Fig 1.
Entamoeba histolytica promotes the degradation of cullin-1/5 from THP-1 derived macrophages, colonic epithelial cells, and proximal colonic loop tissues in a time- and dose-dependent fashion.
(A) Schematic representation of Skp-1-Cullin-1-F-box (SCF) protein E3 complex (Skp-1: S-phase kinase-associated protein 1; Rbx: RING [Really Interesting New Gene]-box1; E2: Ubiquitin-loaded E2). (B, C) THP-1 macrophages were incubated with different Eh:macrophage ratios, or (D, E) for increasing times (5 to 30 min) with Eh (20:1 ratio) and Eh (10:1 ratio). (F, G) T84 colonic epithelial cells were grown in 12-well plates and stimulated with Eh (10:1 ratio) for 5 to 30 min. (H, I) Proximal colonic loops were inoculated with Eh and cell lysates were prepared. Post Eh stimulations, cells were washed and cytoplasmic extracts were prepared, and an equal amount of protein was loaded on to the SDS- PAGE gel (7.5%) and immunoblotted with the anti-cullin-1, anti-cullin-5 and anti-GAPDH antibody. Highlighted boxed areas on the figures show point of interest for cullin-1/5 as described in text. (J) Protein-protein interaction using STRING v11 showed direct interaction between cullin-1, cullin-5, cullin-4A, cullin-4B and Nedd8. Data (B, C, D, E, H, and I) are representative of three independent experiments and data (F and G) are representative of two independent experiment and statistical significance was carried out with Students t- test. Bar represent mean ± SEM. **P<0.01, ***P<0.001.
Fig 2.
Cullin-1/5 degradation requires Gal-lectin-mediated contact with macrophage.
(A, B) THP-1 macrophages were stimulated with either Eh (10:1 ratio) alone or with Eh preincubated with 55mM Galactose or Glucose for 10 min at room temperature and the degradation of the cullin-1/5 determined. (C, D) THP-1 macrophages were stimulated with native Eh Gal-lectin (500ng/ml) for 1h and 2h. Post incubation, cells were washed and lysed. Equal amount of protein was loaded onto 7.5% SDS-PAGE gel and immunoblotted with anti-cullin-1, anti-cullin-5 and anti-GAPDH antibody. Highlighted boxed areas on the figures show point of interest for cullin-1/5 as described in text. Data are representative of three independent experiments and statistical significance was carried out with Students t- test. Bar represent mean ± SEM. **P<0.01, ***P<0.001.
Fig 3.
Cullin-1/5 degradation requires EhCP-A1 and EhCP-A4 but independent of EhCP-A5.
(A, B) THP-1 derived macrophages were incubated with wild type Eh (10:1 ratio), EhCP-A5 deficient Eh (EhCP-A5-), E64 inhibitor-treated Eh, Eh pre-incubated with WRR483, (inhibitor for EhCP-A1), WRR 605 (inhibitor for EhCP-A4) or Eh pre-treated with both WRR483 and WRR605 in combination for 30 min at 37° C for 10 min. Post incubation, cells were washed and lysed and equal amount of protein was loaded onto SDS- PAGE gel and immunoblotted with anti-cullin-1 (A), anti-cullin-5 (B) and anti-GAPDH antibody. Highlighted boxed areas on the figures show point of interest for cullin-1/5 as described in text. Results are representative of two independent experiments.
Fig 4.
Potential cleavage site prediction by caspases in different cullin using the software peptide cutter.
The software Peptide Cutter (ExPASy) that predicts potential cleavage sites by different chemicals and proteases against a given query (Protein sequence) was used, for determining the potential cleavage site in different cullin protein’s (cullin-1, cullin-2, cullin-3, cullin-4A, cullin-4B and cullin-5). (A) The yellow color at D239, aspartic acid residue (arrow) represents the potential site for cullin-1 cleavage by caspase-1. The sequence in red represents a fragment of 27 kDa and the sequence in green represents a fragment of 62 KDa. (B) The yellow color at D524, aspartic acid residue (arrow) denotes the potential cleavage site for cullin-5 by caspase-1. The sequence in blue represents a fragment of 60 kDa, while the sequence marked in red represents a 30 kDa fragment. (C-F) No potential cleavage sites for caspase-1 were noted for cullin-2, 3, 4A and 4B.
Fig 5.
Caspase-1 activation during Eh-macrophage contact degrades cullin-1/5.
(A, B) THP-1 derived macrophages were pre-incubated with the pan-caspase inhibitor Z-VAD-fmk (100μM) and caspase-1 specific inhibitor Z-YVAD-fmk (100μM) for 1 h followed by stimulation with Eh (10:1 ratio) for 10 min. Cleavage of the cullin-1/5 were assessed by western blot. (C, D) Wild type (WT) THP-1 and CASP1 CRISPR/Cas9-KO macrophages were stimulated with Eh (10:1 ratio) for 10 min. (E, F) WT, THP-1 def ASC and NLRP3 CRISPR/Cas9-KO THP-1 macrophages were stimulated with Eh (10:1 ratio) for 10 min. After incubation, cells were washed and lysed. Equal amount of protein was loaded on to the SDS-PAGE (7.5%) and immunoblotted with anti-cullin-1 (Panel: A, C, and E), anti-cullin-5 (Panel: B, D and F) and anti-GAPDH antibody. (G) Cullin-1 was immunoprecipitated (Cul-1 IP) using anti cullin-1 antibody and post immunoprecipitation it was incubated with recombinant caspase-1 (C-1) for 4h or overnight (O/N). (H) Immunoprecipitated cullin-1 was incubated with recombinant caspase-1 or with Y-VAD-fmk (50μM or 100μM) or Z-VAD-fmk (100μM). (I) Immunoprecipitated cullin-5 was incubated with recombinant caspase-1 or with Z-VAD-fmk (100μM) or Y-VAD-fmk (100μM). (J) Recombinant cullin-1 (rec Cul-1) was incubated with recombinant caspase-1 overnight at 37° and was immunoblotted with anti-cullin-1 antibody. Note, recombinant cullin-1 (rec Cul-1) has a molecular weight of ~118 kDa due to the N-terminus GST tag (~28kDa). The highlighted boxed areas in the figures show point of interest for cullin-1/5 as described in text. (K) Protein-protein interaction using STRING v11 showed direct interaction between cullin-1, cullin-5 and Nedd8 demonstrated that cullin-1/5 are novel substrates for caspase-1. Data (A, B, C and D) are representative of three independent experiments while data (E, F, G, I, and J) are representative of two independent experiments.
Fig 6.
Cullin-1/5 are required for NF-κB signaling during Eh-macrophage contact.
(A) THP-1 cells were transfected with 100nM of cullin-1 siRNA and scrambled control (SC siRNA) and in (B) 100nM of cullin-5 siRNA and scrambled control (SC siRNA). After 72h, cells were stimulated with Eh for 1-, 2-, 5- and 10-min. Recombinant human TNF-α (hTNF-α) was used as a positive control for the experiment. Post incubation with Eh, cells were washed and lysed, and an equal amount of lysate was loaded onto the SDS-PAGE gel (12%) and immunoblotted with anti-pIκ-Bα, anti-Iκ-B, anti-p65 and anti-GAPDH antibody. Highlighted boxed areas on the figures show decrease in pIκ-Bα and p65. (C) Protein-protein interaction using STRING v11 showed direct interactions between cullin-1 and Rel and direct interaction between cullin-1/5. Data are representative of two independent experiment.
Fig 7.
Silencing cullin-1/5 decreased mRNA and protein expression of NF-κB dependent cytokines.
(A, B) THP-1 cells were transfected with scrambled control (SC siRNA), 100nM of cullin-1 siRNA (A), and 100nM of cullin-5 siRNA (B), respectively. After 72h, transfected cells were washed and lysed and immunoblotted against the indicated antibody to verify silencing. (C-D) THP-1 cells silenced for cullin-1/5 by siRNA were stimulated with Eh for 2h and TNF-α mRNA expression was measured using real-time PCR. Post silencing of cullin-1/5 genes, cells were incubated with Eh for 2h and pro-inflammatory cytokines levels measured using human cytokine array pro-inflammatory focused 15-plex discovery assay. LPS was used as a positive control for the experiment. Silencing of cullin-1/5 downregulated MCP-1 (E, F) and TNF-α (G, H) in response to LPS stimulation. Data are representative of three independent experiments. Bars represent mean ± SEM. * P <0.05, ** P <0.01, ***P <0.001.
Fig 8.
Eh Gal-lectin initiates contact with macrophages allowing surface bound EhCP-A5 and vesicle bound cysteine proteinases, EhCP-A1 and EhCP-A4 to interact with macrophage membranes at the intercellular junction. EhCP-A1 and EhCP-A4 degradation of cytoskeletal proteins trigger the NLRP3 inflammasome to activate caspase-1 through an unknown mechanism that subsequently degrades cullin-1/5, that in turn decreased the phosphorylation of Iκ-Bα inhibiting the transcription and secretion of NF-κB dependent pro-inflammatory cytokines. Eh-macrophage interaction also led to the degradation of cullin-4A which was partially dependent on caspase-1 but independent in the degradation of cullin-4B. Cullin-2/3 were not degraded in response to Eh.