Figure 1.
The eukaryotic translation initiation factor eIF4G3 is degraded by GrB (1 µg/ml) in Jurkat (Jkt) cells.
(A) Representative Western blot analysis of detectable full-length eIF4G3 and Bid activation in Jurkat cells from time 0 (no GrB treatment) to 30 min post GrB/AD treatment (left panel) and in the presence of zVAD-fmk (right panel). (B) Densitometric analysis of eIF4G3 (A) where the eIF4G3/α-tubulin ratio was calculated and the highest densitometric value was 100%. Other values were plotted as a percentage of the maximum densitometric value. Statistical significance: p<0.01 (**); (n = 3 of 3 independent experiments).
Figure 2.
GrB-mediated cleave of eIF4G3 occurs at position D1408.
(A) Autoradiography of eIF4G3 treated with 30 nM GrB for 30 min. Treatment of eIF4G3 (∼200 kDa) with 30 nM GrB generates a proteolytic fragment; (n = 3 of 3 independent experiments). (B) The eIF4G3 isoform, eIF4G1, is not degraded by GrB. Loaded protein levels for eIF4G1 and eIF4G3 lanes are identical to their corresponding GrB treatment lanes; (n = 3 of 3 independent experiments). To visualize eIF4G3 fragments, eIF4G3 lanes were loaded at a higher concentration. (C) Putative P1 residue D1408 of eIF4G3 was mutagenized to alanine to generate the mutant eIF4G3Δ. eIF4G3 and eIF4G3Δ were synthesized in vitro and labeled with MetS35. These two proteins were treated with 30 and 300 nM GrB. Detectable degradation products are indicated with an arrow; (n = 3 of 3 independent experiments).
Figure 3.
GrB inhibits in vitro translation of Luc.
IVTT was used to synthesize Luc in the presence of MetS35. Labeled Luc protein was visualized by autoradiography. (A) A range of GrB concentrations (0–30 nM) were added to the IVTT master mix to test their effect on transcription/translation; (n = 3 of 3 independent experiments). (B) Detectable Luc was compared in the absence of GrB, and in the presence of 30 nM GrB added before or after IVTT; (n = 3 of 3 independent experiments). (C) Purified Luc mRNA (10 µg/ml) was used to synthesize MetS35-labeled Luc. 30 nM GrB prevents translation; (n = 3 of 3 independent experiments). (D) Two different concentrations of proteases (3 and 30 nM) GrB, granzyme A (GrA), granzyme K (GrK), Caspase-3 (Casp-3), Caspase-7 (Casp-7) and Caspase-8 (Casp-8) were tested in IVTT of Luc; (n = 3 of 3 independent experiments). (E) Luminescence generated by Luc in IVT reactions treated with 30 nM proteases, where sham-treated IVT reactions were considered 100%; Statistical significance: p<0.01 (**) and p<0.005 (***); (n = 3 of 3 independent experiments).
Figure 4.
GrB resistant eIF4G3Δ can restore in vitro translation of Luc.
(A) Translation of Luc mRNA was monitored by autoradiography. In the presence of 30 nM GrB, translation of Luc is blocked. Purified eIF4G3Δ -HA (0.01–2 µg/ml) was added the reaction after GrB treatment to recover Luc in vitro synthesis. Luc shows as a band at 61 kDa and radio-labeled eIF4G3Δ -HA shows as a band at ∼200 kDa; (n = 3 of 3 independent experiments). (B) Same experiments as in (A) but with purified wild-type eIF4G3-HA; (n = 3 of 3 independent experiments). (C) Luminescence generated by Luc IVT in (A) was measured and expressed as %Luc luminescence, where expression of Luc with no treatment was considered 100%. Densitometric analysis was conducted for eIF4G3Δ -HA, and expressed as %eIF4G3Δ -HA densitometry, where densitometric values for Luc with no treatment was considered 100%; (n = 3 of 3 independent experiments). (D) GrB activity colorimetric assay. GrB inh and purified serpina 3n were used as positive controls to inhibit GrB activity. Three different concentrations of eIF4G3Δ (0.1, 1 and 2 µg/ml) did not inhibit GrB activity; (n = 3 of 3 independent experiments).
Figure 5.
GrB or CTL treatment decrease translational rate in Jurkat cells independently of caspase-mediated DNA fragmentation.
(A and B) Jurkat cells were treated with GrB (1 µg/ml) for 300 min. At 15 min interval, cells were assessed for DNA fragmentation (%[3H]-Thymidine Release) and translational rate (% Translated Protein). (B) The pan-caspase inhibitor zVAD-fmk (zV) was used to evaluate the role of caspases. (For (A) and (B); n = 4 of 4 independent experiments). (C) Jurkat cells were treated with CTL for 180 min. As in (A) and (B), DNA fragmentation and translational rate was assessed at 15 min intervals. Statistical significance: p<0.05 (* and φ) p<0.01 (** and φφ); n = 3, where * compares statistical significance of % translated protein of Jurkat cells+zVAD-fmk (Jkt+zV) vs. Jurkat cells+zV+CTL; and φ compares Jkt vs. Jkt+CTL; (n = 3 of 3 independent experiments). (D) Jurkat cells were treated with CTL for 90 min in the presence of the GrB inhibitor L-038587-00Y001 (GrB inh). %Translated protein was assessed at 15 min intervals. Statistical significance: p<0.05 (* and φ), where * compares statistical significance of % translated protein of Jurkat cells+CTL vs. Jurkat cells+CTL+L−038587−00Y001; (n = 3 of 3 independent experiments). (E) qPCR analysis of β-actin (β-act) relative expression at 1 and 2 hr post GrB/AD treatment; (n = 3 of 3 independent experiments).
Figure 6.
Inhibition of VV production by GrB is mediated by cleavage of eIF4G3.
(A) Jurkat cells were infected with VV for 0 to 18 hr. Metabolic labeling was assessed by MetS35 incorporation and viral protein synthesis was monitored by autoradiography analysis. Arrowheads on the left hand side of the radiogram indicate host proteins and arrowheads on the right hand side of the radiogram indicate VV proteins. Late VV proteins P4a, 4a, 4b and early VV protein E are shown. Western blot analysis of eIF4G3 from each time point is shown below the radiogram. (B) Jurkat cells were infected for 12 hr with no virus (mock) or with VV. Infected cells were co-treated with AD, GrB/AD, GrB/AD+zVAD−fmk (zV), GrB/AD+GrB inhibitor (inh) and heat inactivated GrB+untreated AD (ΔGrB/AD). Western blot analysis of eIF4G3 from each experimental condition is shown below the radiogram. Arrowheads in the Western blot image point to eIF4G3 degradation product; (n = 4 of 4 independent experiments). (C) Jurkat cells, Jurkat cells transformed with wild-type eIF4G3 (JurkateIF4G3-WT) and Jurkat cells transformed with GrB-resistant mutant (JurkateIF4G3-Δ) were infected with VV for 12 hr (D) The effect of GrB concentration (1–4 µg/ml) on VV replication was tested. AD and zV concentrations were the same in all treatments; Statistical significance: p<0.05 (*) and p<0.01 (**); where * compares 2 and 4 µg/ml GrB vs. 0 µg/ml GrB; (n = 3 of 3 independent experiments). (E) VV replication in untreated Jurkat cells, JurkateIF4G3-WT, and JurkateIF4G3Δ, was measured by plaque assay. Statistical significance: p<0.01 (**); (n = 3 of 3 independent experiments).