Journal: Cell Death and Differentiation

Article Title: Site-specific ubiquitination of MLKL targets it to endosomes and targets Listeria and Yersinia to the lysosomes

doi: 10.1038/s41418-021-00924-7

Figure Lengend Snippet: A , B Time-dependent MLKL ubiquitination and phosphorylation in human HT-29 cells in response to treatment with TBZ (TNF, 1000 U/ml, the bivalent IAP antagonist BV6, 1 μM, and the caspase inhibitor z-VAD-fmk, 20 μM. These are the concentrations at which these three reagents were applied in all other experiments). A Western blot analysis (WB) of 15 μg of the proteins of the total cellular lysates (bottom) and of the proteins pulled down (PD) from these amounts of lysates using TUBE1-agarose (top). Unless otherwise specified, arrows pointing to western blots that were probed with anti-MLKL antibodies indicate non-ubiquitinated MLKL. B Comparison of the extents of ubiquitination and of cell death at different time points. Cell death was assessed twice in duplicates ( n = 4), 6with practically identical results. C – E Assessments of MLKL ubiquitination and phosphorylation: in C MEFs in response to TBZ; in D HT-29 cells in which FADD was knocked out by CRISPR/Cas9 in response to treatment with just TB, i.e., TNF (1000 U/ml) and Bv6 (1 μM); and in E L929 cells in response to TNF alone (1000 U/ml in this and in all other experiments). F , G MLKL ubiquitination in response to TBZ depends on its phosphorylation and oligomerization. The extents of MLKL ubiquitination (top), aggregation (middle) and phosphorylation (bottom) were determined in MLKL-knockout (KO) HT-29 cells constitutively expressing wild-type MLKL and F a non-phosphorylatable MLKL mutant (T357A/S358A), or G the coiled-coil domain mutant (L162G/L165G). H Comparison of the in vitro effects of the indicated deubiquitinases on HA-tagged polyubiquitin chains that were conjugated to MLKL in HT-29 cells by their treatment for 3 h with TBZ. I Effects of these deubiquitinases on synthetic K63-linked and K48-linked ubiquitin chains. J Conjugation of wild-type and K63R-mutated HA-tagged ubiquitin to MLKL in HT-29 cells. K Accumulation of ubiquitinated MLKL in response to TBZ is not enhanced by treatment for 3 h with MG132 (10 μM) or bafilomycin A1 (Baf A1, 0.1 μM).

Article Snippet: Recombinant human MLKL (CSB-EP850851HU) and mouse MLKL (CSB-EP861529MO) were from Cusabio Technology and recombinant ITCH (E3-260) was from R&D Systems.

Techniques: Ubiquitin Proteomics, Phospho-proteomics, Western Blot, Comparison, CRISPR, Knock-Out, Expressing, Mutagenesis, In Vitro, Conjugation Assay

Journal: Cell Death and Differentiation

Article Title: Site-specific ubiquitination of MLKL targets it to endosomes and targets Listeria and Yersinia to the lysosomes

doi: 10.1038/s41418-021-00924-7

Figure Lengend Snippet: A Results of quantitative mass spectroscopic analysis of MLKL ubiquitination in HT-29 cells and of its increase by treatment for 3 h with TBZ. The K50-containing peptide is VLGLIKPLEMLQDQGKR, the K66-containing peptide is FKAALEEANGEIEK and the K331-containing peptide is LHHSEAPELHGKIR. Both in untreated and in TBZ-treated cells, the amounts of K50-containing peptide in which the methionine residue was oxidized were less than 1% of the total amounts of this peptide. B Assessment of MLKL ubiquitination in TBZ-treated HT-29 cells in which endogenous MLKL had been knocked out, and either the wild-type or the K50R-mutated human MLKL was inducibly re-expressed under control of the GEV16/pF5x UAS system. C TBZ-treated MLKL KO HT-29 cells that inducibly express wild-type MLKL, but not cells expressing the K50R MLKL mutant, contain ubiquitinated MLKL molecules that bind K63-TUBE (a reagent that specifically recognizes K63-linked polyubiquitin chains). D K63-linked polyubiquitination of wild-type MLKL, but not of its K50R mutant, can be discerned by an antibody that specifically recognizes the K63 link. Western blot analysis was done using MLKL preparations that were affinity-purified from lysates of TBZ-treated HT-29 cells, as described in Materials and methods. E Assessment of MLKL ubiquitination in TBZ-treated MLKL KO MEFs expressing the indicated mutants of mouse MLKL as in B . F Assessment of MLKL ubiquitination in TNF-treated MLKL KO L929 cells expressing either wild-type or K50,51R-mutated mouse MLKL as in B . The arrowhead points to phosphorylated mouse MLKL. G Site of MLKL ubiquitination within the HeLo domain structure. Shown are ribbon diagrams of the HeLo domains in human MLKL (amino acids 4–120) and mouse MLKL (amino acids 2−115), drawn using PyMOL software based on the solution structure of the human MLKL N-terminal region, as determined using nuclear magnetic resonance (PDB 2MSV), and the crystal structure of full-length mouse MLKL, as determined by X-ray diffraction (PDB 4BTF). The helices forming the four-helix bundle are denoted and colored, as are the lysine residues that we found to be ubiquitinated (blue) and arginine 51 in human MLKL (green).

Article Snippet: Recombinant human MLKL (CSB-EP850851HU) and mouse MLKL (CSB-EP861529MO) were from Cusabio Technology and recombinant ITCH (E3-260) was from R&D Systems.

Techniques: Ubiquitin Proteomics, Residue, Control, Expressing, Mutagenesis, Western Blot, Affinity Purification, Software, Nuclear Magnetic Resonance

Journal: Cell Death and Differentiation

Article Title: Site-specific ubiquitination of MLKL targets it to endosomes and targets Listeria and Yersinia to the lysosomes

doi: 10.1038/s41418-021-00924-7

Figure Lengend Snippet: A Assessment of the occurrence of ubiquitinated and phosphorylated MLKL, induced by treatment for 3 h with TBZ, in the indicated subcellular fractions of HT-29 cells. Samples of 15 μg per well of the proteins in the lysates of each fraction were subjected to western blot analysis, and samples of 170 μg per well of the proteins were assessed for MLKL ubiquitination. B – I Microscopic analysis of the effect of the K50R mutation in MLKL on the association of MLKL ( B − E ) and of both MLKL and ubiquitin ( F − I ) with the early and late endosomes (visualized by constitutive expression of Rab5 or Rab7 fused to mCherry) in MLKL KO HT-29 cells inducibly expressing MLKL or its K50R mutant fused to GFP, observed after treatment for 2.5 h with TBZ. Ubiquitin was detected by immunostaining with its specific antibody. B , C , F , G Examples of the fluorescence images. D , E , H , I Quantification of the data (~110 cells). Arrows point to MLKL-GFP + Rab5 or Rab7 fused to mCherry in B and C , and to MLKL-GFP + Rab5 or Rab7 fused to mCherry + ubiquitin in F and G . Scale bar, 10 μm. J Comparison of the amounts of MLKL and of ubiquitinated MLKL in 60 μg of proteins of the total cellular lysate and of EVs released from HT-29 cells during treatment for 3 h with TBZ. K Effect of the K50R mutation in MLKL on MLKL release in EVs from HT-29 cells treated for 2.5 h with TBZ. Samples of 15 μg of cellular lysates and of EVs derived from 4 × 10 7 cells were applied to each lane.

Article Snippet: Recombinant human MLKL (CSB-EP850851HU) and mouse MLKL (CSB-EP861529MO) were from Cusabio Technology and recombinant ITCH (E3-260) was from R&D Systems.

Techniques: Western Blot, Ubiquitin Proteomics, Mutagenesis, Expressing, Immunostaining, Fluorescence, Comparison, Derivative Assay

Journal: Cell Death and Differentiation

Article Title: Site-specific ubiquitination of MLKL targets it to endosomes and targets Listeria and Yersinia to the lysosomes

doi: 10.1038/s41418-021-00924-7

Figure Lengend Snippet: A Comparison of the amounts of ITCH in 15 μg of proteins of the total cellular lysate and of EVs released from HT-29 cells during treatment for 3 h with TBZ. B ITCH binds to MLKL in HEK293T cells transiently overexpressing the two proteins. Samples were subjected to western blot analysis following precipitation of MLKL via a fused Strep-tag. C In response to TBZ, ITCH binds inducibly to MLKL in MLKL knocked-down HT-29 cells constitutively expressing wild-type MLKL, but not to its T357A/S358A or L162G/L165G mutants. MLKL (wild-type and mutants) were precipitated using an anti-MLKL antibody (ab184718 from Abcam). D , E Immunocytochemical evidence for the colocalization of ITCH and activated MLKL in association with endosomes. D Immunofluorescence images. Arrows point to MLKL-GFP + Rab5 or 7 fused to mCherry + ITCH. ITCH was detected by immunostaining with its specific antibody. Scale bar, 10 μm. E Quantification of the data (75 cells). F , G Deletion analysis for identification of the region in the ITCH molecule to which MLKL binds. F Western blot analysis of the binding of various ITCH deletion mutants to MLKL fused C-terminally to the double Strep-tag upon their transient expression in HEK293T cells. MLKL was eluted by biotin after its precipitation by Strep-Tactin beads. The arrowhead points to non-specific bands. G Diagram showing the deletion mutants used and the location of WW motifs in ITCH. H Recombinant ITCH catalyzes the in vitro ubiquitination of MLKL isolated from HT-29 cells, but not of its K50R mutant. I Enhancement of TBZ-induced ubiquitination of MLKL, but not of its K50R mutant, when inducibly expressed in MLKL KO HT-29 cells, by inducibly expressed ITCH. J Enhancement of the association of MLKL (expressed inducibly in MLKL KO HT-29 cells) with ESCRT proteins, in response to TBZ treatment, by transient expression of ITCH. (Arrows indicate MVB12B.) K TBZ-induced MLKL ubiquitination in HT-29 cells is compromised by CRISPR/Cas9-facilitated KO of ITCH, but not by KO of NEDD4. Two distinct clones of HT-29 ITCH KO were analyzed, with identical results.

Article Snippet: Recombinant human MLKL (CSB-EP850851HU) and mouse MLKL (CSB-EP861529MO) were from Cusabio Technology and recombinant ITCH (E3-260) was from R&D Systems.

Techniques: Comparison, Western Blot, Strep-tag, Expressing, Immunofluorescence, Immunostaining, Binding Assay, Recombinant, In Vitro, Ubiquitin Proteomics, Isolation, Mutagenesis, CRISPR, Clone Assay

Journal: Cell Death and Differentiation

Article Title: Site-specific ubiquitination of MLKL targets it to endosomes and targets Listeria and Yersinia to the lysosomes

doi: 10.1038/s41418-021-00924-7

Figure Lengend Snippet: A , B Kinetic analysis of the effects of TBZ treatment of HT-29 cells expressing the wild-type and the K50R mutant MLKL ( A ), and the effects of TNF treatment of L929 cells expressing wild-type and K50,51R mutant MLKL ( B ), on the amounts of viable Listeria in the cells and on the extent of cell death. As described in Supplementary Fig. , TNF was applied to HT-29 cells 3 h after initiation of infection, and to L929 cells 2 h after initiation of infection. Timings of bacterial yield quantification presented in the figure, therefore, correspond to 3, 5, and 7 h of infection period in A , and 2, 6, and 8 h in B . At the multiplicity of infection employed in this study (20 in HT-29 cells and 15 in L929 cells) Listeria had no effect on the extent of cell death. Expression of identical amounts of wild-type and mutant MLKL in the tested cells was confirmed by western blot analysis (Supplementary Fig. ). C Effect of ITCH knockdown on the amount of viable Listeria in infected HT-29 cells 5 h after infection, and on its modulation by TBZ treatment. D Effects of various MLKL mutants constitutively expressed in MLKL knocked-down HT-29 cells, and of treatment with TBZ, on the amounts of viable Listeria in the cells. E – G Comparison of the impacts of TBZ treatment, the K50 mutation in MLKL, and the KO of MLKL on the amounts of viable Listeria in HT-29 cells, 5 h after infection with E wild-type L. monocytogenes , F the LLO mutant strain of L. monocytogenes DP-L2161, and G the latter strain in which LLO expression was reconstituted by transformation with LLO-expressing cDNA. Unless otherwise stated, in this and in all other presented experiments the durations of cellular infection by the indicated bacteria and the timing and duration of treatment with TBZ or with TNF were as specified in Supplementary Fig. . Each of the experiments presented in panels A − G was carried out three times, with duplicates of the samples ( n = 6). H , I Immunocytochemical analysis of the intracellular location of Listeria compared to that of the endosomal marker Rab7, at 5 h after infection of HT-29 cells with the wild-type bacteria ( H ) or with the DP-L2161 strain ( I ). Arrows point to Listeria colocalized with Rab7 (as determined by staining of the two with their specific antibodies). In all experiments presented in this figure, MLKL and its mutants were re-expressed inducibly in MLKL KO cells.

Article Snippet: Recombinant human MLKL (CSB-EP850851HU) and mouse MLKL (CSB-EP861529MO) were from Cusabio Technology and recombinant ITCH (E3-260) was from R&D Systems.

Techniques: Expressing, Mutagenesis, Infection, Western Blot, Knockdown, Comparison, Transformation Assay, Bacteria, Marker, Staining

Journal: Cell Death and Differentiation

Article Title: Site-specific ubiquitination of MLKL targets it to endosomes and targets Listeria and Yersinia to the lysosomes

doi: 10.1038/s41418-021-00924-7

Figure Lengend Snippet: A − C Yields of the indicated bacteria 5 h after infection of HT-29 cells or their HT-29 K50R mutant, and 6 h after infection of L929 cells or their K50,51R mutant, and the modulation of these yields obtained by treatment of the HT-29 cells with TBZ and treatment of the L929 cells with TNF. D Yields of E. coli 8 h after infection of L929 cells or their K50,51R mutant, and the modulation of these yields obtained by treatment of the L929 cells with TNF. E Yield of S. typhimurium 5 h after infection of HT-29 cells, and modulation of the yield by TBZ treatment. In A and B , three independent tests were performed ( n = 6). C and D were done twice ( n = 4). F Yield of S. typhimurium 8 h after infection of L929 cells, and modulation of the yield by TNF treatment. E and F were performed twice ( n = 4). In all experiments presented in this figure, MLKL and its mutants were re-expressed inducibly in MLKL KO cells.

Article Snippet: Recombinant human MLKL (CSB-EP850851HU) and mouse MLKL (CSB-EP861529MO) were from Cusabio Technology and recombinant ITCH (E3-260) was from R&D Systems.

Techniques: Bacteria, Infection, Mutagenesis

Journal: Cell Death and Differentiation

Article Title: Site-specific ubiquitination of MLKL targets it to endosomes and targets Listeria and Yersinia to the lysosomes

doi: 10.1038/s41418-021-00924-7

Figure Lengend Snippet: A , B Effects of chloroquine (CQ, 25 μM), applied throughout the times of infection of HT-29 cells by Listeria on the bacterial yield and on its modulation by MLKL. Comparison of the amounts of viable Listeria at 5 h ( A ) and at 24 h ( B ) post infection (h.p.i), and of the effects of TBZ treatment for 2 h at the end of the infection period, in MLKL KO HT-29 cells and in KO cells that inducibly express either wild-type MLKL or its K50R mutant. C Effect of chloroquine (CQ, 25 μM) and of pepstatin A (PepA, 10 μg/ml), applied to L929 cells as in A and B , on the TNF-induced reduction in Listeria yields. TNF was applied to the cells for 6 or 8 h following their infection with Listeria . D Effects of chloroquine and of pepstatin A, applied to HT-29 cells as in A and B , on the TBZ-induced reduction in Y. enterocolitica yields. E Effects of chloroquine and of pepstatin A, applied for 4 h to L929 cells together with TNF, at 6 h after infection of the cells with Y. enterocolitica , on the TNF-induced reduction of the bacterial yield in these cells. In A − E , three independent tests were performed ( n = 6). F , H Immunocytological analysis of the location of Listeria , 5 h after infection, in early and late endosomes, in lysosomes and in the cytosol, and its TBZ-induced modulation, in MLKL KO HT-29 cells inducibly expressing wild-type and K50R-mutated MLKL fused C-terminally to GFP. F Typical immunofluorescence images. Arrows point to Listeria colocalized with MLKL-GFP + Rab7 fused to mCherry + Lamp1. H Ratios of the numbers of Listeria in the indicated compartments (analysis of 130 infected cells). G , I Immunocytological analysis of the location of Listeria in early and late endosomes, in lysosomes and in the cytosol, 6 h after infection, and their TNF-induced modulation, in MLKL KO L929 cells inducibly expressing wild-type and K50,51R-mutated MLKL. G Typical immunofluorescence images. Arrows point to Listeria colocalized with Rab7 + Lamp1. I Ratios of the numbers of Listeria in the indicated compartments (analysis of 120 infected cells).

Article Snippet: Recombinant human MLKL (CSB-EP850851HU) and mouse MLKL (CSB-EP861529MO) were from Cusabio Technology and recombinant ITCH (E3-260) was from R&D Systems.

Techniques: Infection, Comparison, Mutagenesis, Expressing, Immunofluorescence

Journal: Cureus

Article Title: Favipiravir Protects Enterocytes From Cell Death After Inflammatory Storm

doi: 10.7759/cureus.47417

Figure Lengend Snippet: Levels of IL8 (A) and TNF-α (B) in culture mediums from control, inflammation model, before (prophylactic) or after favipiravir applied (treatment) inflammation model groups after ELISA. The results are presented as mean ± SD. IL-8 and TNF-α levels were highest in the treatment group ( p =0.0266 and p =0.0266 respectively). ELISA: Enzyme-linked immunosorbent assay.

Article Snippet: The cells from all groups were then treated with blocking solution (TA-125-UB, ThermoFisher) for an hour at room temperature and were incubated with anti-caspase 1 (BT-AP01191, BT-LAB), anti-caspase 3 (BT-AP01199, BT-LAB), IL6 (SC-1265, SantaCruz), IL8 (BT-AP04514, BT-LAB), MLKL (BS-5513R, Bioss), RIPK1 (BS-5805R, Bioss) and TNF-α (BT-AP09103, BT-LAB) overnight at 4˚C.

Techniques: Control, Enzyme-linked Immunosorbent Assay

Journal: Cureus

Article Title: Favipiravir Protects Enterocytes From Cell Death After Inflammatory Storm

doi: 10.7759/cureus.47417

Figure Lengend Snippet: Immunoreactivity of IL6 (A-D), IL8 (E-H), and TNF-α (I-L) in control (A, E, I), inflammation model (B, F, J), before (prophylactic) (C, G, K) or after favipiravir applied (treatment) (D, H, L) inflammation model groups. Scale bars: 10 µm.

Article Snippet: The cells from all groups were then treated with blocking solution (TA-125-UB, ThermoFisher) for an hour at room temperature and were incubated with anti-caspase 1 (BT-AP01191, BT-LAB), anti-caspase 3 (BT-AP01199, BT-LAB), IL6 (SC-1265, SantaCruz), IL8 (BT-AP04514, BT-LAB), MLKL (BS-5513R, Bioss), RIPK1 (BS-5805R, Bioss) and TNF-α (BT-AP09103, BT-LAB) overnight at 4˚C.

Techniques: Control

Journal: Cureus

Article Title: Favipiravir Protects Enterocytes From Cell Death After Inflammatory Storm

doi: 10.7759/cureus.47417

Figure Lengend Snippet: IL6 (A), IL8 (B), and TNF-α (C) immunoreactivity measurements and statistical analysis of them in all experimental groups. The results are presented as mean ± SD and significance of difference indicated with asterisk (*) as p value smaller than 0.05 ( p <0.05). Two asterisks (**) indicate p value smaller than 0.01 ( p <0.01).

Article Snippet: The cells from all groups were then treated with blocking solution (TA-125-UB, ThermoFisher) for an hour at room temperature and were incubated with anti-caspase 1 (BT-AP01191, BT-LAB), anti-caspase 3 (BT-AP01199, BT-LAB), IL6 (SC-1265, SantaCruz), IL8 (BT-AP04514, BT-LAB), MLKL (BS-5513R, Bioss), RIPK1 (BS-5805R, Bioss) and TNF-α (BT-AP09103, BT-LAB) overnight at 4˚C.

Techniques:

Journal: Immunity

Article Title: The gut microbiome regulates psychological stress-induced inflammation

doi: 10.1016/j.immuni.2020.06.025

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-mouse Lineage Biotin , BD Bioscience , Cat# 559971.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Software