EMBO/soda-nel huggingface dataset:
Deciphering preferential interactions within supramolecular protein complexes: the proteasome case. (2015)
Figure 1

Protein correlation profiling (PCP) analysis of glycerol density gradient-separated proteasome complexes

PCP-MS strategy to identify proteins interacting with specific proteasome subtypes. U937 cells were cross-linked with formaldehyde and lysed, and proteins were concentrated and ultrafiltrated on a 100 kDa cutoff device. Protein complexes were then separated on a 15–40% glycerol gradient. Each fraction of the gradient was analyzed by nano-LC-MS/MS. Protein quantification was performed using the mean XIC of the three most intense validated peptides for each protein, after internal standard calibration using a mix of 8 isotopically labeled peptides. The PCP analysis was performed as described in the Materials and Methods section.

PCP analysis of the 19S regulatory complex. Protein abundance profiles of 16 proteins of the 19S RP (Rpt1–6, Rpn1–3, Rpn5, 7–9, 11–13, gray lanes) and of their median abundance (black lane) (left panel). PCP analysis is performed by plotting the χ2 values (representing the Euclidian distance between the abundance profile of each protein and the reference profile) of the experimental replicate 2 as a function of the χ2 values of the experimental replicate 1 (middle left panel). The median profile of the 19S complex subunits was used as the reference profile for the calculation of the χ2 values. Different zooms of the graph are represented (middle right and right panels). Light gray dots represent the proteins quantified in all the fractions of the density gradient and blue dots represent 19S subunits (right panel).

PCP analysis of proteasome 20S complex. Protein abundance profiles of 17 proteins of the 20S CP (α1–α7, β1–β7, β1i, β2i, β5i, gray lanes) and of their median abundance (black lane) (left panel). PCP analysis is performed by plotting the χ2 values of the experimental replicate 2 as a function of the χ2 values of the experimental replicate 1 (middle left panel). The median profile of the 20S complex subunits was used as the reference profile for the calculation of the χ2 values. Different zooms of the graph are represented (middle right and right panels). Light gray dots represent the proteins quantified in all the fractions of the density gradient and red dots represent 20S subunits.
Deciphering preferential interactions within supramolecular protein complexes: the proteasome case. (2015)
Figure 2

Protein correlation profiling (PCP) analysis using the median profile of the PA28αβ regulator as the reference profile

Profiles of the PA28α, PA28β, and the β2i proteins (blue, red and green lines, respectively).

Plot of the χ2 values of the experimental replicate 2 as a function of the χ2 values of the experimental replicate 1.

A zoom of the graph in (B) is represented and χ2 coordinates for PA28α, PA28β, and β2i proteins are highlighted as blue, red, and green dots, respectively. Light gray dots represent the χ2 coordinates of the proteins quantified in all the fractions of the gradient. The median profile of the PA28α and PA28β subunits was used as the reference profile for the calculation of the χ2 values.
Deciphering preferential interactions within supramolecular protein complexes: the proteasome case. (2015)
Figure 3

Protein abundance correlation of affinity-purified complexes analyzed by mass spectrometry strategy applied to proteasome complexes

A Proteasome complexes were immunopurified from nine formaldehyde-crosslinked human cell lines and analyzed by nano-LC-MS/MS. Protein abundance indexes (PAIs) were used to represent the abundance of proteins in purified proteasome samples. The correlation between two different proteins was quantified using coefficients of determination (R2).

B–E Correlations of abundances of α7 and α6 (B), Rpn3 and Rpn1 (C), PA28β and PA28α (D), and Rpn3 and PA28β (E).
Deciphering preferential interactions within supramolecular protein complexes: the proteasome case. (2015)
Figure 5

Changes in the expression of 20S proteasome catalytic subunits modulate 20S-associated regulators

The two HEK EBNA cell lines express only standard proteasome or immunoproteasome subunits. Western blots against the immuno- (β1i, β2i, β5i) and standard (β1, β2, β5) catalytic subunits of the 20S proteasome. Calnexin is used as a loading control. Black lines delineate the boundary between vertically sliced images that juxtapose lanes that were non-adjacent in the gel. Importantly, the bands were assembled from the same blot.

Relative normalized abundance indexes of proteasome regulators in HEK EBNA cells containing only immunoproteasome compared to HEK EBNA cells containing only standard proteasome. The normalized abundance indexes for each regulator were set to 1 for standard proteasome conditions (n = 4).

Kinetics of IFN-γ treatment on HeLa cells. HeLa cells were stimulated for 0, 24, 48, or 72 h with IFN-γ. Western blots were performed on total cell lysates with antibodies against the β2i, α2, and α5 subunits. IRF-1 was used to control IFN-γ treatment efficiency, and GAPDH was used as a loading control.

For each time point of the IFN-γ treatment, proteasome complexes were purified and analyzed by LC-MS/MS. Proteasome complexes dynamics was measured by label-free quantitative proteomics. The normalized abundance index of each protein or protein complex obtained at each time point was compared to the one obtained at the 0 h time point to obtain a regulator relative normalized PAI (n = 3).

Data information: *P < 0.05; **P < 0.01; ***P < 0.001 (Student t-test).

Source data are available online for this figure
Mitophagy Regulates Integrity of Mitochondria at Synapses and is Critical for Synaptic Maintenance (2020)
Figure 1

Figure 1. Rheb initiates mitophagy within axons upon mitochondrial damage.

A-C Representative dual-channel kymographs (A) and quantitative analysis (C) showing that Rheb is recruited to mitochondria in axons and co-migrates with mitochondria in a predominant retrograde direction upon mitochondrial membrane potential (∆ψm) dissipation. The motility of mitochondria (Mito) and Rheb-associated mitochondria (Rheb-Mito) in axons were quantified, respectively. Vertical lines represent stationary organelles. Slanted lines or curves to the right (negative slope) represent anterograde movement; those to the left (positive slope) indicate retrograde movement. An organelle was considered stationary if it remained immotile (displacement ≤ 5 μm). Time-lapse images (B) showing colocalization and comigration (white arrows) of Rheb with mitochondria in a retrograde direction along axons during a 148-s recording period. Cortical neurons were transfected with GFP-Rheb and DsRed-Mito at 5-7 DIV and incubated with DMSO or 10 μM CCCP for 24 hr prior to imaging at 10-12 DIV.

D,E Quantitative analysis (D) and representative images (E) showing robust association of Rheb with depolarized mitochondria within axons treated with CCCP. Rheb-SSVM, a farnesylation deficient mutant of Rheb, fails to be recruited to mitochondria upon dissipating ∆ψm. Mitochondria associated with Rheb were marked by white arrows. The percentages of mitochondria targeted by Rheb or Rheb-SSVM in the presence or absence of CCCP were quantified, respectively (D).

F-H Representative dual-channel kymographs (F) and quantitative analysis (G, H) showing that Rheb-mediated mitophagy activation depends on Rheb recruitment to mitochondria through Rheb farnesylation. Note that Rheb, but not Rheb-SSVM, is associated with autophagic vacuoles (AVs) within axons upon ∆ψm dissipation. Rheb-associated AVs move in a predominant retrograde direction along axons. The number of Rheb-associated AVs (Rheb-AVs) with or without CCCP treatment and the relative motilities of AVs and Rheb-AVs in CCCP-treated axons were quantified, respectively (G, H).

Data information: Data were quantified from the total number of neurons (n) indicated in parentheses (D, G) from greater than four experiments. Scale bars: 10 μm. Error bars represent SEM. Student's t test: ***P < 0.001.
Mitophagy Regulates Integrity of Mitochondria at Synapses and is Critical for Synaptic Maintenance (2020)
Figure 2

Figure 2. Rheb-mediated mitophagy initiation in axons requires Nix but not Parkin.

A-C Representative dual-channel kymographs (A) and quantitative analysis (B, C) showing that Rheb enhances mitophagy in CCCP-treated axons. The numbers of mitochondria (Mito), AVs, and Mito-AVs/mitophagosomes, the percentage of Mito within AVs, and the motilities of AVs and Mito-AVs along axons with and without elevated Rheb expression were quantified, respectively (B, C).

D,E Representative images (D) and quantitative analysis (E) showing that mitophagy in axons requires Rheb and Nix, but not Parkin. The number of mitophagosomes indicated by white arrows and the percentage of Mito within AVs in CCCP-treated axons expressing scrambled shRNA, Rheb shRNA, Nix shRNA, or Parkin shRNA were quantified, respectively (E).

F,G Quantitative analysis (F) and representative blots (G) showing increased mitochondrial association with Rheb and LC3-II in mature cortical neurons upon ∆ψm depolarization. Cortical neurons at 14 DIV were incubated with DMSO or 10 μM CCCP for 24 hr and then subjected to fractionation into post-nuclear supernatant (P), mitochondria-enriched membrane fraction (M), and cytosolic supernatant (S). Equal amounts of protein (20 μg) were sequentially immunoblotted with antibodies against mitophagic/autophagic proteins Rheb, LC3, and Rab7, mitochondrial protein VDAC, and cytosolic protein GAPDH on the same membranes after stripping between each antibody application. The purity of mitochondrial fractions was confirmed by enrichment of VDAC and less abundance of GAPDH, compared to post-nuclear supernatant. Protein levels in mitochondrial fractions of CCCP-treated neurons were normalized to those in neurons incubated with DMSO. Data were quantified from four independent repeats.

H,I Quantitative analysis (H) and representative images (I) showing that Rheb RNAi augments axonal retention of oxidized mitochondria upon ∆ψm depolarization. The fluorescence of MitoROGFP was emitted at 510 nm and excited at 405 nm or 488 nm, respectively. Ratiometric images were generated from fluorescence excited by 405 nm light relative to that excited by 488 nm light. The ratio has been false colored with the indicated heat map, with high intensity indicative of ROGFP fluorescence in a more oxidative level (I). Mean fluorescence intensity ratios evoked by the two excitation wavelengths at individual mitochondria in the CCCP-treated axons expressing scrambled shRNA or Rheb shRNA were quantified and normalized to those of control neurons transfected with scrambled shRNA (H).

Data information: Data were quantified from the total number of neurons (n) indicated in parentheses (B, E) or on the top of bars (H) from at least four experiments. Scale bars: 10 μm. Error bars represent SEM. Student's t test: ***P < 0.001; **P < 0.01.
Mitophagy Regulates Integrity of Mitochondria at Synapses and is Critical for Synaptic Maintenance (2020)
Figure 3

Figure 3. Presynaptic mitophagy stress in AD-related mutant hAPP Tg mouse brains.

A,B Aberrant accumulation of mitophagosomes co-labeled by antibodies against LC3 and cytochrome c (Cyto c) in the hippocampal mossy fibers of mutant hAPP Tg mouse brains. Note that mitophagosomes indicated by white arrows are not readily observed in WT mouse brains (A). The average number of mitophagosomes per section (320 μm × 320 μm) was quantified from the total number of brain slice sections (n) indicated on the top of the bars (B).

C Clustering of mitophagosomes (white arrows) co-labeled by antibodies against LC3 and Cyto c or p62 and HSP60 within swollen/dystrophic presynaptic terminals indicated by synaptophysin (SYP) surrounding amyloid plaques in AD mouse brains.

D,E Quantitative analysis (D) and representative TEM images (E) showing presynaptic retention of mitophagosome-like structures in the hippocampal regions of mutant hAPP mouse brains. White arrow-labeled mitophagosomes are not readily detected in WT mouse brains. Black arrows marked amphisomes or degradative autophagic vacuoles (AVd). Data were quantified from the total number of the imaging fields (n) indicated in parentheses (D).

F,G Mitophagic accumulation at Aβ-enriched synapses in AD mouse brains. Note that the levels of mitophagic/autophagic proteins (red box) along with Aβ (green box) are increased in synaptic fractions isolated from AD mouse brains. Equal amounts (10 μg) of synaptosomal preparations (Syn) and post-nuclear supernatants (PNS) from WT and mutant hAPP mouse brains were sequentially immunoblotted with antibodies against hAPP/Aβ, Rheb, LC3, p62, and Rab7, as well as mitochondrial proteins TOM20 and SOD2 on the same membrane after stripping between each antibody application. The purity of synaptosomal fractions was confirmed by relative enrichment of SYP and less abundance of p115 and GAPDH (F). Relative protein levels in the synaptosomal preparations from mutant hAPP mice were compared to those in WT littermate controls. Data were quantified from five repeats (G).

H,I Representative blots (H) and quantitative analysis (I) showing increased levels of Rheb along with LC3-II, p62, and Rab7 (red box) in Mito fractions purified from AD mouse brains. The purity of Mito fractions was confirmed by enrichment of mitochondrial proteins VDAC and TOM20 and by the absence of GAPDH and p115, relative to PNS fractions. Data were quantified from four repeats.

Data information: Scale bars: 25 μm (A, C); 10 μm (C at high magnification); 200 nm (E). Error bars represent SEM. Student's t test: ***P < 0.001; **P < 0.01.
Mitophagy Regulates Integrity of Mitochondria at Synapses and is Critical for Synaptic Maintenance (2020)
Figure 4

Figure 4. Increased mitophagy initiation coupled with defective retrograde transport in AD axons.

A,B Representative images (A) and quantitative analysis (B) showing increased mitochondrial association with Rheb in AD axons, relative to that of WT littermate controls. Note that CCCP treatment enhances Rheb localization to mitochondria. The percentages of white arrow-marked mitochondria that were targeted by Rheb in WT and mutant hAPP axons in the absence and presence of CCCP were quantified, respectively (B).

C-E Representative dual-channel kymographs (C) and quantitative analysis (D,E) showing defective retrograde transport and mitophagic accumulation within AD axons. Relative motility of AVs and Mito-AVs/mitophagosomes, the numbers of Mito, AVs, and Mito-AVs, and the percentage of mitochondria within AVs in WT or mutant hAPP axons with and without CCCP were quantified, respectively (D, E).

F,G Quantitative analysis (F) and representative blots (G) showing that ∆ψm depolarization augments mitophagy activation in cortical neurons cultured from AD mouse brains. Note increased localization of Rheb along with LC3-II and Rab7 to mitochondria in mutant hAPP Tg neurons after CCCP treatment (red box). Data were quantified from three independent repeats. P: post-nuclear supernatants; M: mitochondria-enriched membrane fractions; S: cytosolic supernatants.

H,I Quantitative analysis (H) and representative images (I) showing abnormal accumulation of oxidatively damaged mitochondria in AD axons, which is exacerbated after dissipating ∆ψm. Mean fluorescence intensity ratios evoked by the two excitation wavelengths (405 nm and 488 nm) at individual mitochondria in mutant hAPP axons with and without CCCP treatment were quantified and normalized to those of WT controls (H).

Data information: Data were quantified from the total number of neurons (n) indicated on the top of the bars (B, H) or in parentheses (D) from more than four independent experiments. Scale bars: 10 μm. Error bars represent SEM. Student's t test: ***P < 0.001; *P < 0.05.
Mitophagy Regulates Integrity of Mitochondria at Synapses and is Critical for Synaptic Maintenance (2020)
Figure 5

Figure 5. Deficiency in Snapin-mediated retrograde transport recapitulates presynaptic mitophagy stress and leads to synaptic defects in mouse brains.

A,B Representative images (A) and quantitative analysis (B) showing aberrant accumulation of mitophagosomes labeled by LC3 and Cyto c antibodies in the hippocampal mossy fibers of snapin-cKO mouse brains. Note that white arrow-labeled mitophagosomes are not readily observed in WT mouse brains. The average number of mitophagosomes per section (320 μm × 320 μm) was quantified.

C,D Representative blots (C) and quantitative analysis (D) showing aberrant mitophagic retention at synaptic terminals in snapin-mutant mouse brains. The levels of Rheb along with LC3-II, p62, and Rab7 are significantly elevated in the synaptosomal fractions (Syn) purified from snapin-deficient mouse brains, relative to those of WT littermate controls (red box). Equal amounts (10 μg) of Syn and post-nuclear supernatants (PNS) from WT and snapin-mutant mouse brains were sequentially immunoblotted with antibodies against Snapin, mitophagic/autophagic proteins, and mitochondrial proteins VDAC and TOM20 on the same membrane after stripping between each antibody application. The purity of synaptosomal fractions was confirmed by relative enrichment of SYP and PSD95 and less abundance of GAPDH. Data were quantified from five independent repeats.

E,F Representative blots (E) and quantitative analysis (F) showing mitophagic accumulation in the brains of snapin conditional KO (cKO) mice by crossing with Thy1-cre Tg mice. Note increased levels of Rheb along with LC3-II, p62, and Rab7 in mitochondrial (Mito) fractions isolated from snapin-deficient mouse brains, relative to those of WT littermate controls (red box). Data were quantified from five independent repeats. PNS: post-nuclear supernatants.

G,H Representative TEM images (G) and quantitative analysis (H) showing abnormal presynaptic retention of mitophagosome-like structures in the hippocampal regions of snapin-deficient mouse brains. Mitophagosomes, indicated by black arrows, are not readily detected in WT mouse brains. Data were quantified from the total number of the imaging fields (n) indicated in parentheses (H).

I,J Representative images (I) and quantitative analysis (J) showing that deletion of snapin in mouse brains induces synaptic damage. Note that the density of presynaptic terminals in the hippocampal mossy fibers revealed by SYP immunostaining is significantly reduced in snapin-mutant mice crossed with CamKII-cre Tg mice at the age of 8 months, relative to that of WT littermate controls.

Data information: Data were quantified from the total number of brain slice sections (n) indicated on the top of the bars (B, J). Scale bars: 25 μm (A); 10 μm (I); 200 nm (G). Error bars represent SEM. Student's t test: ***P < 0.001; **P < 0.01; *P < 0.05.
Mitophagy Regulates Integrity of Mitochondria at Synapses and is Critical for Synaptic Maintenance (2020)
Figure 6

Figure 6. Snapin-enhanced retrograde transport reduces mitophagic accumulation and attenuates mitochondrial defects in mutant hAPP Tg axons.

A-C Representative dual-channel kymographs (A) and quantitative analysis (B, C) showing that overexpression of Snapin, but not Snapin-L99K, a Snapin mutant deficient in dynein motor binding, enhances retrograde transport of mitophagosomes and reduces mitophagic accumulation within AD axons incubated with CCCP.

D Mean fluorescence intensity ratios evoked by 405 nm and 488 nm excitation wavelengths at individual mitochondria in mutant hAPP axons expressing Snapin or Snapin-L99K were quantified and normalized to those of mutant hAPP controls. Note that oxidatively stressed mitochondria are reduced within AD axons expressing Snapin after CCCP treatment.

Data information: Data were quantified from the total number of neurons (n) indicated in parentheses (B, C, D) from more than four independent experiments. Scale bars: 10 μm. Error bars represent SEM. Student's t test: ***P < 0.001.
Mitophagy Regulates Integrity of Mitochondria at Synapses and is Critical for Synaptic Maintenance (2020)
Figure 7

Figure 7. Overexpression of Snapin ameliorates mitophagy stress and mitigates synapse loss in mutant hAPP Tg mouse brains.

A Representative images showing gene delivery into the hippocampus of AD mice injected with AAV-mCherry or AAV-mCherry-Snapin. mCherry fluorescence indicated by white arrows is present in the soma and processes of transduced neurons in the hippocampal CA3 and dentate gyrus (DG) of mutant hAPP mouse brains. NeuN, a neuronal nuclear marker, indicates neurons.

B,C Attenuation of mitophagic accumulation in the hippocampal mossy fibers of AD mice transduced with AAV-mCherry-Snapin. Note a marked reduction of mitophagosomes (white arrows) in mutant hAPP Tg mouse brains with elevated Snapin expression (C). Cyto c*: color is converted from blue to red for better contrast (B).

D,E Quantitative analysis (D) and representative images (E) showing increased density of presynaptic terminals in the hippocampal mossy fibers of AD mice infected with AAV-mCherry-Snapin. Blue indicates the signal of DAPI staining. The percentage area of SYP-labeled presynaptic terminals was quantified and normalized to that of control AD mice injected with AAV-mCherry (D).

Data information: Data were quantified from a total number of 40-44 imaging slice sections of three pairs of mutant hAPP Tg mice with AAV injection. Scale bars: 10 μm. Error bars represent SEM. Student's t test: ***P < 0.001.

Expanded View figure legends
Macrophages inhibit Coxiella burnetii by the ACOD1-itaconate pathway for containment of Q fever (2022)
Figure 3

Figure 3: Increased bacterial burden in ACOD1-deficient mice infected with C. burnetii. (A) Mice were infected by the i.t. route with 106 NMII. Bacterial load in lung, spleen and liver on d7 after i.t. infection. n=12 for Acod1-/-, n=13 for B6, Acod1+/- (pooled from three independent experiments).

(B) Bacterial load on d11 after i.t. infection. n=10-11 for Acod1-/-, n=8 for B6, Acod1+/ (pooled from two independent experiments).

(C) Immunohistochemistry for C. burnetii in the lung on d7 after i.t. infection. Representative images and quantitation of lung tissue positive for C. burnetii (n=12-13 mice, pooled from three experiments, each normalized to the mean of WT or Acod1+/- infected mice). Each dot represents one mouse. Bars show mean and SD. (D, F) Bacterial load in lung, spleen and liver on d7 (D) and d11 (F) after i.p. infection with 5x107 NMII. n=6-9 mice per genotype, pooled from two independent experiments. (E) Immunohistochemistry for C. burnetii on day 7 after intraperitoneal infection. Each dot represents one mouse. Bars show mean with SD.

(G) Radiation chimeric mice were infected i.t. with NMII. Each dot represents one mouse, data are pooled from two experiments. Bacterial burden in the tissues was determined after 7 days, except for one experiment with CD45.1 recipients when mice were sacrificed after 5 days because of weight loss. n=4-8 mice.

Data information: Statistical analyses comparing Acod1-/- to B6 or Acod1+/- controls with Mann-Whitney test, * p<0.05, ** p<0.01, *** p<0.001.