EMBO/soda-nel huggingface dataset:
Sensor NLR immune proteins activate oligomerization of their NRC helpers in response to plant pathogens (2022)
Figure 1

Figure 1:

Figure 1: Activation of Rx with Potato virus X coat protein leads to NRC2 oligomerization.

(A) Structure of the AtZAR1 resistosome, highlighting the N-terminal α1 helix. Much like AtZAR1 and NRC4, the N-terminal α1 helix encodes a MADA motif. Alignment of NRC2, NRC4 and AtZAR1 N-terminal MADA motifs along with the consensus sequence pattern for the motif and the HMM score for MADA prediction of each sequence. Residues mutated in NRC2EEE mutant are highlighted with red asterisks (positions 9, 13 and 17 respectively). (B) Schematic representation of the experimental pipeline used. Agrobacterium tumefaciens strains were used to transiently express proteins of interest in leaves of nrc/2/3/4 Nicotiana benthamiana CRISPR mutant lines by agroinfiltration. Leaf tissue was harvested 3 days post-infiltration and total protein extracts were used for BN and SDS-PAGE assays. (C) BN-PAGE and SDS-PAGE assays with inactive and activated Rx-NRC2. C-terminally 6xHA tagged Rx and C-terminally 4xMyc-tagged NRC2EEE were co-expressed with either free GFP or C-terminally GFP-tagged CP. Free mCherry-4xMyc and mCherry-6xHA fusions were used as controls in the treatments without NRC2 and Rx, respectively. Total protein was extracted with a Tris-HCl-based buffer, as described in materials and methods. Extracts were run on native and denaturing PAGE assays in parallel and immunoblotted with the appropriate antisera labelled on the left. Approximate molecular weights (kDa) of the proteins are shown on the right. Red asterisks indicate bands corresponding to the activated NRC2 complex. Rubisco loading control was carried out using Ponceau stain (PS). The experiment was repeated three times.
Sensor NLR immune proteins activate oligomerization of their NRC helpers in response to plant pathogens (2022)
Figure 2

Figure 2:

Figure 2: Rx and NRC2 do not form a stable complex upon activation.

(A) Schematic representation of NRC2EEE and Rx with differently sized tags. The C-terminally 4xMyc-tagged NRC2EEE and C-terminally 6xHA tagged Rx used in previous experiments were termed "light" versions, while the new C-terminally tandem mCherry-4xMyc-tagged NRC2EEE and C-terminally mCherry-6xHA-tagged Rx variants were termed "heavy" versions. (B) BN-PAGE and SDS-PAGE assays with inactive and activated Rx-NRC2 using different heavy and light sensor-helper combinations. Total protein was extracted with a Tris-HCl-based buffer, as described in materials and methods. Extracts were run on native and denaturing PAGE assays in parallel and immunoblotted with the appropriate antisera labelled on the left. Approximate molecular weights (kDa) of the proteins are shown on the right. Red asterisk indicates bands corresponding to the activated NRC2 complex. Rubisco loading control was carried out using Ponceau stain (PS). The experiment was repeated three times.
Sensor NLR immune proteins activate oligomerization of their NRC helpers in response to plant pathogens (2022)
Figure 3

Figure 3:

Figure 3: The activated NRC2EEE complex is composed of multiple NRC2 units.

(A) Schematic representation of NRC2EEE with differently sized tags. The C-terminally 3xFLAG-tagged NRC2EEE was termed the "light" version and C-terminally mCherry-4xMyc tagged NRC2EEE was termed the "heavy" variant. If NRC2 forms homo-oligomers, active complexes with different proportions of "heavy" and "light" NRC2 monomers should in theory exhibit different molecular weights. (B) BN-PAGE and SDS-PAGE assays with inactive and activated Rx-NRC2 using different "heavy" and "light" helper combinations. Total protein was extracted with a Tris-HCl-based buffer, as described in materials and methods. Extracts were run on native and denaturing PAGE assays in parallel and immunoblotted with the appropriate antisera labelled on the left. Approximate molecular weights (kDa) of the proteins are shown on the right. A non-specific band was observed at ~110 kDa for the anti-FLAG antibody. Red asterisks indicate bands corresponding to the activated "heavy" NRC2 complexes. Blue asterisks indicate bands corresponding to "light" NRC2 complexes. Purple bands indicate intermediate molecular weight complexes combining "heavy" and "light" NRC2. Rubisco loading control was carried out using Ponceau stain (PS). The experiment was repeated three times.
Sensor NLR immune proteins activate oligomerization of their NRC helpers in response to plant pathogens (2022)
Figure 4

Figure 4:

Figure 4: Bs2, another NRC2-dependent sensor NLR, triggers oligomerization of NRC2EEE.

(A) Schematic representation of the NRC genetic dependencies of sensors used in this experiment. (B) BN-PAGE and SDS-PAGE assays with inactive and activated NRC-dependent sensors and NRC2. Total protein was extracted with a Tris-HCl-based buffer, as described in materials and methods. Extracts were run on native and denaturing PAGE assays in parallel and immunoblotted with the appropriate antisera labelled on the left. Approximate molecular weights (kDa) of the proteins are shown on the right. Red asterisk indicates bands corresponding to the activated NRC2 complexes. Rubisco loading control was carried out using Ponceau stain (PS). The experiment was repeated three times.
Sensor NLR immune proteins activate oligomerization of their NRC helpers in response to plant pathogens (2022)
Figure 5

Figure 5:

Figure 5: Bs2, Rx and Rpi-blb2 trigger oligomerization of another helper NLR, NRC4.

BN-PAGE and SDS-PAGE assays with inactive and activated NRC-dependent sensors and NRC4AAA. Total protein was extracted with a HEPES-based buffer, as described in materials and methods. Extracts were run on native and denaturing PAGE assays in parallel and immunoblotted with the appropriate antisera labelled on the left. Approximate molecular weights (kDa) of the proteins are shown on the right. Red asterisk indicates bands corresponding to the activated NRC4 complexes. Rubisco loading control was carried out using Ponceau stain (PS). The experiment was repeated three times.
Sensor NLR immune proteins activate oligomerization of their NRC helpers in response to plant pathogens (2022)
Figure 6

Figure 6:

Figure 6: Potato virus X coat protein activated NRC2 forms plasma membrane-associated puncta, unlike Rx.

C-terminally GFP-tagged NRC2EEE and C-terminally RFP-tagged Rx were co-expressed either with an EV-4xMyc construct or a CP-4xMyc construct in leaves of nrc2/3/4 N. benthamiana CRISPR mutant lines. (A-B) Single-plane confocal micrographs show the localization of both components of the inactive and active Rx-NRC2 system, together with PM marker RPW8.2-BFP. Scale bars represent 10 µm. (A) NRC2EEE-GFP and Rx-RFP co-localize in the cytoplasm prior to activation. (B) Upon co-expression of CP and activation of the system, NRC2EEE forms puncta associated with the PM while Rx remains in the cytoplasm. (C) Membrane enrichment assays are consistent with microscopy, showing that inactive NRC2EEE-GFP is mostly present in the soluble (cytoplasmic) fraction, whereas activated NRC2EEE-GFP exhibits equal distribution across soluble and membrane fractions. Rx is mostly present in the soluble fraction and exhibits no change upon activation of the system with CP. T = total, S = soluble, M = membrane. ATPase was used as a membrane marker. Rubisco was used as a marker for total and soluble fractions and visualized by Ponceau staining (PS). Red asterisks indicate bands matching the expected MW for each protein. The experiment was repeated two times.
Sensor NLR immune proteins activate oligomerization of their NRC helpers in response to plant pathogens (2022)
Figure 7

Figure 7:

Figure 7: Potato virus X infection triggers Rx-mediated oligomerization of NRC2.

(A) Schematic representation of the experimental pipeline used. A. tumefaciens strains were used to transiently express proteins of interest in leaves of nrc/2/3/4 N. benthamiana CRISPR mutant lines by agroinfiltration. Simultaneously, the same leaves were infected with PVX by using an A. tumefaciens carrying GFP expressing PVX. Free GFP was used as a negative control for PVX-GFP infection. Free mCherry-4xMyc and mCherry-6xHA fusions were used as controls in the treatments without NRC2 and Rx, respectively. Leaf tissue was harvested 3 days post-infiltration and total protein extracts were used for BN and SDS-PAGE assays. (B) BN-PAGE and SDS-PAGE assays with infected and uninfected leaves expressing Rx and NRC2. Total protein was extracted with a Tris-HCl-based buffer, as described in materials and methods. Extracts were run on native and denaturing PAGE assays in parallel and immunoblotted with the appropriate antisera labelled on the left. Approximate molecular weights (kDa) of the proteins are shown on the right. Red asterisks indicate bands corresponding to the activated NRC2 complexes. Rubisco loading control was carried out using Ponceau stain (PS). The experiment was repeated three times.
Sensor NLR immune proteins activate oligomerization of their NRC helpers in response to plant pathogens (2022)
Figure 8

Figure 8:

Figure 8: Potato virus X infection triggers Rx-dependent NRC2 membrane-associated puncta.

(A-B) Single-plane confocal micrographs show the localization of NRC2 together with Rx or free RFP during PVX infection. C-terminally GFP-tagged NRC2EEE was co-infiltrated with either EV-RFP or Rx-RFP in leaves of nrc2/3/4 N. benthamiana CRISPR mutant lines and infected with PVX by agroinfection. Scale bars represent 10 µm. (A) In the absence of Rx, NRC2EEE-GFP is localized to the cytoplasm during PVX infection. (B) When Rx is present, NRC2EEE forms puncta associated with the PM during PVX infection.
Spatial and temporal control of mitochondrial H2O2 release in intact human cells (2022)
Figure 5

Figure 5. Increased activity of the cytosolic thioredoxin system limits mitochondrial H2O2 release during increased use of the respiratory chain

A. Protein levels in cells grown on glucose and galactose. Cell lysates were analyzed by quantitative mass spectrometry (n = 4 biological replicates, Dataset EV1). Levels of antioxidative enzymes (especially peroxiredoxins, PRDX in blue) are not changed except for cytosolic thioredoxin reductase (TXNRD1).

B. Protein levels in cells grown on glucose and galactose. Cell lysates were analyzed by immunoblot (n = 2 biological replicates). Cytosolic thioredoxin reductase (TXNRD1) is increased by more than two-fold in cells grown on galactose compared to glucose-grown cells.The dotted line depicts 1, meaning no change in protein levels.

C. Mechanisms of H2O2 degradation by peroxiredoxins. Auranofin inhibits thioredoxin reductase and thus the reductive half reaction in the detoxification of H2O2. Arrows indicate the "flux" of electrons.

D,E. Response of HyPer7 (D) and roGFP2-scPrx1 (E) probes targeted to indicated compartments to treatment of mtDAO-expressing HEK293 cells with 2 mM D-Ala (cells generated with the Flp-In T-REX-system). HEK293 cells were incubated either with 1 µM auranofin (red curve and data points) or DMSO (black curve and data points). Solid line represents average, points colored in the lighter version of the respective color are the corresponding ratios measured in individual cells. The numbers of cells per experiment for each dataset can be found in Appendix Table S6. As most of the data were not normal distributed, instead of a t-test, a Wilcoxon/Mann-Whitney-U-test was performed and samples were compared in pairs.***, p ≤ 0.001
Spatial and temporal control of mitochondrial H2O2 release in intact human cells (2022)
Figure 7

Figure 7. Cytosolic peroxiredoxins cooperate in controlling mitochondrial H2O2 release

A,B. Peroxiredoxin levels in peroxiredoxin 1 (PRDX1 KO) and peroxiredoxin 2 (PRDX2 KO) knockout cells. The upper subpanel depictes the guides used to generated the KO cell lines. Lysates of the indicated cell lines grown in glucose-containing medium were analysed by immunoblot (A) and quantitative proteomics (n = 4, technical replicates) (B). The right subpanel of B lists the identified proteins belonging to cellular antioxidative systems which are not altered for PRDX1 KO or PRDX2 KO, respectively. In both quantitative proteomics experiments 4403 proteins were detected in total.

C. HyPer7 steady state ratios of the indicated cell lines. HyPer7 probes were targeted to the indicated compartments of the respective cell lines. Cells were grown in glucose-containing medium. The numbers of cells per experiment for each dataset can be found in Appendix Table S6. In the boxplot, the central band is the median. The lower and upper hinges correspond to the first and third quartiles. The upper whisker extends from the hinge to the largest value no further than 1.5 times the inter-quartile range, whereas the lower whisker extends from the hinge to the smallest value no further than 1.5 times the inter-quartile range.

D. Response of HyPer7 probes targeted to indicated compartments to treatment of mtDAO-expressing cell lines with 4 mM D-Ala (black, wild type; red, PRDX1 KO; blue, PRDX2 KO; cell generated with the piggyBAC system). Cells were grown in glucose-containing medium. Solid line represents average, points colored in the lighter version of the respective color are the corresponding ratios measured in individual cells.

E. Response of HyPer7 probes targeted to indicated compartments to treatment with 25 µM of exogenous H2O2. Indicated cell lines (black, wild type; red, PRDX1 KO; blue, PRDX2 KO) were grown in glucose-containing medium. Solid line represents average, points colored in the lighter version of the respective color are the corresponding ratios measured in individual cells.

Data information: The numbers of cells per experiment for each dataset can be found in Appendix Table S6. As most of the data were not normal distributed, instead of a t-test, a Wilcoxon/Mann-Whitney-U-test was performed and samples were compared in pairs. *, p ≤ 0.05, **, p ≤ 0.01, ***, p ≤ 0.001
Spatial and temporal control of mitochondrial H2O2 release in intact human cells (2022)
Figure 8

Figure 8. Peroxiredoxin 1 and 2 double knockout cells are strongly impaired in handling of mitochondrial H2O2 release

A,B. Peroxiredoxin levels in peroxiredoxin 1 and 2 double knockout cells (PRDX1/2 DKO). Lysates of the indicated cell lines grown in glucose-containing medium were analysed by immunoblot (A) and quantitative proteomics (B).The right subpanel in (B) lists all detected but not altered proteins belonging to cellular antioxidative systems.

C. HyPer7 steady state ratios of the indicated cell lines. HyPer7 probes were targeted to the indicated compartments of the respective cell lines. Cells were grown in glucose-containing medium. The numbers of cells per experiment for each dataset can be found in Appendix Table S6. In the boxplot, the central band is the median. The lower and upper hinges correspond to the first and third quartiles. The upper whisker extends from the hinge to the largest value no further than 1.5 times the inter-quartile range, whereas the lower whisker extends from the hinge to the smallest value no further than 1.5 times the inter-quartile range

D. Response of HyPer7 probes targeted to indicated compartments to treatment with 25 µM of exogenous H2O2. Indicated cell lines (black, wild type; red, PRDX1/2 DKO) were grown in glucose-containing medium. Solid line represents average, points colored in the lighter version of the respective color are the corresponding ratios measured in individual cells.

E. Response of HyPer7 probes targeted to indicated compartments to treatment of mtDAO-expressing cell lines with 4 mM D-Ala (black, wild type; red, PRDX1/2 DKO; cell generated with the piggyBAC system). Cells were grown in glucose-containing medium. Solid line represents average, points colored in the lighter version of the respective color are the corresponding ratios measured in individual cells.

F. Model. See discussion for details

Data information: The numbers of cells per experiment for each dataset can be found in Appendix Table S6. Statistical analysis was only performed to compare the PRDX1/2 DKO with the wildtype. As most of the data were not normal distributed, instead of a t-test, a Wilcoxon/Mann-Whitney-U-test was performed and samples were compared in pairs. *, p ≤ 0.05, **, p ≤ 0.01, ***, p ≤ 0.001
Phospholipid oxidation generates potent anti-inflammatory lipid mediators that mimic structurally related pro-resolving eicosanoids by activating Nrf2. (2015)
Figure 1

Oxidized phospholipids are potent inhibitors of the pro-inflammatory response of myeloid cells

A, B BMDCs were treated with OxPAPC or DPPC (40 μg/ml) for 60 min followed by R837 stimulation (5 μg/ml). (A) Supernatants were harvested after 18 h, and concentrations of IL-6 and IL-12 were quantified by ELISA. One-way ANOVA adjusted by Dunnett's multiple comparisons test. Mean ± SD of triplicate determinations from > 3 independent experiments are shown. (B) mRNA was harvested after 2 h, and expression of IL-6 and IL-12 was measured by real-time PCR and normalized to G6pdx. Unpaired two-tailed t-test. Data (mean ± SD) are representative of three independent experiments.

C Bioactive OxPAPC mixtures were obtained by various oxidation protocols from highly pure PAPC. Periods of oxidation were 24 h for CuSO4 (10 μM), 48 h for FeSO4 (10 μM), and 72 h for air. BMDCs were treated for 60 min with lipids prior stimulation with R837 (5 μg/ml) for 18 h. Cytokine concentrations in supernatants were quantified by ELISA. One-way ANOVA adjusted by Dunnett's multiple comparisons test. Data represent mean ± SEM of triplicate determinations.

D PAPC (40 μg/ml) oxidized with CuSO4 (10 μM) for the indicated times was used for treatment of BMDCs prior to R837 (5 μg/ml) stimulation and measurement of IL-12 secretion. Mean ± SEM of triplicate determinations from three different oxidation series are shown.

E, F Treatment of BMDCs with the indicated concentrations of CuSO4-oxidized PAPC suppressed IL-6 (E) and IL-12 (F) secretion triggered by a variety of different TLR agonists. After OxPAPC treatment for 60 min, BMDCs were stimulated for 18 h with LTA (500 ng/ml), Poly I:C (50 μg/ml), LPS (10 ng/ml), R837 (5 μg/ml), and CpG (100 nM). Cytokine concentrations in the supernatant were quantified by ELISA. Data are shown as mean ± SD of triplicate determinations from three independent experiments and were analyzed by one-way ANOVA adjusted by Dunnett's multiple comparisons test.

G–I Splenic dendritic cells were treated with OxPAPC or DPPC (40 μg/ml) before co-culturing with naïve transgenic SMARTA CD4 T cells in the presence of the specific peptide gp61. (G) After 4 days of cell culture, T-cell polarization was assessed by intracellular staining for the cytokines IL-4 (Th2) and IFN-γ (Th1). (H) Bar graphs represent the frequencies of IL-4- and IFN-γ-producing T cells after 4 days of co-culture with OxPAPC-treated and DPPC-treated splenic dendritic cells. Bars represent mean ± SD of duplicate experiments. *P ≤ 0.05 by unpaired two-tailed t-test. (I) IFN-γ production in supernatants of SMARTA CD4 T cells (stimulated with 1,000 nM gp61) and co-cultured with OxPAPC- and DPPC-treated splenic dendritic cells for 4 days. One-way ANOVA adjusted by Dunnett's multiple comparisons test. Bars represent mean ± SD.

Data information: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant.
Phospholipid oxidation generates potent anti-inflammatory lipid mediators that mimic structurally related pro-resolving eicosanoids by activating Nrf2. (2015)
Figure 2

In vitro generated OxPAPC preparations represent complex mixtures of OxPL species with distinct bioactivities

Mass spectrometric quantification of a variety of OxPL species obtained by CuSO4-catalyzed oxidation of PAPC for 2 and 24 h. Mean ± SEM of duplicate determinations are shown.

Correlation between the abundance of individual OxPL species detected in mixtures of differentially oxidized OxPL preparations and the capacity of the respective overall OxPL mixtures to suppress the IL-12 secretion of thioglycollate-elicited macrophages. Dots represent data of individual OxPAPC preparations.
Phospholipid oxidation generates potent anti-inflammatory lipid mediators that mimic structurally related pro-resolving eicosanoids by activating Nrf2. (2015)
Figure 3

An OxPL containing a fatty acid epoxycyclopentenone mediates the anti-inflammatory bioactivity of OxPAPC

Selected candidate lipids were tested for their inhibitory activity on R837-induced (5 μg/ml; 18 h) cytokine secretion in BMDCs. Concentrations of indicated lipids: PECPC (10 μM), PEIPC (10 μM), OxPAPC (40 μg/ml), DPPC (40 μg/ml), POVPC (40 μM), PGPC (40 μM), and KOdiAPC (40 μM). Representative data (mean ± SD of triplicate determinations) from one of three independent experiments are shown. ****P < 0.0001; ns, not significant; as determined by one-way ANOVA adjusted by Sidak's multiple comparisons test.

Structures of the cyclopentenone-containing arachidonic acid-derived lipids EC and 15d-PGJ2.

Dose–response curves of IL-12 secretion in BMDCs pulsed for 60 min with the indicated lipids, followed by stimulation with R837 (5 μg/ml) for 18 h. Toxic concentrations of lipids were excluded from analysis. Mean ± SEM of triplicate determinations are shown.

EC50 values of anti-inflammatory lipid products normalized to the capacity of 15d-PGJ2 to suppress IL-12 production in BMDCs (left panel) and their respective bioactivities (right panel) depicted as the fold increase relative to 15d-PGJ2. Mean ± SEM of triplicate determinations are shown.

IL-12 production of BMDC stimulated via TLR 4 (LPS; 100 ng/ml), TLR 9 (CpG; 100 nM), and TLR 7 (R837; 5 μg/ml) after pretreatment with the indicated free and esterified versions of EC and 15d-PGJ2. Lipids were used at 1 μM (EC), 10 μM (PECPC), or 20 μM (15d-PGJ2 and 15d-PGJ2PC). Data (mean ± SEM) are representative of 3 independent experiments. **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; determined by one-way ANOVA adjusted by Dunnett's multiple comparisons test.

Analysis of the capacity of various OxPL-derived species to license splenic dendritic cells to polarize naïve CD4 T cells toward the Th2 subset. Concentrations of indicated lipids: EC (1 μM), PECPC (10 μM), 15d-PGJ2 (20 μM), OxPAPC (40 μg/ml), DPPC (40 μg/ml), POVPC (40 μM), PGPC (40 μM), KOdiAPC (40 μM).