EMBO/soda-nel huggingface dataset:
BI-ALLELIC PATHOGENIC VARIANTS IN NDUFC2 CAUSE EARLY-ONSET LEIGH SYNDROME AND STALLED BIOGENESIS OF COMPLEX I (2020)
Figure 4

Figure 4. Complexome Profiling of Fibroblasts from Subjects 1 and 3 confirm complex I deficiency.

A Complexome profiling identified assembly intermediates in subject cells with NDUFC2 variants. Complexes from mitochondrial membranes of Control, Subject 1 and Subject 3 fibroblasts were separated by BN-PAGE (Fig EV2) and analysed by complexome profiling. Sample preparation, mass spectrometry, data processing and raw data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (Vizcaíno et al., 2013) with the dataset identifier <PXD014936>. Assignment of complexes: III, complex III; IV, complex IV; III/IV, supercomplex containing dimer of complex III and 1-2 copies of complex IV; S, supercomplex containing complex I, dimer of complex III and 1-4 copies of complex IV. Assembly intermediates are highlighted in dashed boxes indicating stalled complex I assembly. In subject complexomes, various stalled complex I intermediates are assembled: ND4 module (blue dashed box); Q-TIMMDC1-NDUFA13 complex I intermediate (orange dashed box); and Q-TIMMDC1-NDUFA13-MCIA (green dashed box). In control complexome, the Q module (orange dashed box) is assembled without TIMMDC1 or NDUFA13. Complex I modules are denoted according to Stroud and colleagues (Stroud et al., 2016).

B Abundance data for all the subunits of complex I were averaged to plot an abundance profile of complex I. Heatmap (upper panel) and profile plot (lower panel) compare complex I between control (black), Subject 1 (red) and Subject 3 (blue).

C Profile plot of complex III (average of all subunits). Black arrows indicate shifted complex III to the position of the individual complex and small supercomplex (III/IV) in Subjects 1 and 3.

D Profile plot of complex IV (average of all subunits) indicate less complex IV at the position of the supercomplexes for Subject 1 and Subject 3, but higher amounts of individual complex IV and small supercomplex (III/IV, black arrow).

E Details of Q module-containing assembly intermediates of Subject 3. Intensity based absolute quantification (iBAQ) values of assembly factors, single subunit and averaged Q-module subunits were plotted to show abundance profiles. The cartoon (lower panel) indicate assembled intermediate complexes relative to the plot.

F Details about ND4 module assembly intermediate of Subject 3. Abundance profile of averaged iBAQ values of ND4 subunits and assembly factors show accumulation of a complex containing the ND4 module, FOXRED1 and TMEM70.
Compartment‐specific aggregases direct distinct nuclear and cytoplasmic aggregate deposition (2015)
Figure 1

Cytosolic and nuclear misfolded proteins share INQ (JUNQ) as a common deposition site

S. cerevisiae wt or hsp42Δ cells expressing mCherry‐VHL (red) and GFP‐luciferase‐DM‐NLS (green) were grown at 30°C and shifted to 37°C for 90 min. MG132 was added prior to temperature upshift. Changes in protein localizations were recorded. DNA was stained by DAPI (blue).
S. cerevisiae wt cells or hsp42Δ cells expressing GFP‐VHL (green) were treated as in (A). Nuclear membranes were visualized by Nsp1 immunofluorescence labeling (red).
S. cerevisiae wt cells or hsp42Δ cells expressing mCherry‐VHL (red) and GFP‐Nup49 (green) were treated as in (A). Three‐dimensional reconstructions of respective cells are given.
Time‐lapse microscopy of wt cells expressing mCherry‐VHL (green) and GFP‐Nup49 (red) upon temperature upshift from 30°C to 37°C. * indicates collision of peripheral foci leading to fusion, and an arrow indicates apparent encounters of peripheral foci and nuclear INQ, followed by foci separation.

Data information: Scale bars, 2 μm.
Compartment‐specific aggregases direct distinct nuclear and cytoplasmic aggregate deposition (2015)
Figure 2

INQ is located inside the nucleus

A–D Cryo‐sections of (A) S. cerevisiae wt cells expressing GFP‐luciferase‐DM‐NLS, (B) hsp42Δ cells expressing GFP‐VHL, and (C, D) wt or hsp42Δ cells expressing Hsp104‐GFP. Sections were immunogold‐labeled with GFP‐specific antibodies. Gold particles are marked (black arrows). Electron‐dense regions represent protein aggregates (A). Locations of cytosol (C), nuclear envelope (NE, orange arrows), and nucleus (N) are given. Scale bars, 200 nm.
E, F Average numbers of gold particles associated with nuclear and cytosolic protein aggregates or distributed throughout the cytosol or nucleus were determined.
Compartment‐specific aggregases direct distinct nuclear and cytoplasmic aggregate deposition (2015)
Figure 3

INQ formation does not involve ubiquitination

S. cerevisiae wt cells expressing GFP‐VHL (green) were grown at 30°C and shifted to 37°C for 90 min in the presence of MG132. Ubiquitin (red) was stained by immunofluorescence using specific antibodies. DNA was stained by DAPI (blue). Changes in protein localizations were recorded. Scale bars, 2 μm.
tGnd1‐GFP is stabilized in ubr1Δ san1Δ cells. S. cerevisiae wt and ubr1Δ san1Δ mutant cells expressing tGnd1‐GFP were grown at 30°C. Cycloheximide was added, and protein levels were determined at the indicated time points by Western blot using GFP‐specific antibodies. Zwf1 levels are given as a loading control.
Ubiquitination status of tGnd1‐GFP expressed in S. cerevisiae wt and ubr1Δ san1Δ cells was determined by GFP pull‐down and Western blot analysis using ubiquitin‐specific antibodies. Levels of isolated tGnd1‐GFP after GFP pull‐down were determined by Western blot using GFP‐specific antibodies and are given as a control.
S. cerevisisae wt, ubr1Δ san1Δ, and ubr1Δ san1Δ btn2Δ cells expressing tGnd1‐GFP (green) were grown at 30°C. DNA and nuclear envelopes were visualized by coexpressing Htb1‐mCherry or Nic96‐mCherry, respectively (red). The fraction of cells showing specific tGnd1‐GFP foci numbers per cell was determined. INQ formation in cells containing tGnd1‐GFP foci was determined (% cells with INQ) based on close vicinity of tGnd1‐GFP foci to Htb1‐mCherry or localization inside Nic96‐mCherry signals, yielding comparable results. Scale bars, 2 μm.
Compartment‐specific aggregases direct distinct nuclear and cytoplasmic aggregate deposition (2015)
Figure 4

Defect in nuclear import delays INQ formation

A, B S. cerevisiae hsp42Δ and hsp42Δ nup42Δ cells expressing mCherry‐VHL (red) were grown at 30°C and shifted to 37°C in the presence of MG132. Changes in protein localization were monitored. Scale bars, 2 μm. The total number of mCherry‐VHL foci per cell was determined.
C Dynamic exchange of a misfolded model protein between cytosol and nucleus. FLIP measurements of mCherry‐VHL were performed in S. cerevisiae wild‐type cells at 30°C. Nuclear or cytosolic areas were bleached, and loss of fluorescence intensities in non‐bleached compartments was determined. Bleaching and acquisition controls are given. Error bars: SEM.
Compartment‐specific aggregases direct distinct nuclear and cytoplasmic aggregate deposition (2015)
Figure 5

Hsp70 co‐chaperones Sis1 and Sti1 modulate protein aggregation

S. cerevisiae hsp42Δ and hsp42Δ tet‐off sis1 cells expressing GFP‐VHL were grown for 20 h in the absence (−Dox) or presence (+Dox) of doxycycline at 30°C and shifted for 90 min to 37°C in the presence of MG132. Changes in protein localization were monitored. DNA was stained by DAPI (blue). The total number of GFP‐VHL foci per cell and frequencies (%) of INQ formation were determined. Scale bars, 2 μm.
S. cerevisiae wt, hsp42Δ, sti1Δ, and hsp42Δ sti1Δ cells expressing GFP‐VHL were grown at 30°C and shifted to 37°C in the presence of MG132. Changes in protein localization were monitored. The total number of GFP‐VHL foci per cell and frequencies (%) of INQ formation were determined. Scale bars, 2 μm.
Compartment‐specific aggregases direct distinct nuclear and cytoplasmic aggregate deposition (2015)
Figure 6

Btn2 triggers protein aggregation inside the nucleus

A, B S. cerevisiae wt, hsp42Δ, btn2Δ, and hsp42Δ btn2Δ cells expressing GFP‐VHL (green, A) or GFP‐luciferase‐DM‐NLS (green, B) were grown at 30°C and shifted to 37°C for 90 min in the presence of MG132, and protein localizations were recorded. DNA was stained by DAPI (blue), and the nuclear envelope was stained by Nsp1 immunofluorescence (red). Solubilities of GFP‐VHL and GFP‐luciferase‐DM‐NLS were determined after stress application by Western blot using GFP‐specific antibodies. Solubility of actin was determined as a control. T, total fraction; S, soluble fraction; P, pellet fraction. Scale bars, 2 μm.
C S. cerevisiae wt, hsp42Δ, btn2Δ, and hsp42Δ btn2Δ cells expressing mCherry‐VHL were heat‐shocked to 38°C. Upon return to 30°C, protein synthesis was stopped by cycloheximide (CHX) addition and degradation of mCherry‐VHL was monitored by Western blot analysis. The stability of GFP‐luciferase‐DM‐NLS expressed in S. cerevisiae wt and btn2Δ cells was determined accordingly. Ccs1 or Zwf1 levels are given as loading controls.
D S. cerevisiae hsp42Δ btn2Δ cells expressing either GFP or LuciDM‐GFP and Htb1‐mCherry were grown at 30°C and heat‐shocked to 37°C for 30 min. Line intensity plots of luciferase‐DM‐GFP and Htb1‐mCherry before and after heat shock are given. The ratio of nuclear and cytosolic luciferase‐DM‐GFP fluorescence intensity was determined at 30°C and 37°C (n > 25). Scale bars, 2 μm.
Compartment‐specific aggregases direct distinct nuclear and cytoplasmic aggregate deposition (2015)
Figure 7

Hsp42 and Btn2 are compartment‐specific aggregases

A, B Cellular localizations of Hsp42 and Btn2 were determined at the indicated temperature by immunofluorescence. DNA was stained by either Htb1‐mCherry or DAPI. Scale bars, 2 μm. In case of Hsp42 immunofluorescence, a line intensity plot of a deconvoluted widefield image is given.
C S. cerevisiae btn2Δ hsp42Δ Hsp42‐NLS cells expressing GFP‐VHL (green) were grown at 30°C and heat‐shocked to 37°C for 30 min in the presence of MG132. Changes in protein localizations were recorded. DNA was stained by DAPI (blue). The total number of GFP‐VHL foci per cell and frequencies (%) of INQ formation were determined. Scale bars, 2 μm.
D S. cerevisiae btn2Δ hsp42Δ Hsp42‐NLS cells expressing mCherry‐VHL (red) were treated as described in (C). The nuclear membrane was visualized by Nsp1 immunofluorescence (green) and colocalization of mCherry‐VHL foci with Hsp42 was probed by Hsp42 immunofluorescence (green). DNA was stained with DAPI (blue). Scale bars, 2 μm.
E Hsp42 and Btn2 levels were determined prior and post‐heat shock at the indicated time points by Western blot. The numbers of molecules/cell were calculated using purified proteins as standard.
Compartment‐specific aggregases direct distinct nuclear and cytoplasmic aggregate deposition (2015)
Figure 8

MMS treatment leads to formation of Btn2‐dependent non‐canonical DNA stress foci at the INQ

A S. cerevisiae wt cells were treated with MMS and Btn2 levels were determined at the indicated time points. Zwf1 levels are given as a loading control.
B, C S. cerevisiae wt and btn2Δ cells expressing Hos2‐GFP (green, B) or GFP‐VHL (green, C) were grown at 30°C and treated with MMS. Changes in protein localizations were recorded at the indicated time points. DNA was stained by DAPI (blue). The total number of GFP‐VHL foci per cell and frequencies (%) of INQ formation were determined.
Systemic inflammation impairs microglial Aβ clearance through NLRP3 inflammasome (2019)
Figure 2

Figure 2. Nlpr3 knock out mice are refractory to peripheral immune challenge or age-associated changes.

a) Schematic representation of two-photon microscopy experimental design

b) Two-photon representative images of wild-type and Nlrp3-/- mice (5 and 15 months old). Scale bar: 20µm.

c) Quantification of morphological parameters for wild-type and Nlpr3-/- mice after LPS injection (mean of 5-6±SEM; two-way ANOVA followed by Tukey's post hoc test, *p<0.05, **p<0.01).
Systemic inflammation impairs microglial Aβ clearance through NLRP3 inflammasome (2019)
Figure 3

Figure 3. Peripheral immune challenge affects amyloid deposition in APP/PS1 mice.

a) Representative cortical images of MXO4 staining for APP and APP/Nlrp3-/- 15 months old mice. Scale bar: 50µm.

b) Cortical amyloid plaque number and size quantification, and Amyloid-beta1-40 and 1-42 ELISA quantification for APP and APP/Nlrp3-/- mice (5 and 15 month old) mean of 8±SEM; two-way ANOVA followed by Tukey's post hoc test, *p<0.05, **p<0.01, ***p<0.001).
Systemic inflammation impairs microglial Aβ clearance through NLRP3 inflammasome (2019)
Figure 4

Figure 4. Microglia dynamics depends on distance to Aβ deposition in APP/PS1 mice.

a) Two-photon images of microglia (e-GFP) cells clustering around amyloid plaque for APP and APP/Nlrp3-/- (5 and 15 months old). Scale bar: 20µm b) Quantification of morphological parameters in (a) (mean of 5-6±SEM).

c) Flow cytometry plots from APP and APP/Nlrp3-/- mice (15 months old), cells were gated on CD11b and MXO4 after microglia isolation. d) Relative Aβ microglia uptake quantification (mean of 5±SEM; two-way ANOVA followed by Tukey's post hoc test, *p<0.05).
Systemic inflammation impairs microglial Aβ clearance through NLRP3 inflammasome (2019)
Figure 5

Figure 5. Microglia dynamics depends on distance to Aβ deposition in APP/PS1 mice.

a) Two-photon microglia (e-GFP) images from APP and APP/Nlrp3-/- in areas free of plaque (5 and 15 months old mice). Scale bar: 20µm

b) Quantification of APP and APP/Nlrp3-/- morphological parameters for microglia in areas free of plaque (mean of 5±SEM; two-way ANOVA followed by Tukey's post hoc test, *p<0.05, **p<0.01).
Systemic inflammation impairs microglial Aβ clearance through NLRP3 inflammasome (2019)
Figure 6

Figure 6. Peripheral myeloid cells infiltrates into APP/PS1 mice upon LPS injection.

a,b) Iba-1 (green), CD169 (red) and MXO4 staining in plaque-associated areas (cortex and hippocampus) of 5 and 15 months old of APP and APP/Nlrp3-/-. Note the colocalization between Iba-1 and CD169 (white arrows) in APP 15-month-old mice 2 days post LPS injection. Scale bar: 20µm
Systemic inflammation impairs microglial Aβ clearance through NLRP3 inflammasome (2019)
Figure 7

Figure 7. Microglia proliferates in non-APP mice upon peripheral immune challenge.

a,b) Iba-1, Ki67 and DAPI staining in the cortex and hippocampus of wild-type and Nlrp3-/- (5 and 15 months old). Microglia proliferates upon LPS injection (white arrows). Non-microglia cells proliferation was observed as well (yellow arrows) Scale bar: 20µm.

c) Quantification of microglial proliferation in cortex (left panel) and hippocampus (right panel) (mean of 5±SEM; two-way ANOVA followed by Tukey's post hoc test, *p<0.05, **p<0.01, ***p<0.001).
Systemic inflammation impairs microglial Aβ clearance through NLRP3 inflammasome (2019)
Figure 8

Figure 8. Microglia proliferates in APP/PS1 mice upon peripheral immune challenge.

a,b) Iba-1, Ki67 and MXO4 staining in the cortex and hippocampus of APP and APP/Nlrp3-/- (5 and 15 months old). Microglia proliferates upon LPS injection (white arrows). Non-microglia cells proliferation was observed as well (yellow arrows). Scale bar: 20µm

c) Quantification of microglial proliferation in cortex (left panel) and hippocampus (right panel) (mean of 5±SEM; two-way ANOVA followed by Tukey's post hoc test, *p<0.05, **p<0.01, ***p<0.001).
Functional conservation and divergence of the helix-turn-helix motif of E2 ubiquitin-conjugating enzymes (2021)
Figure 1

Figure 1 - Specific charge-swap variants of the UBE2SHTH increase enzymatic activity and retain Ub linkage specificity.

A Structural overview of an E2 (green)~Ub (yellow) undergoing a conformational change from the "open" (PDB: 3UGB) to the "closed" (PDB: 4AUQ) state upon E3 binding, resulting in substrate ubiquitination (Dou et al., 2012; Page et al., 2012).

B UBE2SHTH (green, PDB: 1ZDN) charged residues (spheres) that were mutated and tested in autoubiquitination assays.

C E139R, E143R, and the combination ER/ER mutant substitutions in UBE2S increase the amount of automodification, monitored by the presence of *Ub (* denotes fluorescently labeled protein) and using SDS-PAGE and fluorescent scanning.

D ER/ER substitutions increase Ub chain formation in multiple UBE2S constructs (WT, UBC only, and UBC-UBD fusion), monitored by Coomassie-staining of non-reduced SDS-PAGE gels.

E UBE2S WT and ER/ER form primarily K11-linked Ub chains, determined by quantifying the total detected diGly linkages (left) and calculating the percentage of specific chain topologies (right) using mass spectrometry.
Functional conservation and divergence of the helix-turn-helix motif of E2 ubiquitin-conjugating enzymes (2021)
Figure 2

Figure 2 - The charge at the E2HTH modulates the intrinsic activity of the E2.

A Cartoon of pulse-chase diUb synthesis assays, related to (E-H), where the donor Ub is fluorescently labeled and transferred to unlabeled acceptor UbΔGG.

B Crystal structure of UBE2CHTH (yellow) aligned to UBE2SHTH (green, PDB: 1ZDN) (Lin et al, 2002; Sheng et al, 2012).

C Crystal structure of UBE2D2HTH (blue) aligned to UBE2SHTH (green, PDB: 1ZDN) (Ozkan et al, 2005; Sheng et al., 2012).

D Crystal structure of UBE2R2HTH (red) aligned to UBE2SHTH (green, PDB: 1ZDN) (Sheng et al., 2012; Williams et al, 2019).

E, F, G, H Pulse-chase diUb synthesis assays using different E2s, UBE2S (E), UBE2C (F), UBE2D2 (G), or UBE2R2 (H). DiUb formation was monitored by fluorescent scanning (top), quantitated (bottom), and assessed for statistical significance by either an unpaired Welch's t-test (UBE2C and UBE2R2) or one-way ANOVA (UBE2S and UBE2D2) (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, n=3 independent, technical replicates for all E2s). Error bars: SEM. Data normalized to the maximal WT diUb formation. # denotes a contaminant band.

I Weblogos of the HTH motifs of UBE2S, UBE2C, UBE2D, and UBE2R. Residues colored according to their average net negative (red) or positive (blue) charge. Residues of interest on UBE2S, UBE2C, UBE2D, and UBE2R are labeled. UBE2R is extended to show conservation of D143, used in Fig 4.

J Negative charges at the E2HTH weakens the diUb synthesis activity of the E2 family. Electrostatic models of the UBE2S, UBE2C, UBE2D2, and UBE2R2 HTH motifs were generated using the PBEQ solver (Jo et al, 2008). Active site cysteine is indicated as a yellow sphere. Negative to positive charge represented as a red to blue gradient.
Functional conservation and divergence of the helix-turn-helix motif of E2 ubiquitin-conjugating enzymes (2021)
Figure 3

Figure 3 - UBE2SHTH and UBE2R2HTH charged residues contribute to different steps during Ub chain elongation.

A Schematic of a lysine discharge assay. The acceptor Ub is replaced with free lysine to monitor the stability of the E2~Ub.

B Schematic of a *LRLRGG (C-terminus of Ub) peptide transfer to an acceptor UbΔGG, eliminating interactions with the donor Ub.

C The E139R substitution decreases the stability of UBE2S~Ub compared to WT, revealed by fluorescent scanning of a lysine discharge assay (top) and its quantification (bottom). Statistical comparison was performed using one-way ANOVA (* p ≤ 0.05, ** p ≤ 0.01, n=4 independent, technical replicates). Error bars: SEM. Data normalized to the initial timepoint of each respective E2~Ub band.

D UBE2R2 R149E has a similar E2~Ub stability as WT, monitored by fluorescent scanning (top) of the lysine discharge assay and its quantification through an unpaired Welch's t-test (bottom) (n=3 independent, technical replicates). Error bars: SEM. Data normalized to the initial timepoint of each respective E2~Ub band.

E Fluorescent scans (top) and quantification (bottom) of a *LRLRGG peptide transfer assay comparing UBE2S WT and ER/ER reveals no significant difference through an unpaired Welch's t-test (n=3 independent, technical replicates). Error bars: SEM. Data normalized to the maximal *LRLRGG-Ub formation by WT UBE2S.

F UBE2R2 R149E is still defective in the transfer of the *LRLRGG peptide to an acceptor Ub compared to WT, probed by fluorescent scanning (top) and its quantitation (bottom). Statistical comparison was performed using an unpaired Welch's t-test (** p ≤ 0.01, n=5 independent, technical replicates). Error bars: SEM. Data normalized to the maximal *LRLRGG-Ub formation by WT UBE2R2.
Functional conservation and divergence of the helix-turn-helix motif of E2 ubiquitin-conjugating enzymes (2021)
Figure 4

Figure 4 - The UBE2R2 R149E variant disrupts acceptor Ub binding and gating loop flexibility.

A Dissociation constants of UBE2R2 and its variants binding to acceptor Ub as determined by BLI and fitted data from METRIS. Data from n≥3 independent experiments.

B Schematic of METRIS assay set-up and parameter analysis to assess the binding between UBE2R2 and Ub. In brief, biotinylated Ub is linked to a magnetic bead that is introduced to a magnetic field while on a UBE2R2 functionalized coverslip. The movement of each bead is quantified, and the rolling parameter correlates to binding affinities between Ub and E2.

C Plot of the METRIS rolling parameters of UBE2R2 WT, D143K, and R149E compared using one-way ANOVA (**** p ≤ 0.0001, WT: 63 beads x 17 rolls n=1071, R149E: 46 beads x 17 rolls n=782, D143K: 47 beads x 17 rolls n=799; technical replicates), indicating a weaker interaction with Ub between the UBE2R2 variants compared to WT. Error bars: SEM.

D Docking model of the UBE2R2 (grey) - acceptor Ub (orange) interaction with E2-Ub contacts (blue), UBE2R2 gating loop (yellow), and R149 (purple) highlighted. Dashed line indicates distance measured for Fig 4E between position 149 and T136 (Hill et al., 2016).

E UBE2R2 R149E restricts the movement of the gating loop compared to WT as shown by the distances of the Cα atoms from T136 and R149 in molecular dynamics simulations.

F Plot of METRIS rolling parameters of WT and the R149E substituted variant of UBE2R2 isopeptide-linked to the donor Ub compared using one-way ANOVA (* p ≤ 0.05, WT-Ub: 47 beads x 17 rolls n=799, R149E-Ub: 114 beads x 17 rolls n=1938; technical replicates). Error bars: SEM.

G, H, I Role of the UBE2RHTH in diUb formation is determined from kinetic parameters (G-H) for mutants upon titrating the acceptor Ub in pulse-chase reactions following UBE2R2~*Ub, monitored by fluorescent scanning of nonreducing SDS-PAGE gels (I). Apparent Km and Vmax were determined using the Michaelis-Menten equation (n≥3 independent, technical replicates). Error bars: SEM. Data normalized to the WT apparent Vmax.