EMBO/soda-nel huggingface dataset:
Inhibition of proteasome rescues a pathogenic variant of respiratory chain assembly factor COA7 (2019)
Figure 4

Figure 4. MIA40 facilitates COA7 import into mitochondria.

(A, B) Radiolabeled [35S]COA7 (A) and [35S]TIMM8A (B) precursors were imported into mitochondria that were isolated from Flp-In T-REx 293 cells induced to express MIA40FLAG. The samples were analyzed by reducing SDS-PAGE and autoradiography. The results of three biological replicates were analyzed, quantified, and normalized to control mitochondria at 45 min. The data are expressed as a mean ± SEM (n=3). IAA, iodoacetamide.

(C) Mitochondria were isolated from Flp-In T-REx 293 cells induced to express MIA40FLAG and control cells. The samples were analyzed by SDS-PAGE and Western blot. Mitos, mitochondria.

(D, E) Radiolabeled [35S] COA7 (D) and [35S] TIMM8A (E) precursors were imported into mitochondria that were isolated from Flp-In T-REx 293 cells induced to express ALRFLAG and control cells. The samples were analyzed by reducing SDS-PAGE and autoradiography. The results of three biological experiments were analyzed, quantified, and normalized to control mitochondria at 45 min. The data are expressed as a mean ± SEM (n=3).

(F) Mitochondria were isolated from Flp-In T-REx 293 cells induced to express ALRFLAG and control cells. The samples were analyzed by SDS-PAGE and Western blot.
Inhibition of proteasome rescues a pathogenic variant of respiratory chain assembly factor COA7 (2019)
Figure 5

Figure 5. MIA40 is involved in the import and biogenesis of COA7.

(A) Cellular protein extracts were isolated from HeLa cells transfected with oligonucleotides that targeted different regions of MIA40 mRNA or with control oligonucleotides. The samples were analyzed by reducing SDS-PAGE and Western blot.

(B) Cellular protein extracts were isolated from HeLa cells transfected with oligonucleotides that targeted different regions of ALR mRNA or with control oligonucleotides. The samples were analyzed by reducing SDS-PAGE and Western blot.

(C) Schematic representation of MIA40 sequence containing the CPC motif in HEK293 MIA40 WT/WT and HEK293 MIA40 WT/Del53-60 cells.

(D) Cellular protein extracts were isolated from HEK293 MIA40 WT/WT and WT/Del53-60 cells. The samples were analyzed by SDS-PAGE and Western blot.

(E) Cellular protein extracts were isolated from HEK293 MIA40 WT/WT and WT/Del53-60 cells transfected with a plasmid that encoded MIA40 or an empty vector. The samples were subjected to reducing SDS-PAGE and Western blot.

(F, G) Radiolabeled [35S]COA7 (F) and [35S]TIMM8A (G) precursors were imported into mitochondria that were isolated from HEK293 MIA40 WT/WT and WT/Del53-60 cells. The samples were analyzed by reducing SDS-PAGE and autoradiography. The results of three biological replicates were analyzed, quantified, and normalized to control mitochondria at 45 min. The data are expressed as a mean ± SEM (n=3). IAA, iodoacetamide.
Inhibition of proteasome rescues a pathogenic variant of respiratory chain assembly factor COA7 (2019)
Figure 6

Figure 6. COA7 pathological variants are import-defective.

(A) Schematic representation of wild type and mutant COA7.

(B) Cellular protein extracts were isolated from HEK293 cells that were transfected with a plasmid that encoded wildtype or mutant COA7. The samples were analyzed by reducing SDS-PAGE and Western blot.

(C) Cellular fractions were prepared from HEK293 cells that were transfected with a plasmid that encoded wildtype or mutant COA7. The fractions were analyzed by reducing SDS-PAGE and Western blot. T, total; C, cytosol; M, mitochondria.

(D) HEK293 cells that transiently expressed wildtype or mutant COA7HIS were solubilized, and the affinity purification of COA7HIS was performed. The samples were analyzed by reducing and non-reducing SDS-PAGE and Western blot. Load: 3 %. Eluate: 100 %.

(E) Equal amounts of wildtype and mutant radiolabeled [35S]COA7 precursors were imported into mitochondria isolated from HEK293 cells. The samples were analyzed by reducing SDS-PAGE and autoradiography. The results of three biological replicates were analyzed, quantified, and normalized to wildtype COA7 at 45 min. The data are expressed as a mean ± SEM (n=3). IAA, iodoacetamide.
Inhibition of proteasome rescues a pathogenic variant of respiratory chain assembly factor COA7 (2019)
Figure 7

Figure 7. The proteasome degrades cytosol-localized COA7 variants.

(A) Schematic representation of protein stability assay.

(B) HEK293 cells that transiently expressed wildtype or mutant COA7HIS were treated with CHX for the indicated times, and protein extracts were isolated. The samples were analyzed by reducing SDS-PAGE and Western blot.

(C) Schematic representation of combined translation and proteasome inhibition assay.

(D) HEK293 cells that transiently expressed wildtype or mutant COA7HIS were treated with CHX and/or MG132 for the indicated times, and protein extracts were isolated. The samples were analyzed by reducing SDS-PAGE and Western blot. CHX, cycloheximide.

(E) Schematic representation of the cellular localization assay.

(F) Cellular fractions were prepared from HEK293 cells that transiently expressed mutant COA7HIS and were treated with MG132. The samples were analyzed by reducing SDS-PAGE and Western blot.
Inhibition of proteasome rescues a pathogenic variant of respiratory chain assembly factor COA7 (2019)
Figure 9

Figure 9. Inhibition of the proteasome rescues the activity and assembly of the respiratory complexes.

(A) Complex IV activity was assessed by oxidation of cytochrome c in digitonized cellular extracts from the immortalized patient-derived skin fibroblasts and control healthy fibroblasts The results of three biological replicates were quantified, and normalized to control fibroblasts. Data are expressed as a mean ± SEM (n=3, *p=0.01).

(B) Complex IV activity was assessed by oxidation of cytochrome c in digitonized cellular extracts from immortalized patient-derived skin fibroblasts treated with MG132 (1 μM), bortezomib (20 nM), or carfilzomib (50 nM) for 12 h and recovered for another 6 h. The results of three biological replicates were quantified and normalized to DMSO treated samples and the data are expressed as a mean ± SEM (n=3, *p=0.04 [DMSO vs MG132], **p=0.0025 [DMSO vs Bortezomib], *p=0.02 [DMSO vs Carfilzomib]).

(C) Immortalized patient-derived skin fibroblasts transiently expressing wildtype or mutant COA7 (COA7-Y137C and COA7-Ex2∆) for 48 h were treated with bortezomib (20 nM) during the last 12 h. The fibroblasts were harvested, and complex IV activity was measured in digitonized cellular extracts. The results of three biological replicates were quantified and normalized to empty vector-bortezomib treated samples and the data are expressed as a mean ± SEM (n=3, *p=0.01 [Empty vector vs COA7- Wt], *p=0.005 [Empty vector vs COA7 - Y137C]).

(D) Mitochondria isolated from control and patient fibroblast were solubilized in digitonin buffer and analyzed by 4-13 % gel BN-PAGE and Western blot. SC, supercomplexes.

(E) Immortalized patient-derived skin fibroblasts transfected with plasmid encoding wtCOA7HIS and COA7-Y137CHIS were solubilized in DDM buffer and analyzed by 4-13 % gel BN-PAGE and Western blot.

(F) Mitochondria were isolated from immortalized patient-derived skin fibroblasts treated with bortezomib (10 nM) for 12 h and recovered for another 6 h. Mitochondria were solubilized in digitonin buffer and analysed by 4-13 % gel BN-PAGE and Western blot. SC, supercomplexes.
Drug-induced increase in lysobisphosphatidic acid reduces the cholesterol overload in Niemann-Pick type C cells and mice (2019)
Figure 4

Figure 4. Thioperamide targets the histamine receptor HRH3

(A) Effects of compounds that target histamine receptor family members. The outline shows the compounds (listed in Dataset EV3) that target histamine receptor family members as antagonists (HRH1, HRH2, HRH3 and HRH4) or agonists. These include the compounds originally present in the Prestwick library as well as additional compounds selected from the litterature. Cells treated with this small library of compounds were analysed by automated microscopy after labeling with DAPI and antibodies against LBPA. Compounds that increased LBPA staining intensity, like thioperamide (Fig 1) or pitolisant (Fig 4B) are green, while compounds without effects are red. (n=2 independent experiments with 96 images acquired and analysed automatically).

(B) Effect of Pitolisant on LBPA. HeLa MZ cells treated or not with pitolisant 10µM for 18h at 37°C were labeled with DAPI and anti-LBPA antibodies and analysed by fluorescence microscopy (quantification in fig EV3A).

(C, D) LBPA intensity in a mixed population of cells expressing HRH3-GFP. After transfection with HRH3-GFP, uncloned stably expressing cells (C) were labelled with anti-LBPA antibodies and analysed by automated microscopy (C). Unbiased quantification (D) shows the inverse correlation between HRH3-GFP expression (high expressing cells in green) and the endosome integrated intensity of LBPA staining (high LBPA labelling in red). (n=2 independent experiments with 500,000 cells analysed automatically, error bars = SD).

(E-G) LBPA intensity in cells expressing or not HRH3-GFP. HeLa MZ cells stably expressing HRH3-GFP grown in 96-well plates were separately treated, with 4 different siRNAs that target HRH3 (siHRH3#1; siHRH3#2; siHRH3#3; siHRH3#4) or with control non-target (siNT) siRNA (E). HRH3-GFP was analysed by fluorescence microscopy in 4 rows of cells per condition; micrographs are stitched together in the montage. Panel F shows an example of cells treated with siHRH3#2 or siNT, and then labelled with DAPI and antibodies against LBPA. Cells treated as in (E) and labelled with DAPI and antibodies against LBPA as in (F), were analysed by automated microscopy and the integrated intensities of LBPA (left panel) and HRH3-GFP (right panel) signals are compared (G). (n=2 independent experiments with 192 images acquired and analysed automatically, error bars = SD).
Drug-induced increase in lysobisphosphatidic acid reduces the cholesterol overload in Niemann-Pick type C cells and mice (2019)
Figure 5

Figure 5. Effects of thioperamide in NPC cells

(A) Treatment of NPC fibroblasts and NPC null mice with thioperamide. Fibroblast lines obtained from patients with well-established heterozygote mutations in the NPC1 (GM17912 line: NPC1 P1007A / T1036M; GM17911 line: NPC1 I1061T / T1036M) or with a homozygote mutation in the NPC2 gene (GM17910 line: C93F / C93F) were treated or not for 72h with thioperamide 10µM, stained with filipin (cholesterol) and analysed by fluorescence automated microscopy.

(B) Quantification of cholesterol and LBPA staining in NPC fibroblasts. Cells as in (A) were treated or not with thioperamide for 48h or 72h, labelled with filipin (cholesterol) and anti-LBPA antibodies, and analysed by fluorescence automated microscopy. The panels show the integrated intensity of the filipin (left) and LBPA (right) signals were quantified after 48 (top) and 72h (bottom). The colour code of each fibroblast cell line is as in (A). (n=3 independent experiments with 144 images acquired and analysed automatically, >2000 cells per experiment, error bars = SD, one-way ANOVA, *=p<0.05; **=p<0.005;***=p<0.001).

(C) Quantification of cholesterol in NPC fibroblasts by mass spectrometry. NPC cell lines (A) were treated or not with thioperamide or 0.1% (2-Hydroxypropyl)-ß-cyclodextrin for 72h. After extraction, lipids were analysed and quantified by mass spectrometry. (n=3; error bars = SD, one-way ANOVA, *=p<0.05; **=p<0.005;***=p<0.001)

(D, E) Quantification of cell counts in the 3 NPC cell lines treated with thioperamide. The experiment was as in (A-C) except that nuclei were labelled with DAPI and quantified by automated microscopy. Data are normalized to DMSO control (n=2 independent experiments, 75 images per experiments)
Drug-induced increase in lysobisphosphatidic acid reduces the cholesterol overload in Niemann-Pick type C cells and mice (2019)
Figure 6

Figure 6. Effects of thioperamide in NPC cells and mice

(A) Effects of thioperamide on cholesterol-dependent transcriptional regulation. The parental HeLa MZ cells, NPC1 KO cells or NPC2 KO cells were treated or not with thioperamide 10 μM for 18h. Total mRNA was extracted and both LDLR mRNA and HMG CoA reductase mRNA were quantified by RT-PCR. (n=3 independent experiments, error bars = SD, one-way ANOVA, *=p<0.05; **=p<0.005)

(B) Quantification of cholesterol in NPC null mice by enzymatic analysis. WT or NPC1 null mice were treated or not with thioperamide for 6 weeks after weaning and sacrificed. The total content of unesterified cholesterol in the corresponding liver extracts was then quantified using an enzymatic assay and is expressed in nmol per mg tissue. The total content of LBPA in the same liver extracts was quantified by mass spectrometry (see Appendix Figure S1), and is therefore expressed as a percentage of total phospholipids. The total LBPA content of WT mice was only marginally increased by the drug, either because the doses were low, or because the contribution from tissue material unaffected by the drug obscured selective changes at the cellular level (despite much effort, we were unable to visualize LBPA in liver samples by immunocytochemistry). The relative ratio of LBPA vs. cholesterol values is shown.
Cytoplasmic control of Rab family small GTPases through BAG6 (2019)
Figure 1

Figure 1. Defective distribution of endosomal proteins in BAG6-suppressed cells.

(A) At 72 h after transfection with siRNA duplexes for BAG6 or control siRNA (10 nM each), the intracellular localization of TfnR in HeLa cells was examined (shown as green). Nuclear DNA was stained with Hoechst 33342 (shown as blue). Control knockdown (left panel), Rab8a knockdown (center panel), and BAG6 knockdown (right panel). Efficacy of endogenous BAG6 knockdown in HeLa cells was verified by Western blot experiments (see Fig EV1D). Scale bar: 10 μm.

(B) Intracellular localization of Ptc1 (green) in HeLa cells. Nuclei were stained with Hoechst 33342 (shown as blue). See also Fig EV1A and EV1B. Scale bar: 10 μm.

(C, D) Knockdown of Rab8a (with Rab8a siRNA#1, #2, and #3) or BAG6 (with BAG6 siRNA#1) stimulated the accumulation and stabilization of Ptc1 protein in HEK293 cells. See also Appendix Fig S1B.
Cytoplasmic control of Rab family small GTPases through BAG6 (2019)
Figure 2

Figure 2. BAG6 physically interacts with Rab8a in a nucleotide-binding specific manner.

(A) Protein interaction network suggested by public databases.

(B) BAG6 protein co-precipitated Rab8a, while neither Rab7 nor Rab11a were co-precipitated with BAG6. Flag-tagged Rab8a, Rab7, Rab11a, and luciferase-CL1 (Lc-CL1; a positive control) were expressed in HeLa cells and the cells were treated with 10 μM MG-132 for 4 h. Flag-immunoprecipitates were blotted with anti-BAG6 and anti-Flag antibodies, respectively. Note that all cells used were treated with 10 μM MG-132 for 4 h.

(C) S-tagged BAG6 pull-down efficiently co-precipitated Rab8a (T22N), a GDP-bound mutant, while BAG6 scarcely co-precipitated Rab8a (Q67L), a constitutively active mutant. Co-precipitation of Rab8a WT with BAG6 was used as a standard. MG-132 (10 μM) was included in the cell culture for 4 h, as indicated. Note that the T22N mutant protein was expressed at lower levels than either WT or Q67L, and that this was partly due to increased degradation, as will be shown later. S-BAG6 stands for N-terminally S-tagged BAG6 protein (see the Materials and Methods).

(D) Anti-Flag signals in (C) were quantified, and relative signal intensities are presented. The value of the WT Rab8a signal with MG-132 was defined as 1.0. Note that all signal intensities of Flag-tag were normalized by that of the Rab8a input signals. The graph represents the mean ± standard deviation (S.D.) calculated from 3 independent biological replicates. An asterisk indicates p < 0.05 (Student's t-test).

(E) A series of Flag-Rab8a mutants were immunoprecipitated and quantified the amount of endogenous BAG6 that were coprecipitated with Flag-Rab8a. Note that all cells used were treated with 10 μM MG-132 for 4 h.

(F) Deficiency of Rabin8, a GEF for Rab8a, enhanced the physical interaction between BAG6 and WT Rab8a proteins. Flag-tagged WT Rab8a was expressed in Rabin8 siRNA-treated cells, and Flag immunoprecipitates were probed with an anti-BAG6 antibody. Note that all cells used were treated with 10 μM MG-132 for 4 h.
Cytoplasmic control of Rab family small GTPases through BAG6 (2019)
Figure 3

Figure 3. GDP-bound form of the Rab8a GTPase domain is critical for its ubiquitin-mediated degradation.

(A) Schematic representation of Rab8a protein. To prevent C-terminal geranylgeranylation, the Cys204 residue was substituted with a serine residue. Alternatively, two truncated proteins (N100 and ∆N100 fragments of Rab8a) were prepared for this experiment.

(B) C-terminal geranylgeranylation of Rab8a was dispensable for BAG6 recognition.

(C) The N-terminal GTPase domain of Rab8a was essential for BAG6 recognition. A series of Flag-tagged truncated fragments of WT Rab8a were expressed in HeLa cells with S-tagged BAG6 and treated with (+) or without (-) protease inhibitors for 4 h. Flag-Rab8a substrates were immunoprecipitated and probed with an anti-BAG6 antibody.

(D) Hydrophobicity recognition domain of BAG6 (N465) was critical for Rab8a recognition. A schematic representation of the BAG6-truncated proteins used in this experiment is shown in the upper panel. Numbers denote the corresponding amino acids of mammalian BAG6. Positions of UBL, BUILD, and DUF3538 domains, which are all linked to hydrophobicity recognition by BAG6 [37], are indicated. WT, wild-type; ∆N465, N-terminal 465 residues-deleted mutant; and Ν465, C-terminal 689 residues-deleted mutant.

(E, F) CHX chase experiments show that GDP-bound Rab8a (T22N) was a highly labile protein, while the GTP-bound active mutant (Q67L) was a stable protein (E). Instability of the T22N mutant was not perturbed by the C204S mutation (F). Actin was used as a loading control.

(G) N-terminal 100 residue fragment of the Rab8a GTPase domain was sensitive to the proteasome inhibitor, while the ∆N100 fragment was not.

(H) Rab8a (T22N) was polyubiquitinated in the presence of the proteasome inhibitor. Asterisks indicate non-specific bands.
Cytoplasmic control of Rab family small GTPases through BAG6 (2019)
Figure 4

Figure 4. The Switch I region of Rab8a is critical for BAG6 recognition and instability.

(A) Schematic representation of the two Switch regions (I and II) within the Rab8a GTPase domain. Numbers denote the corresponding amino acids of human Rab8a (upper panel). Amino acid sequence alignments of the Switch I region of Rab family proteins (lower panel). Three conserved hydrophobic residues (Ile38, Ile41, and Ile43) in this region are indicated in red, while the other hydrophobic residues are indicated in orange. The three hydrophobic residues were substituted with serine and this construct was designated as the T22N-3IS mutant. Note that Ile38 and Ile43 of Rab8a are not conserved in Rab7.

(B) Kyte-Doolittle hydrophobicity plots of the complete amino acid sequence of human WT Rab8a and T22N-3IS mutant. The hydrophobicity peak within WT Switch I was abolished in the 3IS mutant (indicated within the red box). The numbers on the horizontal axis denote the corresponding amino acid positions in these proteins.

(C) Substitution of the hydrophobic residues of Switch I with hydrophilic residues (T22N 3IS) abolished its binding to BAG6 protein.

(D) Rab8a (T22N-3IS) mutant protein was highly stable, while Rab8a (T22N) protein was quite unstable in HeLa cells. Actin was used as a loading control.

(E) Rab8a (T22N-3IS) mutant was not subject to polyubiquitin co-precipitation, while Rab8a (T22N) protein was.
Cytoplasmic control of Rab family small GTPases through BAG6 (2019)
Figure 5

Figure 5. Endogenous BAG6 is necessary for the elimination of cytosolic Rab8a.

(A) Rab8a (T22N) protein accumulated in BAG6-knockdown cells. HeLa cells were transfected with siRNA duplexes for BAG6 or control siRNA. At 48 h after siRNA transfection, Flag tagged-Rab8a (T22N) was expressed in the cells. At 24 h after Rab8a (T22N) transfection, the cells were chased with 50 μg/mL CHX and harvested at the indicated time after CHX addition. Actin was used as a loading control.

(B) Anti-Flag blot signals in the control or BAG6 siRNA-treated cells were quantified, and relative signal intensities after CHX addition were calculated. The value of the Flag signal at 0 h was defined as 1.0. Note that all signal intensities of the Flag-tag were normalized by that of actin, a loading control, in each sample. The graph represents the mean ± S.E. calculated from 6 independent biological replicates. These data were analyzed by Welch's t-test.

(C) Polyubiquitin modification of Rab8a was abolished in BAG6-knockdown cells. Flag-Rab8a (T22N) immunoprecipitates were blotted with an anti-polyubiquitin antibody (FK2, left panel). As a negative control, siRNA#1scr was used. Anti-polyubiquitin signals co-precipitated with Rab8a (T22N) (a representative example is shown in the left panel) were quantified (right panel). Note that the intensities of the co-precipitated polyubiquitin signal were normalized both by the input ubiquitin-signal and bait Flag-signal. The graph represents the mean ± S.D. calculated from 3 independent biological replicates. An asterisk indicates p < 0.05 (Student's t-test).

(D) Defective distribution of the endosomal protein TfnR in HeLa cells with the excess accumulation of the inactive form of Rab8a. Right panel shows a merged image of TfnR staining (shown as green), Rab8a (T22N) staining (magenta), and Hoechst 33342 nuclear staining (blue). Scale bar: 10 μm.
Cytoplasmic control of Rab family small GTPases through BAG6 (2019)
Figure 6

Figure 6. BAG6 possesses a distinct preference for multiple, but specific, Rab family proteins.

(A) A series of major Rab family proteins were immunoprecipitated from HeLa cell extracts and then probed with an anti-BAG6 antibody. Co-precipitation of Rab8a (T22N) with BAG6 was used as a positive control for this experiment.

(B) Quantification of BAG6 co-precipitation with Rab family proteins. The graph shows the quantities of BAG6 protein in the respective anti-Flag precipitates that were normalized by the amount of Flag-Rab bait proteins. The value of BAG6 co-precipitation with Rab8a (T22N) was defined as 1.0. The quantities of the respective immunosignals were also normalized to the actin signal of each sample, and represent the median calculated from at least 3 independent biological replicates. The number of independent biological replicates was as follows: Rab1a, n = 4; Rab1b, n = 5; Rab2b, n = 3; Rab3a, n = 4; Rab4, n = 4; Rab5a, n = 3; Rab5b, n = 4; Rab6a, n = 5; Rab6b, n = 4; Rab7a, n = 3; Rab8a, n = 14; Rab9b, n = 4; Rab10, n = 6; Rab11a, n = 3; Rab11b, n = 3; Rab12, n = 2; Rab13, n = 6; Rab14, n = 3; Rab15, n = 5; Rab35, n = 3.