Article Figures & Data
Figures
- FIG 1
Antiapoptotic effects of ks-vFLIP overexpression in Jurkat cells. Viability assays were conducted with Jurkat cells treated with various concentrations of ETO (A) and TNF-α (B). The ability of ks-vFLIP to protect Jurkat cells against the effects of ETO (C) and TNF-α (D) where FADD and ATG3 have been silenced using siRNA, together with those null for IKKγ, was assessed. (E) (Left image) Immunoblot comparing ks-vFLIP levels in Jurkat vFLIP cells with those in the KSHV-transformed PEL cell line BC3. (Right and middle images) Downregulation of FADD and ATG3 in Jurkat cells using siRNA. Asterisks indicate significant differences (*, P < 0.05, **, P < 0.01, ***, P < 0.001; z test). ns, not significant.
- FIG 2
vFLIP-IKKγ interaction and peptide thermofluor analysis. (A) (Left) Cartoon depiction of the ks-vFLIP–IKKγ heterotetramic complex (PDB code 3CL3). The IKKγ and ks-vFLIP monomers are shown in gray and wheat, respectively. (Right) The ks-vFLIP-IKKγ dimer interface highlighting cleft 1 (white) and cleft 2 (cyan). Residues essential for the formation of the cleft 1 interactions (H235, F238, D242, and K246) are colored magenta, and those that interact with cleft 2 (Q236 and E240) are colored blue. (B) IKKγ ks-vFLIP minimal interacting motif (residues 230 to 248) together with those forming the 30- and 21-mer peptides (pink and violet, respectively) used in the thermofluor studies. (C) Thermal denaturation profiles for ks-vFLIP (left) and IKKγ (right) at various concentrations. The lack of a significant response over the range of concentrations tested for IKKγ is owing to its dimeric coiled configuration that lacks a substantial hydrophobic core. (D) Thermal denaturation of ks-vFLIP in the presence of various concentrations of IKKγ (left) and the 21-mer (pink) and 30-mer (violet) (right) peptides.
- FIG 3
spIKKγ and the crystal structure of the GB1–ks-vFLIP–spIKKγ complex. (A) (Left) Sequence of the in silico-designed IKKγ-mimetic peptide (spIKKγ), together with the position and structure of (S)-2-(2′ pentenyl)alanine used to replace Q236 and E240 (green). The key cleft 1 binding residues are depicted in magenta. (Right) Energy-minimized model of spIKKγ highlighting the aliphatic linker (green) after cyclization and its position relative to the cleft 1 binding residues on the opposite face of the helix. (B) (Left) Refined coordinates of spIKKγ superimposed on the initial 2mFo-DFc electron density map (blue, contoured at 1σ), calculated using the coordinates of ks-vFLIP from PDB entry 3CL3 alone after molecular replacement. (Right) 2mFo-DFc (blue, contoured at 1σ) and mFo-DFc (green, contoured at 3σ) electron densities associated with the C-terminal cysteine of one spIKKγ peptide. (C) (Left) ks-vFLIP–spIKKγ dimer interface (for clarity, one IKKγ monomer is depicted as a cartoon [light gray]). The cleft 1 interactions (magenta for spIKKγ and yellow-orange for cleft 1 in ks-vFLIP) are entirely conserved. The aliphatic staple (green), though partially ordered, projects into the cavity between cleft 1 and cleft 2 (cyan) of the opposite ks-vFLIP monomer alongside F53. (Right) spIKKγ-spIKKγ dimer (gray) superimposed on the analogous interface in the ks-vFLIP-IKKγ(150–272) complex (pink) shows an almost identical configuration and arrangement of residues.
- FIG 4
ITC experiments and the effects of IKKγ-mimetic peptides on NF-κB activation. (A) (Top) ITC data obtained for the following: IKKγ titrated into GB1–ks-vFLIP in which both proteins were dialyzed into deionized water (left), an analogous experiment but with the proteins dialyzed into buffer (see Materials and Methods) (middle), and a competition assay in which spIKKγ was incubated with GB1–ks-vFLIP prior to titration of IKKγ (performed in deionized water) (right). (Bottom) Table showing the corresponding “apparent” Kds and Ki for spIKKγ. (B) Immunoprecipitation assays involving Jurkat cell lysates in which ks-vFLIP was expressed in the presence and absence of GST-IKKγ using anti-IKKγ antiserum (upper image) and normal rabbit serum (lower image). GST-IKKγ prevents the association of ks-vFLIP with IKKγ, evidenced by the absence of ks-vFLIP from the pellet fraction. (C) (Top) Immunoprecipitation trials using Jurkat cell lysates treated with 50 μM spIKKγ in which ks-vFLIP was absent or overexpressed. (Bottom) Results of a kinase assay measuring the levels of phosphorylated IκB in the presence and absence of spIKKγ. (D) Similar to panel C except that immunoprecipitation assays were performed using BC3 cell lysates. The rabbit antiserum control is shown in the leftmost image.
- FIG 5
Capacity of IKKγ-mimetic peptides to kill BC3 and BCP-1 cells in the presence and absence of TNF-α and ETO. (A) Viability assays using BC3 cells coexpressing GST-IKKγ (or GST-IKKγDM harboring the F238A D242R double mutant, known to impair both vFLIP binding and NF-κB activation) and ks-vFLIP alone or following treatment with TNF-α or ETO. (B) Viability assays in which BC3 cells expressing ks-vFLIP were treated with various concentrations of spIKKγ in the presence and absence of TNF-α or ETO at the specified concentrations. (C) Assays in which the viability of BC3 cells treated with spIKKγ, TNF-α, ETO, or combinations of spIKKγ and TNF-α or ETO (at the specified concentrations) were monitored after 24 and 48 h. The curves for spIKKγ plus TNF-α (magenta) and spIKKγ plus ETO (yellow) overlap. (D) Effects of TNF-α and ETO (at the specified concentrations) on the viability of DG75 cells at various concentrations of spIKKγ. (E) Control experiment illustrating the individual effects of spIKKγ, TNF-α, and ETO together with the spIKKγ-plus-TNF-α and spIKKγ-plus-ETO combinations on the viability of DG75 cells (at the specified concentrations) at 24 and 48 h. While spIKKγ and TNF-α failed to have a negative impact (either alone or in combination), a significant reduction in viability was observed for cells treated with ETO (or ETO plus spIKKγ) at 48 h. (F) Viability assays conducted with BCP-1 cells similar to those for panel B but performed at the indicated concentrations of spIKKγ, TNF-α, and ETO.
Tables
- TABLE 1
List of all primers used to produce the various plasmid constructs detailed together with target sequences for the FADD and ATG3 siRNAsa
Primer or target Sequence GB1vFLIP-Fwd TTgaattcGATGGCCACTTACG GB1vFLIP-Rev AActcgagCGCCTATGGTG GST Fwd agatctGCCACCATGTCCCCTATACTAGGTTATTGG GST Rev gtcgacctactactaggatccACGCGGAACCAGACCACCACC GST-IKKγ-Fwd gatccTTGCAGGTGGCCTATCACCAGCTCTTCCAAGAATACGACAACCACATCAAGAGCAGCGTGGTGGGCAGTtag GST-IKKγ-Rev tcgactaACTGCCCACCACGCTGCTCTTGATGTGGTTGTCGTATTCTTGGAAGAGCTGGTGATAGGCCACCTGCAAg GST-IKKγDM-Fwd gatccTTGCAGGTGGCCTATCACCAGCTCcgCCAAGAATACcgCAACCACATCAAGAGCAGCGTGGTGGGCAGTtag GST-IKKγDM-Rev tcgactaACTGCCCACCACGCTGCTCTTGATGTGGTTGcgGTATTCTTGGcgGAGCTGGTGATAGGCCACCTGCAAg FADD target 1 CATGGAACTCAGACGCATCTA FADD target 2 TGAACTCAAGCTGCGTTTATT ATG3 target 1 CCAGAACTTATGACCTTTACA ATG3 target 2 ATGTGACCATTGACCATATTT ↵a The restriction sites, stop codons, and positions mutated in the IKKγ double mutant GST constructs (GST-IKKγDM-Fwd and GST-IKKγDM-Rev) are shown in lowercase and are underlined.
- TABLE 2
Data collection, processing, and refinement statistics
Parameter Value(s)a Data collection statistics Space group P63 Unit cell [a, b, c (Å)] a = b = 90.48, c = 134.75, α = β = 90, γ = 120 Resolution (Å) 78.5–3.3 (3.3–3.6) Total no. of reflections 89,313 No. of unique reflections 9,482 Redundancy 9.4 (9.6) Completeness (%) 99.80 (99.7) 〈I〉/〈σ(I)〉 10.3 (1.3) Rmeasb 0.11 (1.8) Refinement (STARANISO-corrected data) Resolution (Å) 25–3.4 (3.4–3.8) No. of protein/peptide atoms 3,437 Completeness (%) 95.7 (85.2) Rworkc/Rfreed (%) 25.7/29.0 Estimated coordinate error based on Rfree (Å) 0.61 Mean B-factor (Å2) 135 Deviations from ideal stereochemistry RMSD bonds (Å) 0.009 RMSD angles (°) 1.03 Wilson B-factor (Å2) 115 Ramachandran plot analysise Most favored (%) 95.6 Additionally allowed (%) 2.64 Disallowed (%) 1.76 ↵a Values in parentheses are for the highest-resolution shell (3.4–3.8 Å).
↵b R meas = Σ[(N/N − 1)]1/2[(|Ii − 〈I〉|)/Σ(〈I〉)], where the sum is calculated over all observations of a measured reflection (Ii), 〈I〉 is the mean intensity of all the measured observations (Ii), and N is the total number of observations for each reflection.
↵c R work = Σ (|Fobs − Fcalc|)/Σ (Fobs), where Fobs is the observed structure factor amplitude and Fcalc those calculated from the model.
↵d R free is equivalent to Rwork but where 5% of the measured reflections have been excluded from refinement and set aside for cross-validation purposes.
↵e Ramachandran plot analysis was from molprobity (http://molprobity.biochem.duke.edu/ ).
