A revised model of TRAIL ‐R2 DISC assembly explains how FLIP (L) can inhibit or promote apoptosis
2020; Springer Nature; Volume: 21; Issue: 3 Linguagem: Inglês
10.15252/embr.201949254
ISSN1469-3178
AutoresLuke Humphreys, Jennifer Fox, Catherine Higgins, Joanna Majkut, Tamas Sessler, Kirsty M. McLaughlin, Christopher McCann, Jamie Z. Roberts, Nyree Crawford, Simon S. McDade, Christopher J. Scott, Timothy Harrison, Daniel B. Longley,
Tópico(s)Phagocytosis and Immune Regulation
ResumoArticle3 February 2020Open Access Source DataTransparent process A revised model of TRAIL-R2 DISC assembly explains how FLIP(L) can inhibit or promote apoptosis Luke M Humphreys Luke M Humphreys The Patrick G. Johnston Centre for Cancer Research, Queen's University Belfast, Belfast, UK Search for more papers by this author Jennifer P Fox Jennifer P Fox The Patrick G. Johnston Centre for Cancer Research, Queen's University Belfast, Belfast, UK Search for more papers by this author Catherine A Higgins Catherine A Higgins The Patrick G. Johnston Centre for Cancer Research, Queen's University Belfast, Belfast, UK Search for more papers by this author Joanna Majkut Joanna Majkut The Patrick G. Johnston Centre for Cancer Research, Queen's University Belfast, Belfast, UK Search for more papers by this author Tamas Sessler Tamas Sessler The Patrick G. Johnston Centre for Cancer Research, Queen's University Belfast, Belfast, UK Search for more papers by this author Kirsty McLaughlin Kirsty McLaughlin The Patrick G. Johnston Centre for Cancer Research, Queen's University Belfast, Belfast, UK Search for more papers by this author Christopher McCann Christopher McCann The Patrick G. Johnston Centre for Cancer Research, Queen's University Belfast, Belfast, UK Search for more papers by this author Jamie Z Roberts Jamie Z Roberts The Patrick G. Johnston Centre for Cancer Research, Queen's University Belfast, Belfast, UK Search for more papers by this author Nyree T Crawford Nyree T Crawford The Patrick G. Johnston Centre for Cancer Research, Queen's University Belfast, Belfast, UK Search for more papers by this author Simon S McDade Simon S McDade orcid.org/0000-0002-3024-4773 The Patrick G. Johnston Centre for Cancer Research, Queen's University Belfast, Belfast, UK Search for more papers by this author Christopher J Scott Christopher J Scott The Patrick G. Johnston Centre for Cancer Research, Queen's University Belfast, Belfast, UK Search for more papers by this author Timothy Harrison Timothy Harrison The Patrick G. Johnston Centre for Cancer Research, Queen's University Belfast, Belfast, UK Search for more papers by this author Daniel B Longley Corresponding Author Daniel B Longley [email protected] orcid.org/0000-0003-2495-2033 The Patrick G. Johnston Centre for Cancer Research, Queen's University Belfast, Belfast, UK Search for more papers by this author Luke M Humphreys Luke M Humphreys The Patrick G. Johnston Centre for Cancer Research, Queen's University Belfast, Belfast, UK Search for more papers by this author Jennifer P Fox Jennifer P Fox The Patrick G. Johnston Centre for Cancer Research, Queen's University Belfast, Belfast, UK Search for more papers by this author Catherine A Higgins Catherine A Higgins The Patrick G. Johnston Centre for Cancer Research, Queen's University Belfast, Belfast, UK Search for more papers by this author Joanna Majkut Joanna Majkut The Patrick G. Johnston Centre for Cancer Research, Queen's University Belfast, Belfast, UK Search for more papers by this author Tamas Sessler Tamas Sessler The Patrick G. Johnston Centre for Cancer Research, Queen's University Belfast, Belfast, UK Search for more papers by this author Kirsty McLaughlin Kirsty McLaughlin The Patrick G. Johnston Centre for Cancer Research, Queen's University Belfast, Belfast, UK Search for more papers by this author Christopher McCann Christopher McCann The Patrick G. Johnston Centre for Cancer Research, Queen's University Belfast, Belfast, UK Search for more papers by this author Jamie Z Roberts Jamie Z Roberts The Patrick G. Johnston Centre for Cancer Research, Queen's University Belfast, Belfast, UK Search for more papers by this author Nyree T Crawford Nyree T Crawford The Patrick G. Johnston Centre for Cancer Research, Queen's University Belfast, Belfast, UK Search for more papers by this author Simon S McDade Simon S McDade orcid.org/0000-0002-3024-4773 The Patrick G. Johnston Centre for Cancer Research, Queen's University Belfast, Belfast, UK Search for more papers by this author Christopher J Scott Christopher J Scott The Patrick G. Johnston Centre for Cancer Research, Queen's University Belfast, Belfast, UK Search for more papers by this author Timothy Harrison Timothy Harrison The Patrick G. Johnston Centre for Cancer Research, Queen's University Belfast, Belfast, UK Search for more papers by this author Daniel B Longley Corresponding Author Daniel B Longley [email protected] orcid.org/0000-0003-2495-2033 The Patrick G. Johnston Centre for Cancer Research, Queen's University Belfast, Belfast, UK Search for more papers by this author Author Information Luke M Humphreys1, Jennifer P Fox1, Catherine A Higgins1, Joanna Majkut1, Tamas Sessler1, Kirsty McLaughlin1, Christopher McCann1, Jamie Z Roberts1, Nyree T Crawford1, Simon S McDade1, Christopher J Scott1, Timothy Harrison1 and Daniel B Longley *,1 1The Patrick G. Johnston Centre for Cancer Research, Queen's University Belfast, Belfast, UK *Corresponding author. Tel: +44 2890 972647; E-mail: [email protected] EMBO Reports (2020)21:e49254https://doi.org/10.15252/embr.201949254 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The long FLIP splice form FLIP(L) can act as both an inhibitor and promoter of caspase-8 at death-inducing signalling complexes (DISCs) formed by death receptors such as TRAIL-R2 and related intracellular complexes such as the ripoptosome. Herein, we describe a revised DISC assembly model that explains how FLIP(L) can have these opposite effects by defining the stoichiometry (with respect to caspase-8) at which it converts from being anti- to pro-apoptotic at the DISC. We also show that in the complete absence of FLIP(L), procaspase-8 activation at the TRAIL-R2 DISC has significantly slower kinetics, although ultimately the extent of apoptosis is significantly greater. This revised model of DISC assembly also explains why FLIP's recruitment to the TRAIL-R2 DISC is impaired in the absence of caspase-8 despite showing that it can interact with the DISC adaptor protein FADD and why the short FLIP splice form FLIP(S) is the more potent inhibitor of DISC-mediated apoptosis. Synopsis A revised model of TRAIL-R2 DISC assembly and stoichiometry can explain the ability of FLIP(L) to act as either an inhibitor or activator of apoptosis signalling in these complexes. The stoichiometry of the pseudo-caspase FLIP(L) relative to caspase-8 at the TRAIL-R2 death-inducing signalling complex (DISC) was re-examined. When equistochiometric with capase-8, FLIP(L) inhibits apoptosis at the TRAIL-R2 DISC (caspase-8:FLIP(L) ratio ≈ 1:1). Sub-stoichiometric FLIP(L) (caspase-8:FLIP(L) ratio > 1:1) accelerates caspase-8 activation. The revised model of DISC assembly predicts that relatively small changes in FLIP(L)'s DISC recruitment can have profound effects on cell death signalling. Introduction Apoptosis can be activated through the intrinsic (mitochondrial-mediated) and extrinsic (death receptor-mediated) pathways and is frequently dysfunctional in cancer 1, 2. The extrinsic apoptotic pathway is initiated when death ligands such as TRAIL (tumour necrosis factor (TNF)-related apoptosis-inducing ligand) bind their receptors (TRAIL-R1 (DR4)/TRAIL-R2 (DR5)). This binding enables the intracellular death domains (DDs) of the receptors to engage the adaptor protein FADD (Fas-associated protein with death domain) through homotypic interactions with its DD 3-7. Engagement of the FADD DD by the activated receptor enables its other domain, the death effector domain (DED), to engage in homotypic protein–protein interactions with other DED-containing proteins, most importantly the tandem DED-containing procaspase-8 (FADD-like interleukin-1β-converting enzyme, FLICE) and its regulator FLIP (FLICE-inhibitory protein) 8-10. At this complex, known as the death-inducing signalling complex (DISC), procaspase-8 forms homodimers or heterodimers with FLIP via homotypic interactions between their DEDs. Homo-dimerisation is required for activation and processing of procaspase-8, which occurs in a two-step manner: rate-limiting inter-dimer cleavage between adjacent dimers recruited to the DISC that cleaves the linker between its large (p18) and small (p10) catalytic domains, followed by intra-dimer cleavage between the p18-subunit and the DED-containing pro-domain. The 2nd cleavage step releases the active enzyme made up of a hetero-tetramer of two p18 and two p10 subunits from the DISC, which can then propagate an apoptotic signal by cleaving downstream substrates such as BID and procaspases-3/7 11-16. At the DISC, hetero-dimerisation of FLIP(S) with procaspase-8 prevents formation of the caspase-8 active site and thereby inhibits procaspase-8 processing 17. However, FLIP(L) has a pseudo-catalytic domain, and when it hetero-dimerises with procaspase-8, this results in formation of an enzymatically active complex with altered substrate specificity compared to the caspase-8 homodimer 18, which is retained at the DISC 19. The role of FLIP(L) at the DISC is complex, and whether it acts to promote or inhibit caspase-8 activation is a matter of debate. In this report, we determine the stoichiometry of FLIP(L):procaspase-8 at the DISC that results in apoptosis induction versus apoptosis inhibition and provide data that advance our understanding of the interplay between FLIP and caspase-8 in this complex and which supports a revised model of DISC assembly. Results Assessment of TRAIL-R2 DISC stoichiometry In a previous study, we quantified the relative levels of FLIP, caspase-8 and FADD at the TRAIL-R2 DISC using recombinant protein standards 20. The approximate ratio of 2 molecules of every tandem DED protein (caspase-8 and FLIP) for every 1 molecule of FADD was in disagreement with those obtained by two other groups 21, 22, who noted higher ratios of tandem DED proteins to FADD. To reconcile these differing findings, we developed a new quantification approach using Flag-tagged proteins of similar molecular weight to endogenous proteins (expressed and purified from human cells rather than truncated versions derived from bacterial expression systems) to quantitatively evaluate the stoichiometry of FLIP, caspase-8 and FADD at the TRAIL-R2 DISC (Fig EV1) 20. In addition, we used both N- and C-terminal antibodies to detect fully processed caspase-8 (the DED1/2-only pro-domain) retained at the DISC, a form of caspase-8 not quantified in our earlier study. Click here to expand this figure. Figure EV1. Quantitative DISC IP methodology Western blot analysis of Flag-tagged-caspase-8/FLIP and Flag-tagged-caspase-8/FADD standards using protein-specific and anti-Flag primary antibodies and LiCOR mouse (800 nm, green) and rabbit (700 nm, red) secondary antibodies. In the green channel, standard curves for each exogenous protein were used to calculate the abundance of each protein-specific band in DISC IPs (see Figs 1 and 2, and EV3 and EV4). A correction was made to account for any small differences between the procaspase-8 and FLIP (C8/FLIP standards) and procaspase-8 and FADD (C8/FADD standards) bands as detected using the anti-Flag antibody. In the red channel, the standard curves were used to calculate the ratio of p41/43-caspase-8 to p24/26-caspase-8; the amount of p24/26-caspase-8 was then calculated from the amount of p41/43-caspase-8 detected in the green channel. Source data are available online for this figure. Download figure Download PowerPoint Three cancer cell lines representing 3 cancer types, A549 (NSCLC), HCT116 (colon) and DU145 (prostate), were selected to assess TRAIL-R2 DISC stoichiometry. A549 is a relatively TRAIL-resistant NSCLC cell line 23, while HCT116 is a TRAIL-sensitive model of colorectal cancer, and DU145 is a classical model of androgen-independent prostate cancer representing a later stage of the disease and which typically expresses high levels of FLIP 24. Basal protein expression (Fig EV2A) and sensitivity (caspase activation and cell death) to a recombinant multivalent form of recombinant TRAIL (IZ-TRAIL, which can activate both TRAIL-R1 and TRAIL-R2; Fig EV2B) confirmed that these models express the relevant TRAIL-R2 pathway components and that the HCT116 model is more sensitive than the A549 model, with the DU145 model in between, giving us a good range of models with which to study TRAIL-R2 DISC assembly. Click here to expand this figure. Figure EV2. Baseline expression of TRAIL-R2 interactome and sensitivity to TRAIL-R2 agonists A, B. Western blot analysis of basal levels of the TRAIL-R2 DISC components FLIP, procaspase 8, FADD and TRAIL-R2 in A549, HCT116 and DU145 cells. Caspase activity (6 h) and cell death induction (24 h) in A549, HCT116 and DU145 cells treated with (B) IZ-TRAIL. C, D. anti-TRAIL-R2 (AMG655)-coated magnetic beads; and (D) MEDI3039. Specific apoptosis was calculated as treatment-induced apoptosis minus background apoptosis in control untreated cells. E. Relative levels of FADD, total caspase-8 and total FLIP pulled down in the TRAIL-R2 DISC IPs presented in Fig 1. Download figure Download PowerPoint To assess the stoichiometry of FLIP, caspase-8 and FADD, a TRAIL-R2 DISC IP was performed in cells treated with increasing concentrations (1×, 2×, 4×) of anti-TRAIL-R2 antibody (AMG655, conatumumab) conjugated to magnetic beads to enable efficient receptor cross-linking, thereby mimicking the receptor clustering effects of both the endogenous membrane-bound ligand and 2nd generation multivalent TRAIL-R2-targeting therapeutics 20, 25-27. In all 3 cell lines, the cleavage products of procaspase-8 (p41/43 and p24/26) increased from 1× to 2× AMG655 (Fig 1A and C). However, going from 2× to 4×, there was little change, suggesting saturation of binding to available (i.e. cell surface) TRAIL-R2 receptors; this was supported by analysis of TRAIL-R2 in the bound and unbound fractions (Fig 1A and B). A similar pattern of DISC recruitment was observed for FADD and both splice forms of FLIP (Figs 1A and C, and EV2E). Assessment of the unbound fraction revealed increasing levels of the p18-caspase-8 large catalytic subunit (indicative of full procaspase-8 processing to the active hetero-tetramer) from 1× to 2×, which correlated with cleavage of the caspase-3 target PARP (a marker of apoptosis induction; Fig 1B). In line with this, analysis of caspase-8-like (IETDase) activity in the unbound fraction indicated that increasing from 1× to 2× AMG655 led to an increase in activity in all 3 cell lines, but not between the 2× and 4× samples (Fig 1D). The caspase activity and PARP cleavage analyses in the unbound fractions also indicated that, as expected, the HCT116 and DU145 models were more sensitive to the AMG655 beads than the A549 model, and these results were also reflected in cellular caspase activity and apoptosis assays (Fig EV2C). Moreover, these results were recapitulated with the 2nd generation multivalent TRAIL-R2-specific agonist, MEDI3039 (Fig EV2D). Figure 1. Assessment of TRAIL-R2 DISC stoichiometry Western blot analysis of Caspase 8, FLIP and FADD recruitment to the TRAIL-R2 DISC in A549, HCT116 and DU145 cell lines following incubation with increasing concentrations (1×, 2×, or 4×) of anti-TRAIL-R2 (AMG655)-conjugated magnetic beads for 90 min. Western blot analysis of the unbound soluble fraction from (A). An untreated input of each cell line is included for comparison. Quantification of Western blot analysis from panel (A) for FLIP, FADD, procaspase-8 and their respective cleavage fragments at the TRAIL-R2 DISC. Proteins were quantified by densitometry and normalised to known protein standards (see Fig EV1). Caspase-8-like (IETDase) activity assay in the unbound soluble fraction from the DISC IP in panel (A). An untreated control (0) was used for each cell line to show basal caspase-8 activity. Ratios of DED proteins quantified at the TRAIL-R2 DISC from panel (C) in A549, HCT116 and DU145 cells; increasing concentrations of AMG655 are indicated by colour intensity. Correlation between caspase-8 activity and ratio of FADD:FLIP(L). Source data are available online for this figure. Source Data for Figure 1 [embr201949254-sup-0002-SDataFig1.pptx] Download figure Download PowerPoint Quantitative comparison of the relative levels of procaspase-8 and FADD at the DISC indicated that the ratio remained relatively constant within each cell line, regardless of the level of TRAIL-R2 stimulation (Fig 1E). In the most TRAIL-sensitive HCT116 cells, the ratio of caspase-8 (the sum of the p55, p41/43 and p24/26 subunits) to FLIP (p43-FLIP(L) plus FLIP(S)) was highest (~5–6:1), while it was lowest in the more TRAIL-resistant A549 model (close to 1:1). When the ratios of the tandem DED proteins (caspase-8 plus FLIP) were quantified relative to FADD, they were remarkably consistent across all 3 models for all 3 levels of receptor stimulation, with the average ratio calculated to be ~3:1 (Fig 1E); thus, including analysis of the p24/26-caspase-8 pro-domains in our quantitative DISC analyses resulted in ratios of tandem DED proteins to FADD that are higher than those calculated in our previous study (~2:1) 20, although still less than some of the ratios reported by others (up to 9:1) 21, 22, 28. Moreover, excluding the p24/26-caspase-8 pro-domain from these analyses yielded a ratio of tandem DED proteins to FADD of ~2.2:1, similar to our original study, indicating that both DISC quantification approaches (using recombinant proteins or Flag-tagged proteins as standards) are valid. We next calculated how expression of DISC protein components correlated with caspase-8-like activity across all 3 cell line models. Of note, a highly significant correlation was observed between caspase-8 activity and the ratio of FADD:FLIP(L) (Pearson's r = 0.91; P = 0.0006; Fig 1F), while the correlation between caspase-8 activity and the ratio of total caspase-8:FLIP(L) approached significance (Pearson's r = 0.67; P = .0506). This suggests that the relative levels of FLIP(L) recruited to the DISC are the major determinant of the extent of TRAIL-R2-induced caspase-8 activity. To determine whether caspase-8 processing altered DISC stoichiometry, we assessed the effect of the pan-caspase inhibitor zVAD-fmk (Fig EV3A–C). Despite partial inhibition of caspase-8 and FLIP(L) processing, the ratios between the 3 main DISC components were largely unaffected by zVAD-fmk, with the ratio of FADD to total caspase-8 plus total FLIP again averaging around 3 (Fig EV3G). This ratio was observed in several independent experiments, as was the tendency for caspase-8:FADD and caspase-8:FLIP ratios to be highest and FLIP:FADD ratios to be lowest in the TRAIL-sensitive HCT116 model (Fig EV3D–G). Click here to expand this figure. Figure EV3. Impact of pan-caspase inhibition on DISC stoichiometry A. Western blot analysis of FLIP, FADD and procaspase-8 recruitment to the TRAIL-R2 DISC in A549, HCT116 and DU145 cells pre-treated with 20 μM zVAD-fmk or DMSO followed by incubation with AMG655-conjugated magnetic beads for 90 min. B. Caspase-8 (IETDase) activity assay of the soluble unbound fraction from panel (A). C–G. Quantification of FADD, caspase-8, FLIP and their respective cleavage fragments from panel (A). TRAIL-R2 DISC IP ratios calculated from several independent experiments for (D) caspase-8:FADD; (E) caspase-8:FLIP; (F) FLIP:FADD; and (G) {Caspase-8+FLIP}:FADD. Source data are available online for this figure. Download figure Download PowerPoint Importantly, these results are not consistent with the caspase-8 chain model of DISC assembly 21, 22, 28, which proposes formation of caspase-8 DED1/2-dominated chains of variable lengths depending on the relative levels of each DISC component available and extent of receptor activation. Not only did we detect a lower ratio of the tandem DED proteins (caspase-8 plus FLIP) to FADD (meaning shorter "chains"), we also found that these ratios varied little across multiple models with varying levels of DISC component expression (Fig EV2A) and extent of receptor activation (average = 3.0; SD = 0.6; n = 16; Fig EV3G). FLIP(L) inhibits full caspase-8 activation when equi-stoichiometric at the DISC We next used siRNA targeting procaspase-8 to titrate the ratio of procaspase-8 to FLIP in the TRAIL-sensitive HCT116 model. Transfection of siCASP8 resulted in a concentration-dependent reduction in procaspase-8 and its cleavage products at the DISC (Figs 2A–C and EV4A). Surprisingly, as caspase-8 levels at the DISC decreased, no significant reductions in the recruitment of either splice form of FLIP were observed (Fig 2A and B). Quantification of caspase-8 and FLIP revealed a ~7:1 ratio in control (SC) cells in keeping with the results presented above for the HCT116 model (Figs 1E and EV3C and E). Notably, as the ratio of caspase-8 to FLIP approached 1:1, caspase-8 (IETDase) and downstream caspase-3/7 (DEVDase) activity approached the baseline levels in untreated cells (Fig 2D). To avoid potentially confounding effects from the intrinsic apoptotic pathway on DISC processing events and caspase activity measurements, we also used a HCT116 BAX/BAK knockout (KO) model (Fig EV4C–F); again, as the ratio of caspase-8:FLIP approached 1:1, caspase-8 and caspase-3/7 activity approached baseline levels. In addition, we found highly significant correlations between the levels of caspase-8 cleavage products (p41/43 and p24/26) at the DISC and IETDase activity in the unbound soluble fraction (Fig EV4B), indicating that this enzymatic activity reflects caspase-8 activity and providing additional validation of the quantification processes used. Figure 2. Impact of decreasing procaspase-8 on DISC assembly and caspase activation Western blot analysis of FADD, FLIP and caspase-8 at the TRAIL-R2 DISC in HCT116 cells treated with escalating doses (0–30 nM) of caspase-8 siRNA for 48 h. Samples were incubated with anti-TRAIL-R2 (AMG655) beads (4×) for 90 min prior to collection. A scrambled control siRNA (SC) was transfected (30 nM) for comparison. Quantification of caspase-8 (p55, p43/41 and p26/24) and FLIP (FLIP(L), FLIP(S) and p43-FLIP) at the TRAIL-R2 DISC by densitometry and normalised to known protein standards (see Fig EV1). Ratio of caspase-8:FLIP calculated from values presented in (B). Caspase-8 (IETDase) and caspase-3/7 (DEVDase) activity assay in the unbound soluble fraction from panel (A). Western blot analysis of FLIP, caspase-8, and FADD recruitment to the TRAIL-R2 DISC in PC3 cells stably expressing an empty vector (EV) or Flag-tagged FLIP L (FL) treated with 2.5 μM MS-275 for 48 h, followed by a 90-min DISC IP. * modified form of FLIP, potentially mono-ubiquitinated p43-FLIP(L). Table of the relative ratios of caspase-8 (p55, p41/43 and p24/26) and FLIP (endogenous and Flag-tagged) at the TRAIL-R2 DISC in MS-275-treated and untreated cells after a 90-min DISC IP. Caspase-8 (IETDase) activity assays of the unbound soluble fraction from (E). Source data are available online for this figure. Source Data for Figure 2 [embr201949254-sup-0003-SDataFig2.pptx] Download figure Download PowerPoint Click here to expand this figure. Figure EV4. Impact of procaspase-8 downregulation on DISC stoichiometry Western blot analysis of caspase-8, FLIP, FADD and TRAIL-R2 in the unbound soluble fraction from Fig 2A. Correlation of levels of caspase-8 cleavage fragments (p43/41 and p26/24) from Fig 2A with caspase-8 activity quantified in Fig 2D. Western blot analysis of proteins recruited to the TRAIL-R2 DISC following a 48-h treatment with caspase-8-targeting siRNA (0–30 nM) in HCT116 BAX/BAK null cells. Quantification of caspase-8 (p55, p41/43, p24/26), FLIP (FLIP(L), p43-FLIP, FLIP(S)) and FADD at the TRAIL-R2 DISC from panel (C). Caspase activity assays from the unbound soluble fraction from panel (C). Quantification of FLIP, FADD and caspase-8 at the TRAIL-R2 DISC in panel (C). Source data are available online for this figure. Download figure Download PowerPoint As a complementary approach, we assessed the impact of increasing FLIP levels relative to caspase-8 at the DISC using a prostate cancer cell line (PC3) stably expressing exogenous FLIP(L). The ratio of caspase-8:FLIP at the DISC was high in the control EV cell line even compared to the HCT116 model (Fig 2E and F). In the FLIP(L) overexpressing model, this was reduced and correlated with a decrease in caspase-8 activity (Fig 2G). Upon treatment with the HDAC inhibitor MS275, endogenous FLIP proteins were down-regulated (consistent with previous studies 29); however, the exogenous FLIP(L) (not under control of the endogenous promoter) was unchanged. Subsequently, the ratio of caspase-8:FLIP was significantly increased in the EV control model in response to MS275; although despite this increase, there was no change in caspase-8 activity (Fig 2G). In the FLIP(L) overexpressing model treated with MS275, the ratio of caspase-8 to FLIP (now almost entirely FLIP(L) as endogenous FLIP(S) was down-regulated) fell just below 1 and caspase-8 activity was significantly inhibited (Fig 2F). Collectively, these results indicate that when the levels of FLIP(L) and procaspase-8 recruited to the DISC are approximately equal, FLIP(L) acts as an inhibitor of caspase-8 activation. Although FLIP can interact directly with FADD in a caspase-8-independent manner, its DISC recruitment is highly caspase-8-dependent In the siRNA experiments (Fig 2), while procaspase-8 was significantly down-regulated at the highest concentrations of siRNA used, it could still be detected at the DISC. To assess the impact of complete loss of procaspase-8 on DISC assembly, we next generated a number of CRISPR-Cas9 CASP8 deletion models. In agreement with the findings of others for the TRAIL-R1 and Fas/CD95 DISCs reported during the completion of these studies 28, 30, we found that CASP8 deletion markedly inhibited recruitment of FLIP to the TRAIL-R2 DISC (Figs 3A and EV5A). However, at later timepoints (180 min), there were detectable, albeit low, levels of FLIP(L) at the DISC in CASP8 null cells, approximately half of which was in its unprocessed p55-form (Fig 3A; lane 6). In agreement with the findings of others for the Fas/CD95 DISC 30, recruitment of procaspase-8's other paralog, procaspase-10, to the TRAIL-R2 DISC was also inhibited in the absence of procaspase-8; however, low amounts were again detectable at the latest timepoint. In isogenic HCT116 models lacking either procaspase-8, procaspase-10, or both, we further confirmed the importance of procaspase-8 for both FLIP and procaspase-10 recruitment (Figs 3B and EV5B). The absence of procaspase-10 had no significant impact on the levels (relative to TRAIL-R2 and FADD) and processing of FLIP and procaspase-8, indicating that its contribution to overall TRAIL-R2 DISC stoichiometry (at least in the HCT116 model) is minimal. It was however notable that in the absence of both procaspase-8 and procaspase-10, only unprocessed FLIP(L) was detected (Fig 3B; lane 4), suggesting that the low amounts of FLIP(L) detected in the absence of procaspase-8 are cleaved by procaspase-10 and that these low levels of FLIP can be recruited by direct binding to FADD and/or procaspase-10 (in FADD null cells, no FLIP or caspase-8 is recruited to the TRAIL-R2 DISC, DB Longley, unpublished observations). Figure 3. Although FLIP can interact directly with FADD in a caspase-8-independent manner, its DISC recruitment is highly caspase-8-dependent Western Blot analysis of caspase-8, caspase-10, FLIP and FADD recruitment to the TRAIL-R2 DISC in procaspase-8 (WT) or procaspase-8 deficient (Null) A549 cells incubated with AMG655 beads (4×) for either 30, 60 or 180 min. Unbound fractions are shown in Fig EV5A. Western blot analysis of the recruitment of FLIP, caspase-8, caspase-10 and FADD to the TRAIL-R2 DISC in HCT116 parental, caspase-8 null (CASP8), caspase-10 null (CASP10) or CASP 8/10 cells treated with 4× AMG655-conjugated beads for 90 min. Unbound fractions are shown in Fig EV6B. * modified form of FLIP, potentially mono-ubiquitinated p43-FLIP(L). AlphaScreen® assessment of the interaction of recombinant FLIP DED1/2 and recombinant FADD DED. AlphaScreen® assessment of the impact on the FLIP-FADD interaction of unstapled and stapled peptides corresponding to the FADD α1 helix. NanoBiT® assay to quantify FLIP/FADD interactions in a U2OS cell line stably expressing the NanoBiT® constructs, FLIP (LgBiT) and FADD (smBiT) following 48-h silencing of FLIP, FADD or caspase-8. Western blot analysis was used to confirm successful knockdown of target proteins by siRNA. NanoBiT® assay of HCT116 and U20S wild-type (WT) and procaspase 8 null (Null) cells transiently co-transfected with FLIP (LgBiT) and FADD (SmBiT) NanoBiT® constructs for 24 h. Construct expression was analysed by Western blotting. Source data are available online for this figure. Source Data for Figure 3 [embr201949254-sup-0004-SDataFig3.pptx] Download figure Download PowerPoint Click here to expand this figure. Figure EV5. Impact of FLIP depletion on DISC stoichiometry and schematic to explain procaspase-8 and FLIP processing events at the DISC Western blot analysis of FLIP, caspase-8, caspase-10 and FADD in the soluble unbound fraction from Fig 3A. Western blot analysis of FLIP, caspase-8, caspase-10 an
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