Journal: Journal of Cell Science

Article Title: Charcot–Marie–Tooth type 2A variants of mitofusin 2 sensitize cells to apoptotic cell death

doi: 10.1242/jcs.263691

Figure Lengend Snippet: CMT2A variants of MFN2 cause apoptotic cell death. (A) Western blot analysis (upper panel) and quantification (lower panel) of HeLa WT cells or 2KO cells stably expressing FLAG-tagged K357N variant of MFN2, untreated or treated with Act D (1 µM, 6 h), with or without ZVAD (20 µM, 6 h), as indicated, immunoblotted with anti-PARP1 and anti-MFN2 antibodies. Staining of total protein with Ponceau S (PoS) was used as loading control. Bars represent the mean±s.d. intensity of cleaved PARP1, normalized to the PoS staining and shown relative to untreated K357N cells ( n =5 biological repeats). Individual values of each experiment are plotted as open and filled circles, squares, triangles, diamonds and hexagons. One-way ANOVA with Tukey's post-hoc test was applied. P values from left to right: ** P =0.0091; * P =0.0205; **** P <0.0001; ** P =0.0060. (B) Annexin V 568 fluorescence, measured by the Incucyte live imaging system over 48 h and plotted relative to timepoint zero, of HeLa WT or 2KO cells stably expressing FLAG-tagged K357N variant of MFN2, either untreated or treated with ABT+S (BH3 mimetic ABT-737 and the MCL1 inhibitor S63845 , 0.1 µM, 48 h) for apoptosis induction, and in presence or absence of ZVAD (20 µM, 48 h) for inhibition of caspases, as indicated. Values represent the mean±s.d. intensity per object count of three technical replicates each from three biological replicates. (C) Annexin V 568 fluorescence, analysed as in B, of HeLa 2KO cells transiently transfected with WT MFN2, MFN2 R364Q, MFN2 R707W or MFN2 K357N, either untreated or treated with ABT+S (0.1 µM, 24 h). Values represent the mean intensity per object count of a single technical replicate each from three biological replicates.

Article Snippet: The following chemicals were used for cell culture treatments: actinomyin D (Sigma-Aldrich, Schnelldorf, Germany), ABT-737 (MedChemExpress, Sollentuna, Sweden), MCL1 inhibitor S63458 (MedChemExpress Sollentuna, Sweden), ZVAD-FMK (Hölzel, Köln, Germany), ISR inhibitor (ISRIB) (Merck, Hamburg, Germany), thapsigargin (NEB, Frankfurt am Main, Germany), staurosporine (Sigma-Aldrich, Schnelldorf, Germany).

Techniques: Western Blot, Stable Transfection, Expressing, Variant Assay, Staining, Control, Fluorescence, Imaging, Inhibition, Transfection

Journal: Cell Death & Disease

Article Title: Human RIPK3 C-lobe phosphorylation is essential for necroptotic signaling

doi: 10.1038/s41419-022-05009-y

Figure Lengend Snippet: a Evaluation of endogenous RIPK3 S227 autophosphorylation in parental and MLKL − / − HT29 cells. Cells were either left untreated (UT), treated with an apoptotic stimulant (TS; T, TNF; S, Smac mimetic Compound A), a necroptotic stimulant (TSI; I, pan-caspase inhibitor IDN-6556), or intrinsic apoptosis stimulant (A + M; A, ABT-737; M, Mcl-1 inhibitor S63485) for 7.5 h, before lysis and analysis by immunoblotting with antibodies indicated. Data are representative of three independent experiments ( n = 3). b Recombinant RIPK3 kinase domain autophosphorylation is upregulated when co-expressed with the MLKL pseudokinase domain in insect cells. Equimolar amounts of purified recombinant RIPK3:MLKL complex, RIPK3 kinase domain expressed alone, and MLKL pseudokinase domain alone were tryptic digested and analyzed by mass spectrometry. Top , bar plot of log 2 -based peptide intensity values for tryptic peptide (EVELPTEPSLVYEAVCNR) with phosphorylation of T224 and S227. Bottom , bar plot of total RIPK3 (left) or MLKL (right) peptide intensity in each recombinant protein sample.

Article Snippet: HT29 cells, and their RIPK1 − / − , RIPK3 − / − , or MLKL − / − counterparts were treated with extrinsic apoptotic stimulant (TS; TNF and Smac-mimetic Compound A), necroptotic stimulant (TSI; TNF, Smac-mimetic Compound A, and IDN-6556), or intrinsic apoptotic stimulant (A + M; A, 1 μM ABT-737, Abbott Laboratories; M, 0.1 μM Mcl-1 inhibitor S63485, custom synthesized by SYNthesis MedChem) for 7.5 h, before being lysed in ice-cold RIPA buffer (10 mM Tris–HCl pH 8.0, 1 mM EGTA, 2 mM MgCl 2 , 0.5% v/v Triton X-100, 0.1% w/v Na deoxycholate, 0.5% w/v SDS and 90 mM NaCl) supplemented with 1× Protease & Phosphatase Inhibitor Cocktail (Cell Signaling Technology #5872S) and 100 U/mL Benzonase (Sigma #E1014).

Techniques: Lysis, Western Blot, Recombinant, Purification, Mass Spectrometry, Phospho-proteomics

Journal: Cell Death & Disease

Article Title: Human RIPK3 C-lobe phosphorylation is essential for necroptotic signaling

doi: 10.1038/s41419-022-05009-y

Figure Lengend Snippet: a A schematic diagram of the current model of necroptosis signaling. RIPK3 kinase activity is negatively regulated by pS164/pT165, which promotes apoptotic pathways . Under basal conditions, in the absence of inhibitory pS164/pT165, RIPK3 autophosphorylates T224/S227 in a MLKL-dependent manner, allowing MLKL to be recruited to the kinase domain of RIPK3. Upon TNF signaling in the absence of cIAPs and Caspase-8 activity, MLKL is phosphorylated in the necrosome, before disengaging from the RIPK3 kinase domain, undergoing a structural transition [ , ], and assembling into active oligomers, which are then trafficked to the plasma membrane, where it accumulates as hotspots to enact cell death . The skull and crossbones motif (Mycomorphbox_Deadly.png; Sven Manguard) is used under a Creative Commons Attribution-Share Alike 4.0 license. b , c The human RIPK3 crystal structures (wheat; αC helix, pink; activation loop, cyan) rationalize the mechanism by which phosphorylation events regulate RIPK3 functions . Peptide backbones are shown as ribbons. Residues are shown as sticks and colored by chemical element. b In the human RIPK3 kinase domain:MLKL pseudokinase domain complex (gray; αC helix, yellow; activation loop, green; PDB 7MON) , RIPK3 kinase domain adopts an open, inactive conformation. pT224 and pS227 mediate multiple intermolecular salt bridges and electrostatic interactions (dashes, distance labeled in Å). pT224, pS227, and MLKL-interacting residues are shown as thick sticks. c In the closed, active conformer of human RIPK3 kinase domain, S164/T165 reside in a structured region of the activation loop. The side chain of S165 is buried by hydrophobic residues, including the regulatory (R-spine (dots), A63 of αC helix, F166 and L138. S164/T165 and the residues surrounding S164 are shown as thick sticks. d Sequence alignment of RIPK3 from model mammalian species (human, Homo sapiens , Uniprot Q9Y572; mouse, Mus musculus , Uniprot Q9QZL0; rat, Rattus norvegicus , Uniprot Q9Z2P5; horse, Equus caballus , Uniprot F6SCQ8; bovine, Bos taurus , Uniprot E1BKA8; cat, Felis catus , Uniprot M3W4D3; pig, Sus scrofa , Uniprot F1SGQ6). Only regions with highly conserved phosphorylation sites are shown. Phosphorylation sites identified in this study are shown with black box with residue number (according to human RIPK3 sequence) labeled on top. Phosphorylation sites with proposed roles in necroptotic signaling are colored red with a yellow background. Relevant secondary structures are highlighted on the human RIPK3 sequence according to the crystal structure of human RIPK3 kinase domain (shown in b ; PDB 7MX3) . Alignment was performed using Clustal Omega algorithm in Snapgene.

Article Snippet: HT29 cells, and their RIPK1 − / − , RIPK3 − / − , or MLKL − / − counterparts were treated with extrinsic apoptotic stimulant (TS; TNF and Smac-mimetic Compound A), necroptotic stimulant (TSI; TNF, Smac-mimetic Compound A, and IDN-6556), or intrinsic apoptotic stimulant (A + M; A, 1 μM ABT-737, Abbott Laboratories; M, 0.1 μM Mcl-1 inhibitor S63485, custom synthesized by SYNthesis MedChem) for 7.5 h, before being lysed in ice-cold RIPA buffer (10 mM Tris–HCl pH 8.0, 1 mM EGTA, 2 mM MgCl 2 , 0.5% v/v Triton X-100, 0.1% w/v Na deoxycholate, 0.5% w/v SDS and 90 mM NaCl) supplemented with 1× Protease & Phosphatase Inhibitor Cocktail (Cell Signaling Technology #5872S) and 100 U/mL Benzonase (Sigma #E1014).

Techniques: Activity Assay, Clinical Proteomics, Membrane, Activation Assay, Phospho-proteomics, Labeling, Sequencing, Residue

Journal: Clinical and Translational Medicine

Article Title: CD98 defines a metabolically flexible, proinflammatory subset of low‐density neutrophils in systemic lupus erythematosus

doi: 10.1002/ctm2.1150

Figure Lengend Snippet: CD98 + low‐density neutrophils (LDN) are relatively resistant to apoptosis, but equally susceptible to necroptosis. Cell death was evaluated using the IncuCyte S3 system. (A) Spontaneous apoptosis of normal‐density neutrophils (NDN) (white) and LDN (black) was measured over 18 h ( n = 5). (B) Quantification of apoptosis after 12 h induced by Smac‐mimetic compound A ( n = 5). (C) Spontaneous apoptosis ( n = 5), extrinsic apoptosis (stimulated with tumor necrosis factor [TNF] and a Smac‐mimetic, n = 6) or intrinsic apoptosis (stimulated with ABT‐737 and an Mcl‐1 inhibitor, n = 3) quantified after 12 h. (D) Both neutrophil subsets were treated with a necroptotic stimulus (TNF, Smac‐mimetic and IDN‐6556 [TSI]) and cell death was measured over 18 h using the IncuCyte S3 System ( n = 8). (E) The level of necroptotic cell death at 12 h was quantified ( n = 8). Data are mean ± standard error of mean (SEM). * p < .05, ** p < .01, *** p < .001, one‐way analysis of variance (ANOVA)

Article Snippet: Cells were treated with combinations of the following agonists/antagonists: 100 ng/ml recombinant human TNF (produced in‐house ), 500 nM Smac‐mimetic/compound A (Tetralogic Pharmaceuticals ), 5 μM IDN‐6556 (Idun Pharmaceuticals), 100 μg/ml cycloheximide (Sigma), 1 μM ABT‐737 (Abbott) and 0.1 μM Mcl‐1 inhibitor (also known as S63485; SYNthesis MedChem).

Techniques: