Table 1.
ΔN-LmSEPHS2 crystal structure refinement statistics.
Fig 1.
ΔN-LmSEPHS2 crystal structure.
A- Cartoon representation of the monomer structure in the asymmetric unit showing a typical AIRS-like folding. The sulfate ion is represented as sticks. B- Superimposition of AaSEPHS (grey) [38], EcSEPHS (green) [39], HsSEPHS1 (light blue) [22] and LmSEPHS2 (purple). C- Dimeric model generated using PDBePISA [40] depicting amino acid residue conservation. D- Native gel electrophoresis showing the prevalence of dimers in solution for T. brucei (Tb) and L. major (Lm) selenophosphate synthetases at 2 mg/mL. A small amount of tetramers is also observed for both protein preparations (top bands). MW: molecular weight. E- Sedimentation coefficient distribution (S) at increasing protein concentration normalized to the most abundant oligomer (dimer) obtained by sedimentation velocity analytical ultracentrifugation (SV-AUC). The inset displays sedimentation coefficients measured for dimers at increasing total protein concentration. F- Measured and theoretical sedimentation coefficient, molecular weight and relative abundance of dimers and tetramers. ssphere corresponds to the theoretical sedimentation coefficient calculated for a spherical protein. The discrepancy between the experimental sedimentation coefficient for the dimer and its theoretical value (ssphere) suggests that it is elongated.
Fig 2.
ATPase activity and functional complementation assays.
A- ATP hydrolysis in vitro over time measured by HPLC for full length and N-terminally truncated constructs of T. brucei and L. major selenophosphate synthetases. B- Selenophosphate synthetase functional complementation assays in SEPHS deficient E. coli strain (WL400 (DE3)) transformed with different constructs. The purple color indicates a functional formate dehydrogenase H selenoprotein expression. R1 and R2 correspond to biological duplicates.
Fig 3.
Interaction between selenophosphate synthetase and selenocysteine lyase.
A- SEC-MALS profiles for TbSCLY (40 μM; theoretical molecular weight (dimer) = 127 kDa), TbSEPHS2 (40 μM; theoretical molecular weight (dimer) = 89 kDa) and 1 TbSCLY (40 μM): 1 TbSEPHS (40 μM) indicating the formation of a binary complex in vitro. The molecular weight (MW) corresponding to the highest peak centroid is indicated. B- ITC curves obtained by in vitro titration of TbSEPHS2 (200 μM, syringe) to TbSCLY (10 nM, calorimeter cell). C- TbSCLY-PLP (20 μM) fluorescence in the presence of different concentrations of TbSEPHS2 (1:0.5, 1:0.75, 1:1, 1:1.25, 1:1.5). D- SyproRubyTM stained SDS-PAGE of tandem affinity purification (TAP) products of either TbSEPHS2-PTP, TbSCLY-PTP, TbPSTK-PTP, TbSEPSECS-PTP or TbeEFSec-PTP as baits. E- Analysis of tRNASec copurification by RT-PCR. Input: lysate expressing the respective PTP-tagged protein. IP: Immunoprecipitated complex using anti-IgG beads. F- LC-MS/MS analysis of the corresponding SDS-PAGE bands.
Fig 4.
Polysomal profile analysis of selenoprotein synthesis factors.
A- Schematic representation of the selenocysteine pathway in trypanosomatids. Lysates of PCF T. brucei PTP-tagged selenocysteine biosynthesis proteins were fractionated in a sucrose gradient centrifugation (7–47% sucrose) as ribosome-free, monosome (40S, 60S and 80S) and polysome fractions as monitored by UV absorbance at 254 nm. Western blot analyses of tagged proteins, using anti-protein A antibody were carried out to localize selenoprotein synthesis factors (B- TbPSTK-PTP, C- TbSCLY-PTP, D- TbSEPSECS-PTP, and E- TbeEFSec-PTP). BiP and EIF5A were used as ribosome-free and polysome fraction markers, respectively. F- Ribosomes dissociation into monosome units in the presence of EDTA fractionated in a 5–25% sucrose gradient.
Fig 5.
TbSEPHS2-RNAi T. brucei cells sensitivity to DTT and tunicamycin.
Tetracycline non-induced (dark bars) and induced (grey bars) TbSEPHS2 RNAi PCF and BSF T. brucei cells treated with various concentrations of DTT and tunicamycin. The plots show cell concentration relative to untreated control after incubation at 28°C and 37°C for procyclic (PCF) and bloodstream (BSF) T. brucei, respectively. Bars represent the average of 3 independent experiments including standard deviations of experiments proceeded in A- and C- PCF T. brucei cells, and B- and D- BSF T. brucei cells. The asterisks represent significant differences between the stressors of ER treatment (PCF T. brucei, 0.5 mM DTT: ** P = 0.007; 1.0 mM DTT: * P = 0.020; 2.0 mM DTT: P = 0.040; 5μg/mL tunicamycin: * P = 0.033; 10μg/mL tunicamycin: * P = 0.010; 20μg/mL tunicamycin: * P = 0.020; BSF T. brucei, 0.15 mM DTT: * P = 0.020; 0.3 mM DTT: * P = 0.010; two-tailed Student’s t test). Western blot analysis of BiP in whole cell extracts of I- PCF and J- BSF TbSEPHS2 RNAi T. brucei cell (12% SDS-PAGE; α-tubulin as a normalization standard).
Fig 6.
TbSELENOT is dispensable for both procyclic and bloodstream T. brucei.
Growth curves of representative TbSELENOT-RNAi procyclic (PCF) and bloodstream (BSF) T. brucei cultures. A- and C- PCF and B- and D- BSF T. brucei cells induced (black) and non-induced (grey) with tetracycline and real-time qPCR analysis relative to TERT as a normalization standard, respectively.
Fig 7.
TbSELENOT-RNAi T. brucei cells sensitivity to DTT and tunicamycin.
Tetracycline non-induced (black) and induced (grey) T. brucei cells were treated with various concentrations of DTT and tunicamycin. The plots show cell concentration relative to untreated control after a 24h-incubation at 28°C and 37°C for procyclic (PCF) and bloodstream (BSF) T. brucei, respectively. Bars represent the average of 3 independent experiments including standard deviations of experiments proceeded in A- and C- PCF T. brucei cells, and B- and D- BSF T. brucei cells. Asterisks: PCF T. brucei, 1.0 mM DTT: * P = 0.010; 10μg/mL tunicamycin: * P = 0.033; two-tailed Student’s t test. Western blot analysis of BiP in whole cell extracts of E- PCF and F- BSF TbSPS2 RNAi T. brucei cells (12% SDS-PAGE; α-tubulin as a normalization standard).
Fig 8.
TbSELENOT knockdown does not affect procyclic and bloodstream T. brucei sensitivity to H2O2.
Tetracycline non-induced (black) and induced (grey) T. brucei cells were treated with various concentrations of DTT and tunicamycin. The plots show cell concentration relative to untreated control after a 24h-incubation at 28°C and 37°C for procyclic (PCF) and bloodstream (BSF) T. brucei, respectively. TbSELENOT-RNAi A- PCF and B- BSF T. brucei cells were also treated with various concentrations of H202. Bars show the cell concentration relative to untreated controls after an 18h-incubation at 28°C and 37°C for PCF and BSF T. brucei, respectively. The average of 3 independent experiments is shown together with the respective standard deviation.