immunoblotting data were verified in at least two independent experiments. (I) The illustration of the IMPDH2 full length (IMPDH2FL), truncated 41–43 aa of IMPDH2 (IMPDH2Δ41–43) and truncated 279–281 aa of IMPDH2 (IMPDH2Δ279–281). (J) Recombinant proteins, GST- IMPDH2FL, GST-IMPDH2Δ41–43 and GST-IMPDH2Δ279–281, were subjected to biotin and biotin-labeled inositol pull-down assay, followed by immunoblotting with GST antibody. Coomassie blue indicates the equal amount of recombinant proteins in each group. Immunoblotting data were verified in at least two independent experiments. (K) Recombinant GST- IMPDH2FL protein were subjected to 40 μM of biotin and 40 μM of biotin-labeled inositol pull-down assay upon adding 1 mM of IMP, 75 nM of MPA and excessive 500 μM of inositol, followed by immunoblotting with IMPDH2 antibody. Immunoblotting data were verified in at least two independent experiments. (L) 22RV1 cell lysates were subjected to biotin and biotin-labeled inositol pull-down assay, followed by immunoblotting with indicated antibodies. Immunoblotting data were verified in at least two independent experiments. (M) Immunoblotting of various prostate cancer cell lines with indicated antibodies. Immunoblotting data were verified in at least two independent experiments. (N) Schematic representation of purine metabolism with a series of enzymes and metabolites. (O and P) The activity of IMPDH was measured in DU145 (O) and PC3 (P) stably expressing shLuc or shIMPA1 (#1 and #2) upon 10 μM of MPA treatment by using IMPDH activity assay kit. The detailed procedure is described in Materials and methods. The activity was normalized by protein concentration in each experimental group. The data were shown as the mean ± SEM of three independent experiments for each group. *, P < 0.05; **, P < 0.01 by two-tailed unpaired t test. (Q) The activity of IMPDH was measured in DU145 cells stably expressing shLuc or shIMPA1 upon 25 μM of inositol (Ins) treatment by using IMPDH activity assay kit. The data were shown as the mean ± SEM of three independent experiments for each group. *, P < 0.05; **, P < 0.01 by two-tailed unpaired t test. (R) In vitro IMPDH2 activity using IMPDH activity assay kit was determined by incubation recombinant IMPDH2 protein with 40 μM of inositol (Ins) or 10 μM of MPA with indicated time. (S) Targeted metabolomics of PC3 cells expressing shLuc or shIMPA1 upon 25 μM of inositol (Ins) treatment was performed to measure the levels of purine metabolism including guanosine, xanthosine, cGMP, GDP, xanthine, GTP, Urate. The data were shown as the mean ± SEM of three independent experiments for each group. *, P < 0.05; **, P < 0.01 by two-tailed unpaired t test. (T) Heatmap plot from RNA-seq shows the expression levels of reprogramming factors, epigenetic factors, E2F targets, and AR targets in CRPC 22RV1 cells stably expressing shLuc, shIMPA1, or shIMPDH2. Duplicate RNA samples in each group were performed for RNA-seq. Source data are available for this figure: SourceData F6.