Article Figures & Data
Figures
- Figure 1. CSNAP C-terminus binds within a groove formed by CSN3 and CSN8.
(A) The sequence of CSNAP. The C-terminal region of CSNAP is highlighted in yellow. (B) AlphaFold-Multimer prediction of the structure of CSN1, CSN3, CSN8, and the C-terminal region of CSNAP. The surfaces of CSN1, CSN3, and CSN8 are colored in cyan, purple, and pink, respectively. The C-terminal region of CSNAP, which displayed high AF confidence, is in inset close-up represented by yellow cartoon. Polar and charged residues are shown in sticks. CSN3 E113 and E333 were used for cross-linking distance analysis. (C) Close-up of CSNAP’s-binding region. The hydrophobic and aromatic residues of CSNAP (yellow spheres) are largely buried within the groove formed by CSN3 and CSN8. (D) Electrostatic potential distribution maps of CSN3, CSN8, and CSNAP colored by charge (red negative and blue positive). Maps for CSN3/CSN8 and CSNAP were generated separately. The image emphasizes the negative charge of CSNAP and the complementary positive potential of the CSN3/CSN8 groove.
- Figure S1. The C-terminal CSNAP structure displays high AlphaFold confidence parameters.
(A) pLDDT scores for CSNAP. Regions with pLDDT >85 are expected to be modeled to high accuracy which is the case with the C-terminus tail of CSNAP. (B) Predicted aligned error scores for the CSN1, CSN3, CSN8, CSNAP-combined complex. When predicted aligned error is high (red), the relative positions of the residues in the 3D structure is uncertain. The C-terminus domain of CSNAP shows high certainty scores (blue) in the interface of the CSN3 and CSN8 domains.
- Figure 2. The last 16 amino acids of CSNAP efficiently displace the endogenous CSNAP subunit.
(A) The different CSNAP C-terminal sequences that were examined. The 16 amino acid sequence is highlighted in blue, and the N- or C-terminally truncated forms are labelled in green and magenta, respectively (numbering of residues are corresponding to full length CSNAP). (B) Immunoprecipitation experiments of cells transiently expressing full-length CSNAP (black) or truncated versions (green, blue, and magenta) fused to cerulean (Cer). Lysates from HAP1 cells transiently expressing CSNAP variants are shown on the top, and the corresponding anti-GFP pull downs on the bottom. The last 16 amino acids of C-CSNAP fused to Cer (blue) were preferentially incorporated into the COP9 signalosome complex. The efficiency of incorporation of the 16 amino acid C-CSNAP outperforms even that of the full-length CSNAP subunit. (C) Quantification of the densitometry results of at least three independent immunoprecipitation experiments using an anti-GFP antibody, represented as mean ratios of immunoprecipitated CSN5/GFP, followed by normalization to the sample transfected with Cer, plotted as mean ± SEM.
- Figure S2. The C-terminal region of CSNAP is incorporated into the CSN∆CSNAP complex.
(A) The different CSNAP C-terminal sequences that were examined. The 16 amino acid sequence is highlighted in blue, and the N- or C-terminally truncated forms are labelled in green and magenta, respectively. (B) Immunoprecipitation experiments of ΔCSNAP cells transiently expressing full-length CSNAP (black) or truncated versions (green, blue, and magenta) fused to cerulean. Lysates from HAP1 cells transiently expressing CSNAP variants are shown in the top, and the corresponding anti-GFP pull downs in the bottom panel. All C-CSNAP derivatives were efficiently incorporated into the CSNΔCSNAP complex.
- Figure 3. Peptide array screen of modified C-CSNAP peptides.
C-CSNAP peptide and its derivatives were synthesized on a microarray. The array was blocked and incubated with or without the COP9 signalosome complex (CSN) and probed using an anti-CSN3 antibody and HRP-conjugated secondary antibody. To assess non-specific binding of the antibodies to the array, anti-CSN3 and anti-GAR–HRP were used without the addition of the CSN. Signal intensity of each spot was measured and normalized to the intensity of the spot of C-CSNAP (A1). Measured values for each spot were averaged from six arrays and plotted with SEM. Red bars indicate increased binding compared with C-CSNAP (>1.14-fold, [average − SEM] > 1), and blue bars represent weaker interaction (<−1.14-fold, [average + SEM] < 1), all other bars are shown in gray. In (C), results for spot B22 was disregarded because of non-specific CSN binding to this spot. (A) A representative image of the peptide array (left) and the background (right). (B) Elongation of the C-terminal 16 amino acids (green) with one residue at a time based on the full-length sequence. The data highlight stronger binding to CSN when the sequence is 20 amino acids long. (C) The bar plot shows the key positions in which single-residue substitution with alanine significantly reduced the binding to CSN. (D) Phe substitution screen to Trp demonstrates the specificity of the Phe residues in CSN binding, the more Phe are substituted with Trp the interaction decreased. (E) Asp to Glu substitution emphasizes the importance of Asp residues. Single substitution increased slightly the strength of binding to CSN, whereas multiple substitutions significantly weakened interaction in most cases. (F) Single D-amino acid substitution at various positions. Substitution of Phe47 significantly reduced binding to CSN, whereas replacement of Leu50 to Asp55 promoted interaction. (G) Incorporation of non-proteinogenic amino acids disrupted the CSN–C-CSNAP interaction.
- Figure S3. Peptide array screen of modified C-CSNAP peptides.
C-CSNAP peptide and its derivatives were printed on a microarray. The array was blocked and incubated with or without the CSN∆CSNAP complex and probed using an anti-CSN3 antibody and HRP-conjugated secondary antibody. To assess non-specific binding of the antibodies to the array, anti-CSN3 and anti-GAR-HRP were used without the addition of the COP9 signalosome complex (CSN). Signal intensity of each spot was measured and normalized to the intensity of the spot of C-CSNAP (A1). Measured values for each spot were averaged from six arrays and plotted with standard errors SEM. Red bars indicate increased binding compared with C-CSNAP (>1.14-fold, [average − SEM] > 1), and blue bars represent weaker interaction (<−1.14-fold, [average + SEM] < 1). In (C), results for spot B22 was disregarded because of non-specific CSN binding to this spot. (A) A representative image of the peptide array (left) and the background (right). (B) Elongation of the C-terminal 16 amino acids (green) with one residue at a time based on the full-length sequence. (C) The bar plot shows the key positions in which single residue substitution with alanine significantly weakens binding to CSN. (D, E) Phe substitution screen to Trp and (E) Asp to Glu replacements. (F) Single D-amino acid substitution at various positions. (G) Incorporation of non-proteinogenic amino acids.
- Figure 4. C-CSNAP peptides display high affinity towards the CSNΔCSNAP complex.
(A, B, C, D, E) Biacore sensograms for (A) full-length CSNAP, (B) 16 residue C-terminal fragment, 16AA C-CSNAP, (C) the 20 residue C-terminal fragment, 20 AA C-CSNAP, (D) a peptide containing the substitution of Asp to Glu at position 42, D42E C-CSNAP, and (E) a scrambled peptide. Biotinylated CSNAP variant peptides were immobilized onto a Series S SA sensor chip at a concentration of 2 μg/ml and CSNΔCSNAP was injected at concentrations of 1 (pink), 2 (yellow), 4 (green), 6.25 (orange), and 8 (purple) nM. (F) Binding rate constants and affinities were determined via Biacore Evaluation Software analysis of SPR sensograms.
- Figure 5. C-CSNAP peptides displace the full-length subunit.
(A) Schematic representation of the displacement experiments using C-CSNAP peptide variants and recombinant COP9 signalosome complex (CSN). For clarity, CSNAP is colored in blue, C-CSNAP peptides in red, and Csn8 in green. CSN was incubated with 10-fold molar excess of 16AA C-CSNAP or modified peptides before coupling to StrepTactin beads (1) for 2.5 h at 37°C (2). StrepTactin-bound CSN (3) was washed three times (4) to remove free peptides, and CSN was eluted from the beads (5) for mass spectrometry analysis. (B) In this analysis, the CSN was separated into its component subunits, using a monolithic column under denaturing conditions. The eluted subunits are directed straight into the mass spectrometer for intact protein mass measurements. Masses corresponding to all four peptides were detected (highlighted with red, light blue, yellow, and dark purple for 20AA, 16AA, D42E, and dLeu50 C–CSNAP, respectively). Representative spectra of the canonical CSN subunit, CSN8 (green). The two different series of peaks in the ESI-MS spectrum correspond to full-length CSN8 and its alternative translation initiation site isoform (34). (C) The bar plot shows average ratios of intensities corresponding to masses of each peptide and CSNAP after incubation with CSN with SEM. Significance was calculated from a minimum of three experiments for each peptide using one-way ANOVA, yielding a value of P < 0.005, followed by Dunnett’s multiple comparisons test ***P < 0.001. (D) The CSN complex was incubated either with or without the 20AA C-CSNAP peptide at a ratio of 1:2 and then analyzed by tandem MS. MSMS analysis of the intact CSN complex resulted in dissociation of CSNAP (upper panel, dark blue squares). Activation of the CSN complex following preincubation with 20AA C-CSNAP resulted in dissociation of both CSNAP and 20AA C-CSNAP (middle panel, dark blue squares, and red circles, respectively), demonstrating that before MSMS analysis, 20AA C-CSNAP was physically associated with the CSN complex. As a control, C-CSNAP was measured alone and was detected as a doubly charged ion (lower panel, red circle).
- Figure S4. Representative COP9 signalosome complex (CSN) subunits identified in the displacement experiment (Fig 5).
The recombinant CSN complex was bound to StrepTactin beads and after elution separated into its composing subunits, using a monolithic column under denaturing conditions followed by on-line mass spectrometry analysis. Peaks representing the eluted CSN subunits are highlighted in color in the top panel and correlated with the recorded ESI-mass spectrometry spectra below.
- Figure 6. Expression of C-CSNAP reduces cellular proliferation rate.
(A) Immunoprecipitation analysis using anti-GFP antibody recognizing cerulean (Cer) from lysates of cells expressing the Cer-fused 16AA, 20AA, D42E, and scrambled C-CSNAP in HAP1 WT cells. The COP9 signalosome complex was pulled down from all cells, except when Cer is expressed alone or fused to the scrambled C-CSNAP. Similarly, reciprocal immunoprecipitation by an anti-CSN3 antibody pulled down only the Cer-fused 16AA, 20AA, or D42E C-CSNAP. (B) Stable overexpression of 16AA and 20AA C-CSNAP–Cer in HAP1 WT cells reduces their rate of proliferation. The bar plot shows average proliferation rates of each cell line using five independent replicates with SEM. One-way ANOVA (P < 0.05) with Dunnett’s multiple comparisons test was used to compare means *P < 0.05, **P < 0.01.
- Figure S5. Replicas of reciprocal co-immunoprecipitation experiments indicate the integration of the C-CSNAP peptides within COP9 signalosome complex.
The COP9 signalosome complex was immunoprecipitated using anti-GFP antibody recognizing cerulean (Cer) from lysates of cells expressing the Cer-fused 16AA, 20AA, and D42E C-CSNAP in HAP1 WT cells but not when Cer is expressed alone or fused to the scrambled C-CSNAP. Similarly, reciprocal immunoprecipitation by an anti-CSN3 antibody pulled down only the Cer-fused 16AA, 20AA, or D42E C-CSNAP.
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