EMBO/soda-nel huggingface dataset:
Trigger factor is a bona fide secretory pathway chaperone that interacts with SecB and the translocase (2020)
Figure 2

Figure 2. TF is a tight holdase for translocation-competent proOmpA.

A. TF-proOmpA physical interaction in solution. proOmpA1 (5 μM) was incubated (50 μl; Buffer C; < 0.2 M urea; 37°C; 60 min) with the indicated molar excess of TF2. Soluble proteins were analyzed on 10% native-PAGE (4 mA; 16h; 4°C) and Coomassie-blue stained. A representative experiment is shown; n= 4 biological replicates.

B. TF-proOmpA intact Native-MS analysis. Mass spectra of TF2 (20 μM; Buffer G), proOmpA (20 μM; pre-treated with 10 mM DTT; Buffer G) or their mixture were acquired in near native conditions using a Synapt G2 mass spectrometer, analyzed using MassLynX 4.1, masses were deconvoluted using MaxEnt 1 and shown together with the theoretical masses and deduced stoichiometries. n=2 biological replicates.

C. TF solubilizes proOmpA*. proOmpA*1 (5 μM) was incubated (50 μl; Buffer C; < 0.2 M urea; 37°C; 60 min) alone or in the presence of TF. Proteins in the soluble fraction were analyzed on 15% SDS-PAGE and Coomassie-blue stained. Lane 2: 2.84 μg proOmpA*. Representative experiment shown; n= 5 biological replicates.

D. proOmpA preincubated at 37oC fails to stimulate SecA ATPase activity. proOmpA (0.5 μM) was either diluted directly (lane 1) or after preincubation alone (10 min; buffer D; < 0.2 M urea; either at 4°C or 37°C) into reactions containing SecYEG-SecA alone or together with TF2 or SecB4 (2 and 4 x molar excess over proOmpA, respectively). T/M ratios (as in Fig. 1D) are shown (n= 8 biological replicates; mean values ± SEM; Unpaired parametric t-test, 95% confidence interval: **: p = 0.0017; ****: p < 0.0001).

E. proOmpA pre-incubated at 37oC does not interact with TF. proOmpA1 (5 μM) was preincubated (Buffer D; < 0.2 M urea; 37°C; 10 min) or not (lane 3), prior to addition of the indicated molar excess of TF2 (analyzed as in A). Representative experiment shown; n= 4 biological replicates.
Trigger factor is a bona fide secretory pathway chaperone that interacts with SecB and the translocase (2020)
Figure 4

Figure 4. Physical and functional interaction of SecB with proOmpA and TF.

A. SecB maintains soluble translocation-competent states of proOmpA at 37°C. proOmpA1 (0.5 µM) was incubated with a range of SecB4 concentrations (0 - 2 µM; Buffer D; < 0.2 M urea; 10 min, 37°C). Soluble samples (20,000 x g; 10 min; 4°C) were added to translocation ATPase reactions (as in Fig. 1C). Normalized T/M ratios were plotted (n= 2-8 biological replicates; mean values ± SEM) and an apparent solubilization constant (Ksol) determined (as in Fig. 1F).

B. SecB protects proOmpA* from aggregation. proOmpA*1 (5 μM) was incubated (50 μl; Buffer C; 37°C; 60 min) alone or in the presence of the indicated molar excess of SecB4, and proOmpA* in the soluble fraction (after centrifugation) was analyzed by SDS-PAGE (as in Fig. 2C). Lane 2: 2.84 μg proOmpA*. Representative experiment shown; n= 4 biological replicates.

C. SecB physically interacts with proOmpA. proOmpA1 (5 μM) was incubated (50 μl; Buffer C; < 0.2 M urea; 4°C; 60 min), with the indicated molar excess of SecB4 (filled arrow), analyzed by native-PAGE as in Fig. 2A, and stained as indicated. brace: higher order proOmpA species. Representative experiment shown; n= 5 biological replicates.
Trigger factor is a bona fide secretory pathway chaperone that interacts with SecB and the translocase (2020)
Figure 5

Figure 5. TF:proOmpA:SecB super-complex analysis

A. Analysis of proOmpA:SecB and TF:proOmpA:SecB complexes by native-MS. SecB4 (30 μM; Buffer G), SecB4 (40 μM) and proOmpA (20 μM; pre-treated with 10 mM DTT; buffer G) and of SecB4 (40 μM), proOmpA (20 μM) and TF2 (20 μM) in buffer G were analyzed as in Fig. 2B (raw data in Fig. EV3). n=2 biological replicates.

B. Blow up of the ESI-mass spectrum of the TF:proOmpA:SecB4 super-complex from A (complete dataset in Fig. EV3). Left: charge state distributions, at the indicated m/z range. Right: masses deconvoluted from the mass spectra (as in Fig. 2B). Inset cartoon depicts stoichiometry. Representative spectrum shown; n=2 biological replicates.

C. TF:proOmpA:SecB supercomplex analysis using GPC-MALS in Buffer A. TF1:proOmpA1 (10 µM each; green), SecB4 (40 µM; blue) and TF1:proOmpA1:SecB4 (10, 10 and 40 µM, respectively; black). n= 5 biological replicates; mean mass: 137 kDa (± 6 kDa) representative experiment shown; red circles: determined mass (kDa); AU: arbitrary units.
Trigger factor is a bona fide secretory pathway chaperone that interacts with SecB and the translocase (2020)
Figure 6

Figure 6. SecB-mediated, SecA C-tail dependent release of TF-associated proOmpA for translocation

A. SecB fails to restore proOmpA translocation ATPase activity stimulation of SecA(noC-tail) in the presence of TF. proOmpA translocation ATPase activity stimulation (as in Fig. 1C; SecB molar excess over proOmpA indicated). T/M ratio of SecA(noC-tail) in the absence of TF or SecB= 100%; lane 1; all other values were expressed as a % of this value. n= 4 biological replicates; mean values ± SEM. Unpaired parametric t-test, 95% confidence interval: **: p= 0.0048; ****: p < 0.0001.

B. Excess of SecB inhibits the proOmpA-stimulated translocation ATPase activity of SecA(noCtail). ATPase measured as in Fig. 1C; SecB concentration range: 0-6 µM. Values were normalized (effectmax=100%; effectmin=0%) and plotted (n= 3 biological replicates; mean values ± SEM) versus the log10[SecB4]. The Ki was determined as in Fig. 1E.

C. TF and SecB-regulated, SecA-dependent in vitro translocation of proOmpA into SecYEG/A-IMVs, analyzed after proteolytic treatment. Translocated, protease-resistant proOmpA was visualized using immunostaining (α-His) in the absence or presence of TF2 or/and SecB4 (as indicated). Translocation in the absence of TF or SecB was considered 100% (lane 3); all other values were expressed as a percentage of this. A summary of all repeats is presented (mean ± SEM; n= 6 biological replicates; ns: not significant (p = 0.1568 for lane 3 vs 4; p = 0.4835 for lane 3 vs 6); Unpaired parametric t-test, 95% confidence interval: ****: p < 0.0001) with a representative western blot (bottom).
Therapeutic implications of altered cholesterol homeostasis mediated by loss of CYP46A1 in human glioblastoma (2019)
Figure 1

Figure 1. Expression of CYP46A1, a gene involved in brain cholesterol homeostasis, is lost in GBM and associated with increasing tumour grade.

(A) Heatmap of the differentially expressed cholesterol-related genes between normal brain tissues (n = 28) and glioblastomas (n = 217) from the Rembrandt dataset. Gene expression values are z-transformed and colored red for high expression and blue for low expression, as indicated in the scale bar.

(B) Volcano plot showing the fold-change (log2) in cholesterol-related gene levels based on GBM vs normal brain tissue. Data were obtained from the Rembrandt dataset.

(C) Representative images of IHC staining for CYP46A1 protein in normal brain and different pathological grades of gliomas (n = 64). Scale bar = 30 μm.

(D) Quantification of CYP46A1 IHC staining in normal brain (n = 6) and different pathological grades of gliomas (n = 58).

(E) Representative images of CYP46A1 IHC staining in GBM and adjacent brain tissues from one specific case. Scale bar = 30 μm.

(F) CYP46A1 expression levels in tumours from the TCGA dataset using 2016 WHO classification. Data are shown as the mean ± the standard error of the mean (SEM; n = 667). ***P < 0.0001. Statistical significance was determined by one‐way ANOVA.

(G) CYP46A1 expression levels in different molecular subtypes from the Rembrandt GBM dataset. Shown are means and SEM (n = 245). ***P < 0.0001. Statistical significance was determined by one‐way ANOVA.

(H-I) Kaplan-Meier analysis for patient OS and PFS based on high vs low expression of CYP46A1 in LGG and GBM. Data were obtained from the CGGA dataset. P-values were obtained from the log-rank test.
Therapeutic implications of altered cholesterol homeostasis mediated by loss of CYP46A1 in human glioblastoma (2019)
Figure 2

Figure 2. Restoration of CYP46A1 attenuates the growth of GBM cells in vitro and in intracranial GBM xenografts.

(A) qRT-PCR analysis of CYP46A1 mRNA levels in LN229 and GBM#P3 cells transfected with lentivirus expressing CYP46A1 (lenti-CYP46A1) or control sequence (lenti-Ctrl). GAPDH was used as an internal control. Shown are means and SEM (n = 3). LN229: ***P < 0.0001; GBM#P3: ***P < 0.0001. Statistical significance was determined by two-sided Student's t‐test.

(B) Western blot analysis to confirm CYP46A1 overexpression in GBM cells.

(C) Growth curves for GBM cells in vitro infected with lenti-Ctrl or lenti-CYP46A1 derived from trypan blue staining. Shown are means and SEM (n = 3). LN229: **P = 0.003; GBM#P3: **P = 0.002. Statistical significance was determined by two-sided Student's t‐test.

(D) Colony forming assay for GBM cells infected with lenti-Ctrl or lenti-CYP46A1. Shown are means and SEM (n = 3). LN229: *P = 0.013; GBM#P3: *P = 0.012. Statistical significance was determined by two-sided Student's t‐test.

(E) Representative H&E staining of orthotopic tumours derived from GBM cells infected with lenti-Ctrl or lenti-CYP46A1. Scale bar = 2 mm.

(F) Kaplan-Meier survival curve of tumour bearing mice injected with GBM cells infected with lenti-Ctrl or lenti-CYP46A1 (n = 5 per group). A log-rank test was used to assess statistical significance.

(G) IHC for CYP46A1, PCNA and cleaved caspase-3 protein levels in the intracranial tumours. Scale bar = 100 μm.
Therapeutic implications of altered cholesterol homeostasis mediated by loss of CYP46A1 in human glioblastoma (2019)
Figure 3

Figure 3. CYP46A1 inhibits the growth of GBM cells through production of 24OHC.

(A) Schematic model of cholesterol conversion into 24OHC catalyzed by CYP46A1.

(B) 24OHC levels in spent media from LN229 (left) and GBM#P3 (right) cells transduced with lenti-Ctrl or lenti-CYP46A1 measured using targeted LC-MS/MS (see Methods).

(C) IC50 curves for 24OHC in LN229 and GBM#P3 cells using the CCK-8 assay.

(D-E) Flow cytometry to detect annexin V-FITC and PI staining to determine the percentage of LN229 and GBM#P3 cells undergoing apoptosis after exposure to 0 - 20 μM 24OHC for 72 h. Data are shown as the mean ± SEM (n = 3). LN229: ***P = 0.0005, ***P < 0.0001, ***P < 0.0001; GBM#P3: *P = 0.035, ***P = 0.0006, ***P < 0.0001. Statistical significance was determined by one‐way ANOVA.

(F) Colony forming ability of GBM cell lines treated with 24OHC (0 - 20 μM) for 14 days. Data are shown as the mean ± SEM (n = 3). ***P < 0.0001. Statistical significance was determined by one‐way ANOVA.

(G) Western blot analysis of the apoptosis marker c-PARP, c-caspase 3 and PCNA in lysates (20 µg) from GBM cells treated with 24OHC (0 - 20 μM) for 72 h.

(H-I) Tumoursphere formation assays for GSCs treated with different concentrations of 24OHC (0 - 20 μM). Scale bar = 100 μm. Graphic representation of the quantification of tumoursphere formation. Data are shown as the mean ± SEM (n = 3). GBM#P3: *P = 0.0192, ***P = 0.0006, ***P = 0.0002; GBM#BG7: **P = 0.0017, ***P < 0.0001, ***P < 0.0001; GBM#BG5: **P = 0.0027, ***P = 0.0001, ***P < 0.0001. Statistical significance was determined by one‐way ANOVA.
Therapeutic implications of altered cholesterol homeostasis mediated by loss of CYP46A1 in human glioblastoma (2019)
Figure 4

Figure 4. 24OHC suppresses growth of GBM cells by reducing cholesterol content.

(A) Intracellular levels of cholesterol in GBM cells treated with 20 μM of 24OHC or DMSO for 72 h quantified using the Invitrogen™ Amplex™ Red Cholesterol Assay Kit and normalized to total protein. Shown are means and SEM (n = 3). LN229: **P = 0.002; GBM#P3: **P = 0.011. Statistical significance was determined by two-sided Student's t‐test.

(B) Representative images of filipin staining in GBM cells treated with different concentrations of 24OHC (0 - 20 μM) for 72 h. Scale bar = 30 μm.

(C) Growth curves for LN229 cells treated with DMSO, 0.5 μg/mL cholesterol or 10 μM 24OHC in the presence or absence of 0.5 μg/mL cholesterol using trypan blue staining. Data are shown as the mean ± SEM (n = 3). ***P < 0.0001. Statistical significance was determined by one‐way ANOVA.

(D) Flow cytometry to detect annexin V-FITC and PI staining to determine the percentage of GBM cells undergoing apoptosis after exposure to DMSO or 10 μM 24OHC in the presence or absence of 0.5 μg/mL cholesterol for 72 h. Data are shown as the mean ± SEM (n = 3). LN229: **P = 0.0021, **P = 0.0046; GBM#P3: **P = 0.0012, *P = 0.0193. Statistical significance was determined by one‐way ANOVA.

(E) Western blot analysis of c-PARP in GBM cells treated with DMSO or 10 μM 24OHC in the presence or absence of 0.5 μg/mL cholesterol for 72 h.

(F) Tumoursphere formation assays for GBM#P3 and GBM#BG7 treated with DMSO or 10 μM 24OHC in the presence or absence of 0.5 μg/mL cholesterol. Scale bar = 100 μm. Data are shown as the mean ± SEM (n = 3). GBM#P3: **P = 0.0032, *P = 0.031; GBM#BG7: ***P = 0.0008, *P = 0.0242. Statistical significance was determined by one‐way ANOVA.

(G) CCK-8 assay to determine relative cell survival in LN229 or GBM#P3 cells after treatment with vehicle control or different concentrations of MβCD for 72 h. Data are shown as the mean ± SEM (n = 3). LN229: **P = 0.0051, ***P = 0.0002; GBM#P3: **P = 0.0018, ***P < 0.0001. Statistical significance was determined by one‐way ANOVA.
Therapeutic implications of altered cholesterol homeostasis mediated by loss of CYP46A1 in human glioblastoma (2019)
Figure 5

Figure 5. The downstream mechanisms of 24OHC in GBM progression.

(A) Heatmap of differentially expressed transcripts in RNA-seq data from GBM#P3 cells treated with DMSO or 20 μM 24OHC for 72 h. Gene expression data was mean z-transformed for display, and colored red for high expression and blue for low expression.

(B-C) GO and KEGG enrichment analysis of differentially expressed genes based on RNA-seq data with adjusted-P value.

(D) GSEA plot showing normalized enrichment scores (NES) for cholesterol homeostasis, positive regulation of cholesterol efflux, SREBP targets and stem cell proliferation signatures using RNA-seq data from 24OHC and DMSO treated cells.

(E) Volcano plot highlighting up-regulated (red) and down-regulated (blue) genes based on RNA-seq data from GBM#P3 cells after 24OHC treatment. LXR targets, SREBP targets and stemness transcription factors are specifically included. **adjusted-P < 0.01, ***adjusted-P < 0.001.

(F) qRT-PCR analysis of 24OHC-regulated genes in GBM#P3 cells after treatment with 20 μM 24OHC for 72 h. GAPDH was used as an internal control. Data are shown as the mean ± SEM (n = 3). ***P < 0.0001, ***P < 0.0001, *P = 0.0192, ***P = 0.0004, *P = 0.0118, *P = 0.0101, ***P < 0.0001; Statistical significance was determined by two-sided Student's t‐test.

(G) Cell viability assay performed on GBM#P3 cells after 3 days of treatment with DMSO or 5 -10 μM LXR-623 in the presence or absence of 10 μM 24OHC. Data are shown as the mean ± SEM (n = 3). *P = 0.031, *P = 0.050. Statistical significance was determined by one‐way ANOVA.

(H) Western blot analysis of cholesterol metabolism-related proteins in LN229 or GBM#P3 after treatment with 24OHC (0 - 20 μM).

(I) Growth curves for LN229 cells transfected with control vector or SREBP1 plasmid and treated with 24OHC (0 - 40 μM) as assessed by a trypan blue test. Data are shown as the mean ± SEM (n = 3). **P = 0.0039; Statistical significance was determined by two-sided Student's t‐test.

(J) Colony forming ability of GBM cell lines treated with DMSO or 20 μM fatostatin for 14 days. Data are shown as the mean ± SEM (n = 3). ***P < 0.0001. Statistical significance was determined by two-sided Student's t‐test.

(K) Representative images of filipin staining in LN229 (up) or LN18 (down) cells treated with different concentration of fatostatin (0 - 20 μM). Scale bar = 30 μm.
Therapeutic implications of altered cholesterol homeostasis mediated by loss of CYP46A1 in human glioblastoma (2019)
Figure 6

Figure 6. Efavirenz, an activator of CYP46A1, attenuates the growth of GBM cells and prolongs survival of mice bearing intracranial tumours.

(A) The molecular structure of EFV.

(B) Intracellular concentrations of cholesterol in GBM cells after treatment with 20 μM EFV or DMSO and normalized to total protein. Data are shown as the mean ± SEM (n = 3). LN229: *P = 0.013; GBM#P3: **P = 0.0023. Statistical significance was determined by two-sided Student's t‐test.

(C) Filipin staining of GBM cells after treatment with EFV (0 - 20 μM). Scale bar = 30 μm.

(D) IC50 curves for EFV in GBM cells determined using OD readings (450nm) from the CCK-8 assay.

(E) 24OHC levels in spent media from LN229 (left) or GBM#P3 (right) cells treated with DMSO or 20 μM EFV for 72 h quantified using targeted LC-MS/MS. Data are shown as the mean ± SEM (n = 3).

(F) Images and quantification of colonies formed by GBM cell lines after treatment with different concentrations of EFV (0 - 20 μM). Data are shown as the mean ± SEM (n = 3). LN229: **P = 0.0056, ***P < 0.0001; LN18: ***P < 0.0001. Statistical significance was determined by one‐way ANOVA.

(G) Flow cytometry to detect annexin V-FITC and PI staining to determine the percentage of GBM cells undergoing apoptosis after treatment with EFV (0 - 20 μM) for 72 h. Data are shown as the mean ± SEM (n = 3). LN229: ***P = 0.0006, ***P < 0.0001; GBM#P3: ***P < 0.0001. Statistical significance was determined by one‐way ANOVA.

(H) Tumoursphere formation assays for GSCs treated with different concentrations of EFV (0 - 20 μM). Scale bar = 100 μm. Graphic representation of the quantification of tumoursphere formation (right). Data are shown as the mean ± SEM (n = 3). GBM#P3: **P = 0.0011, ***P = 0.0003, *** P < 0.0001; GBM#BG7: ***P < 0.0001; GBM#BG5: **P = 0.0037, **P = 0.0015, ***P = 0.0002. Statistical significance was determined by one‐way ANOVA.

(I) Western blot analysis of cholesterol homeostasis related proteins (LDLR, ABCA1, P-SREBP1 and N-SREBP1), c-PARP, PCNA and SOX2 in GBM cells treated with EFV (0 - 20 μM) for 72 h.

(J) Schematic diagram of the schedule for implantation and drug treatment in the GBM xenograft model. Seven days after implantation of tumour cells, mice were treated with EFV by gavage (0.1 mg/kg/day). Bioluminescent imaging (BLI) using IVIS was performed at days 4, 7, 14 and 21.

(K) The survival curves of tumour-bearing mice implanted with LN229 or GBM#P3 cells after EFV or PBS treatment (n = 5 per group). A log-rank test was used to assess statistical significance.

(L-M) Bioluminescent images and the corresponding quantification of tumours in mice implanted with GBM#P3 cells at days 7 (n = 5 per group, P = 0.58) and 21 (n = 5 per group, P = 0.005). Statistical significance was determined by two-sided Student's t‐test.

(N) Body weight of tumour-bearing mice after 3 weeks of EFV or PBS treatment. Data are shown as the mean ± SEM. Statistical significance was determined by two-sided Student's t‐test.
Nitrate triggered phosphoproteome changes and a PIN2 phosphosite modulating root system architecture (2021)
Figure 5

Figure 5. Nitrate regulates PIN2 phosphorylation levels.

(A) Schematic representation of the phosphosite (Ser439) identified in PIN2 in our study. PIN2 protein possesses three specific regions: a region with five transmembrane segments (residues 1-163), a central hydrophilic loop extending from residue 164 to 482 and a region with five additional transmembrane segments (residues 483-647). Protein alignment with PIN2 or PIN-like proteins from different species indicates conservation of the serine residue in eudicot, monocot, and gymnosperm species. The red arrow highlights the S439 in the central loop of PIN2 proteins in different plants. The size of each protein in amino acids and the percentage of identity against the Arabidopsis PIN2 are indicated.

(B) Levels of PIN2 phosphopeptide in our experiments. Bars represent the mean plus standard error of replicates (2 biological replicates for nitrate treatments at 20 min (Col-0 roots) and 3 biological replicates for all other experimental conditions). Each independent biological replicate consisted of a pool of 4.500 roots collected from Arabidopsis plants grown independently under the same experimental conditions. The asterisk indicates statistically significant differences in phosphoproteomic analysis (multiple t-test comparison without testing corrections, assuming same variance, p < 0.05).

(C) Detection of phosphorylation PIN2 by Phos-tag Western blotting. Arabidopsis plants (Col-0) were growth in ammonium as only nitrogen source, and treated with 5 mM KNO3 or 5mM KCl as control. Total protein from roots were analyzed in SDS PAGE using Phos-tag to detect changes in phosphorylation status. Immunoblotting was performed with PIN2 antibody. Total proteins isolated from eir1.1 roots were used as a negative control. White and red asterisks indicate a slow- or fast-mobility band corresponding to a more or less phosphorylated PIN2, respectively.

(D) Western blot against PIN2 protein comparing nitrate treated (KNO3) and control (KCl) condition in Arabidopsis roots for all genotypes (eir1-1 mutant background was complemented with PIN2::PIN2wt-GFP (PIN2wt), PIN2::PIN2S439D-GFP (PIN2S439D phospho-mimic point mutation) or PIN2::PIN2S439A-GFP (PIN2S439A, phospho-null point mutation).

(E) Time-course analysis of PIN2 mRNA levels in response to nitrate treatments in Arabidopsis roots. Bars represent the mean plus standard deviation of 3 biological replicates.
Nitrate triggered phosphoproteome changes and a PIN2 phosphosite modulating root system architecture (2021)
Figure 6

Figure 6. PIN2 is essential for nitrate regulation of primary root growth and lateral root density.

(A) Primary root length of Col-0 wild type plants or eir1-1 mutant plants was measured using the ImageJ program 3 days after 5 mM KNO3 or KCl treatments. Tukey box plot show results from 3 independent biological replicates per experimental condition (n = 10-15 roots each replicate). The box plot shows the data within the interquartile range (25th and 75th percentiles) and a solid black line represents the median. Whiskers show maximum and minimum values no further than 1.5x IQR (interquartile range). Outlier data are plotted individually. Asterisk indicates statistically significant difference between analyzed by unpaired, two-tailed and assuming equal variance t-test (** p < 0.01).

(B) Number of initiating and emerging lateral roots of Col-0 or eir1-1 mutant plants were counted using light microscopy 3 days after 5 mM KNO3 or KCl treatments. Tukey box plot show results from 3 independent biological replicates per experimental condition (n = 10-15 roots each replicate). The box plot shows the data within the interquartile range (25th and 75th percentiles) and a solid black line represents the median. Whiskers show maximum and minimum values no further than 1.5x IQR (interquartile range). Asterisk indicates statistically significant difference between analyzed by unpaired, two-tailed and assuming equal variance t-test (** p < 0.01).