Satratoxin G interaction with 40S and 60S ribosomal subunits precedes apoptosis in the macrophage

Abstract

Satratoxin G (SG) and other macrocyclic trichothecene mycotoxins are potent inhibitors of eukaryotic translation that are potentially immunosuppressive. The purpose of this research was to test the hypothesis that SG-induced apoptosis in the macrophage correlates with binding of this toxin to the ribosome. Exposure of RAW 264.7 murine macrophages to SG at concentrations of 10 to 80 ng/ml induced DNA fragmentation within 4 h that was indicative of apoptosis. To relate these findings to ribosome binding of SG, RAW cells were exposed to different toxin concentrations for various time intervals, ribosomal fractions isolated by sucrose density gradient ultracentrifugation and resultant fractions analyzed for SG by competitive ELISA. SG was found to specifically interact with 40S and 60S ribosomal subunits as early as 5 min and that, at high concentrations or extended incubation times, the toxin induced polysome disaggregation. While co-incubation with the simple Type B trichothecene DON had no effect on SG uptake into cell cytoplasm, it inhibited SG binding to the ribosome, suggesting that the two toxins bound to identical sites and that SG binding was reversible. Although both SG and DON induced mobilization of p38 and JNK 1/2 to the ribosome, phosphorylation of ribosomal bound MAPKs occurred only after DON treatment. SG association with the 40S and 60S subunits was also observed in the PC-12 neuronal cell model which is similarly susceptible to apoptosis. To summarize, SG rapidly binds small and large ribosomal subunits in a concentration- and time-dependent manner that was consistent with induction of apoptosis.

Figure 1. SG induces apoptosis in RAW 264.7 cells

Cells were treated with different SG concentrations for various time intervals and then assessed for DNA fragmentation by (A) cell-death ELISA in duplicate or (B) agarose gel electrophoresis.

Figure 2. UV absorption scan for SG

Purified SG was diluted in phosphate buffer (pH 7.0) and absorbance measured over the UV spectrum.

Figure 3. SG binding to ribosomal subunits is concentration-dependent in RAW 264.7 cells

Cells were treated with SG (0, 20, 40, 80 ng/ml) for 1 h and then lysed with polysome extraction buffer (PEB). (A) Ribosomal fractions were separated on a sucrose gradient and the A₂₅₄ monitored and small subunit, large subunit, monosome and ploysomes labeled as 40S, 60S, 80S and P, respectively, (B) Ribosomal subunits and monosome (RS+M) fractions were pooled and analyzed for SG by competitive ELISA and for total protein colorimetrically.

Figure 4. Kinetics of SG binding to ribosomal in RAW 264.7 cells

Cells were untreated or treated (A) with water or SG (10 ng/ml) for 15 (B), 30 (C), 60 (D), 120 (E), and 240 (F) min and then lysed with PEB. Ribosomal fractions were separated on a sucrose gradient A₂₅₄ and monitored. Individual fractions (0.5 ml) were analyzed for SG (open circle) and total protein (vertical bar). Solid arrows indicate increased absorbances with increasing SG concentration. Dotted arrows indicate increased SG is detectable in ribosomal fractions as toxin concentration ins increased.

Figure 5. High SG concentration induces rapid, saturable binding to 40S and 60S ribosomal subunits in RAW 264.7 cells

(A)Cells were untreated or treated with SG (100 ng/ml) for 5, 15, 30 min and then lysed with PEB. Ribosomal fractions were separated on a sucrose gradient system and A₂₅₄ monitored. (B) Cells were treated with SG (100 ng/ml) for 15 min. PEB lysate were separated at high resolution (0.25 ml/fraction) on a sucrose gradient and then fractions analyzed by SG ELISA. Comparable treatment with water vehicle only revealed no detectable SG (data not shown)

Figure 6. DON competitively inhibits SG binding to 40S and 60S ribosomal subunits in RAW 264.7 cells

Cells were treated with vehicle, SG (100 ng/ml), DON (500 ng/ml) or both toxins for 15 min and then lysed with PEB. (A) SG content of cell lysate was analyzed by ELISA. (B) Ribosomal fractions were separated on a sucrose gradient and A₂₅₄ monitored.

Figure 7. SG binding to the ribosome is reversible in RAW 264.7 cells

Cells were incubated with SG (20 ng/ml) for 60 min and ribosomal fractions were separated on sucrose gradient. Pooled RS+M fractions , all containing bound SG, were incubated with additional SG (20 ng/ml) and/or (DON 500 ng/ml) for 1 h at 37°C. RS+M were repeatedly concentrated and washed to remove free SG and then analyzed by competitive SG ELISA.

Figure 8. SG induces modest p38 and JNK phosphorylation in RAW 264.7 cells

Cells were treated with SG (100-500 ng/ml) or DON (100 ng/ml) for 30 min and then lysed with SDS buffer. Total protein was analyzed by Western blotting with specific antibodies to non-phosphorylated p38 and JNK and their phosphorylated forms.

Figure 9. SG and DON induce MAPK interaction with the ribosome

Cells were treated with vehicle, SG (100 ng/ml), DON (500 ng/ml) or both toxins for 15 min, and then lysed with PEB. (A) whole cell lysates were analyzed by Western blotting using specific antibodies to phosphorylated p38 and JNK and their non-phosphorylated forms. (B) Lysates were fractionated on sucrose gradient and pooled ribosomal subunits and monosomes (RS+M) analyzed by Western blotting.

Figure 10. SG binds to 40S and 60S ribosomal protein fractions in PC-12 neuronal cells

Cells were treated with water or SG (10 ng/ml) for 60 min and lysed with PEB. Ribosomal fractions were separated on a sucrose gradient system and A₂₅₄ monitored. Individual fractions were analyzed for SG and protein.