EMBO/soda-nel huggingface dataset:
β-Catenin-RAS interaction serves as a molecular switch for RAS degradation via GSK3β (2018)
Figure 6

Figure 6. Interference of the β-catenin-RAS interaction suppresses the synergistic transformation and tumor growth induced by CTNNB1 and KRAS mutations via RAS degradation.

A, B Effects of KYA1797K and PTD-BBR on RAS degradation and cell growth. IB (A) and MTT (B) assays of MT-HCT116 or SW48 cells treated with indicated conditions for 24 and 96 hr, respectively. Data are presented as the mean ± SD (n = 3).

C-E Effects of overexpression of GFP-BBR-WT or -2E mutant on KYA1797K-induced growth and transformation of CRC cells. MT-HCT116 or SW48 cells were transfected with GFP-BBR-WT, -2E, or GFP-Mock for 24 hr and then treated with KYA1797K via indicated conditions. MTT (C) assays were performed for measurements of cell growth, and foci formation assays (D and E) were performed to detect cellular transformation. Data are presented as the mean ± SD (n = 3).

F Effects of cotreatments with KYA1797K and PTD-BBR on xenografted tumor growth. MT-HCT116 cells were subcutaneously injected in nude mice with subsequent i.p. injection of vehicle, 20 mg/kg of KYA1797K, or a cotreatment of PTD-BBR (25 mg/kg) and KYA1797K (20 mg/kg) for 20 d. Tumor volumes (upper panel) were measured every 4 days, and tumor weights (lower panel) were measured at the times of sacrifice. Data are presented as the mean ± SD (four mice per group).

G WCLs prepared from tumor tissues were immunoprecipitated with anti-β-catenin antibody. WCLs or co-IP samples were subjected to IB analyses.

H IHC analyses of tissue sections incubated with the indicated antibodies and then counterstained with DAPI. Scale bar, 20 µm.

Data information: Statistical significances were assessed by two-sided student's t tests, **P < 0.01 and ***P < 0.001.
TLR2 and TLR7 mediate distinct immunopathological and antiviral plasmacytoid dendritic cell responses to SARS-CoV-2 infection (2022)
Figure 1

Figure 1.

Infection with SARS-CoV-2 is associated with a decrease in plasmacytoid dendritic cells. PBMCs and plasma samples were collected from SARS-CoV-2 infected individuals at time of hospitalization (D1) and after 5 days (D5).

A-B: Percentage of pDCs (A) and mDCs (B) from total PBMCs collected at D1 was quantified by flow cytometry and grouped to self-reported duration of symptoms prior to D1 (A-B; 0-4 n= 22; 5-8 n=39; 9-12 n= 39; ≥13 n=16).

C-D: Percentage of pDCs (C) and mDCs (D) at D1 and D5 matched samples from all patients independent of symptom duration (n=93).

E-J: Matched D1 and D5 plasma samples were available from 55 donors and protein levels were determined for IFNα2a (E, n=30), IL-3 (F, n=38), IL-8 (G, n=44), IL-6 (H, n=51), TNFα (I, n=42) and CXCL10 (J, n=46), note that some samples could not be quantified and were excluded from the analysis.

K-L: Percentage of pDCs grouped by COVID-19 severity, as determined by oxygen supplementation (no oxygen n=43, nasal oxygen n=65 and high flow oxygen n=5). Percentage of pDCs (K) was correlated to COVID-19 disease severity (L) using simple linear regression.

Data information: Each dot represents a patient, lines with error bars show the median values with interquartile ranges (A-K) or mean values with the standard error of the mean (L). Statistical significance was determined using the Kruskal-Wallis test (A-B, K), Wilcoxon matched pairs signed rank test (C-J) and simple linear regression (L). *<p0.05, **<p0.01 ***<p0.001, ns= not significant.
TLR2 and TLR7 mediate distinct immunopathological and antiviral plasmacytoid dendritic cell responses to SARS-CoV-2 infection (2022)
Figure 2

Figure 2. Plasmacytoid DCs can sense SARS-CoV-2 and induce an inflammatory response. A-B: pDCs were either mock treated or exposed to the SARS-CoV-2 FR2020 early Wuhan-like strain or the SARS-CoV-2 alpha variant B.1.1.7 (0.1 MOI). Supernatants were collected at indicated time points and the production of type I IFNα (A) and CXCL10 (B) was quantified.

C- J: The FR2020 strain was used in subsequent experiments where pDC were either mock treated (mock, grey), exposed to SARS-CoV-2 at 1 MOI (SARS-2, purple), TLR7 (2.5 μg/mL R837, blue) or TLR3 agonist (800 ng/mL poly(I:C), pink). Supernatants were collected after 24 hrs and analyzed for type I IFNα (C), IFNβ (D), type II IFNγ (E), type III IFNλ1 (F), IL-6 (G), IL-8 (H), CXCL10 (I) and TNFα (J) expression by ELISA.

K-M: To evaluate the cytokine response to viral titers and exposure duration, pDCs were exposed to increasing viral inoculums (MOI of 0.01, 0.1 and 1) and IFNα2a mRNA expression was quantified at 24 hrs (K) and IFNα protein secretion at 24, 48, 72 and 96 hrs (L). Graph depicting simple linear regression of IFNα protein with time of exposure (M).

Data information: Bars (A-L) and line (M) represent mean values with standard error of the mean (M) and symbols represent individual pDC donors (n=3-4). Equal symbols represent equal donors (A-B and C-L). Statistical significance was determined using the ratio paired student T test and compared the treated condition with the time point-matched mock condition (A-L) and simple linear regression (M). *<p0.05, **<p0.01 ***<p0.001.
TLR2 and TLR7 mediate distinct immunopathological and antiviral plasmacytoid dendritic cell responses to SARS-CoV-2 infection (2022)
Figure 4

Figure 4. SARS-CoV-2 sensing and inflammatory cytokine induction by pDCs is mediated predominantly via MyD88.

A-B: Using CRISPR/Cas9, MyD88 knock-out (KO) and AAVS1KO (control) pDCs were generated. MyD88 protein levels in KO and control pDCs were analyzed by western blotting (A) and cellular DNA was sequenced to perform an Inference of CRISPR Edits (ICE) analysis (B).

C-D: MyD88KO and control pDCs were either mock treated (mock) or exposed to SARS-CoV-2 (SARS-2, 1 MOI), supernatants were collected at indicated time points and analyzed for type I IFNα (C) and CXCL10 (D). Done with n=4

E-F: Type I IFNα production was then determined in cell culture supernatant from SARS-CoV-2 exposed TRIFKO or TRIG+MyD88KO (E) and RIG-IKO or RIG-I+MyD88KO (F) pDCs. Done with n=2

Data information: Bars represent mean values and equal symbols represent equal donors. Statistical significance was determined using Mann-Whitney one-tailed T test in (C) as MyD88-KO data had no variance and values of 0. For (D) a Mann-Whitney two-tailed T test was used for comparison between conditions of mock and MyD88 KO donors.
Uncovering a conserved vulnerability site in SARS-CoV-2 by a human antibody (2021)
Figure 4

Fig 4. FD20 binds RBD at a conserved region previously predicted as an 'ideal' epitope with high functional and structural constraints.

(A) The overall structure of the scFv FD20 (cartoon) in complex with SARS-CoV-2 RBD (green cartoon in semi-transparent surface). VH and VL denote heavy and light chain variable domains, respectively. The VH CDRs and VL CDRs are colored as indicated. The RBD structure resembles a highchair that has a 'seat' region and a short 'backrest' region.

(B) Detailed interactions between residues from the RBD (green) and scFD20 involve VH CDR3 (magenta), VL CDR1 (orange), and VL CDR2 (cyan). FD20 residues are labeled black and RBD residues are labeled grey. A prime symbol indicates residues from VL CDRs.

(C) scFD20 (dark red) binds the RBD (white) at a site distal to that of ACE2 (light blue).

(D) Modest perturbation of scFD20 for RBD-ACE2 interaction. A sensor with RBD immobilized was soaked in 200 nM of scFD20 before being further soaked in scFD20-containing buffer with (red) or without (blue) 25 nM of ACE2 for BLI signal recording. As a control, the ACE2-RBD interaction was monitored in the absence of scFD20 (black).

(E) The FD20 epitope (red) is conserved between SARS-CoV-2, RaTG13, and SARS-CoV. Dots indicate identical residues and dashes indicate gaps. Brackets indicate sequence identity between the aligned RBD and SARS-CoV-2 RBD. NCBI accession codes for the sequences are as follows. RaTG13, QHR63300.2; SARS-CoV, NC_004718.3.

(F) The FD20 epitope (Cα in black sphere) is similarly organized in three-dimension among the coronaviruses that contain an RBD-equivalent region. The PDB accession codes are: SARS-CoV-2, 7CYV (this work); SARS-CoV, 2G75 (Prabakaran et al, 2006); MERS, 5X59 (Yuan et al, 2017); hCoV-HKU1, 5GNB (Ou et al, 2017); and hCoV-OC43, 6OHW (Tortorici et al, 2019).

(G) The predicted 'ideal' epitope (red + magenta) is mostly contained in the FD20 binding site (red + blue). The three CDRs (green for heavy chain and cyan for light chain) that are in close contact with RBD (white) are shown. A green arrow indicates Glu465 which is at the center of the predicted 'ideal' epitope (Starr et al, 2020).
Uncovering a conserved vulnerability site in SARS-CoV-2 by a human antibody (2021)
Figure 5

Fig 5. FD20 acts on Spike to inhibit cell-cell fusion and infection.

(A) Inhibition of cell-cell fusion by mAbs. Cell fusion was quantified by measuring the luciferase expression induced by full cell membrane fusion and mixing of cytoplasm, and the rate of inhibition by indicated antibodies compared to the condition without antibody is reported. Data depict mean ± s.d. (n = 4 biological replicates).

(B) Inhibition of a post-binding step by FD20, but not by other mAbs. Antibodies were added to VeroE6-hACE2 cells during (filled) or after (open), the binding step of SARS-CoV-2 pp. Mean ± s.d. are plotted (n = 3 biological replicates). As control, cells were incubated at each step with an equal volume of PBS or Y112R. p < 0.0001 (****) unless specified otherwise (2-tailed, unpaired student's t-test).

(C) FD20 pre-incubation with SARS-CoV-2 pp in the absence of cells strongly impairs entry. To test direct action on particles, SARS-CoV-2 pp and FD20 were co-incubated for 1 h, then the premixes were used to infect VeroE6-hACE2 cells for 6 h at 37 °C. Mean ± s.d. are plotted (n = 3 biological replicates). Numbers in brackets indicate the gained neutralization (%) by pre-incubation. As control, cells were incubated at each step with an equal volume of PBS or Y112R. Percentages of primary infection were calculated according to viral titers of PBS control conditions. p < 0.0001 (****) unless specified otherwise (2-tailed, unpaired student's t-test). ns, not significant.

(D) Inhibition of cell surface binding of SARS-CoV-2 VLP by FD20. VLP-GFP were preincubated, or not, with FD20 and RBM-targeting mAbs (CB6, REGN10933, REGN10987) for 30 min at 37 °C. Then the VLP premixes were incubated with VeroE6-hACE2 for 1 h at 37 °C and the binding detected by flow cytometry. Control binding assays were performed using soluble ACE2 as a competitor. Data are representatives from three experiments.

Source data for A-C are available online.
Increased levels of mitochondrial import factor Mia40 prevent the aggregation of polyQ proteins in the cytosol (2021)
Figure 2

Figure 2. Temperature-sensitive mia40-3 cells are hypersensitive to polyQ aggregates.

A. Mitochondria were visualized by mitochondrially targeted mCherry (red) in WT cells expressing Q97-GFP for 4 h. Fluorescence micrographs are maximum intensity projections of z stacks subjected to deconvolution. Bar, 5 µm.

B, C. The polyQ proteins were expressed for the indicated time periods. Mitochondria were isolated from wild type strains containing the Q25/97-GFP expressing plasmids and from cells lacking mitochondrial DNA (rho0). The ability of these mitochondria to respire (shown as NADH-induced oxygen consumption) or their activity of cytochrome c oxidase (shown as their capacity to oxidize reduced cytochrome c) was measured.

D. Cells of the temperature-sensitive mia40-3 mutant (Chacinska et al., 2004) or the corresponding wild type were transformed with plasmids expressing the indicated proteins from a low-expression GALL promoter (Mason et al., 2013) and grown on the respective carbon sources at permissive conditions (25°C).

E. Q97-GFP was expressed in mia40-3 and corresponding wild type cells for the times indicated. Cells were harvested, washed and survivors were counted after plating on glucose plates. Mean values and standard deviations of three replicates are shown.
Increased levels of mitochondrial import factor Mia40 prevent the aggregation of polyQ proteins in the cytosol (2021)
Figure 3

Figure 3. Overexpression of Mia40 suppresses polyQ toxicity in yeast.

A. Indicated strains were grown on glucose medium before tenfold serial dilutions were dropped on glucose- or galactose-containing plates.

B. Cell extracts were analyzed by Western blotting after shifting cultures to galactose for 16 h. 25, Q25-GFP; 97, Q97-GFP; ev, empty vector.

C. Microscopy images of the indicated strains 12 h after shifting them to galactose. Note that in GAL-Mia40 cells the form and number of aggregates is very different to WT cells. Bar, 5 µm.

D. The patterns of the Q97-GFP distribution were quantified after different time of expression, n=100. Cell boundaries are indicated by dashed lines.

E. PolyQ proteins were expressed for 24 h before survivors were counted. Mean values and standard deviations from three independent experiments are shown.

F. The GAL-Mia40 strain was much more resistant to Q97-GFP expression than WT cells, indicating that high Mia40 levels can suppress polyQ toxicity.
Increased levels of mitochondrial import factor Mia40 prevent the aggregation of polyQ proteins in the cytosol (2021)
Figure 4

Figure 4. Q97-GFP shows a sporadic aggregation behavior in GAL-Mia40 cells.

A. Cells were grown in microfluidic chambers (see Movies EV1-4). To better visualize the fluorescence signals different intensity settings were used, so that the intensities here are not comparable. Bars, 4 µm.

B. Still frames after different times of growth indicate a uniform fluorescence in Q25-GFP-expressing cells. Q97-GFP expression in wild type leads to many small and scattered aggregates per cell. In GAL-Mia40 cells, Q97-GFP accumulates to much larger intensities but then suddenly collapses into one single aggregate per cell. The arrow depicts aggregate formation. Bars, 4 µm.

C. Quantified signal intensities in cells of the indicated mutants. In top and middle graphs, each curve corresponds to a single cell. Averaged signals (mean + SEM) in bottom graph (N=20 for WT, N=17 cells for GAL-Mia40).

D. Still frames directly before and after the Q97-GFP aggregate formation in GAL-Mia40 cells. Total cellular fluorescence intensity before aggregation and total intensity in the aggregate (red arrow) were quantified and are shown in the graph on the right hand side, indicating that the entire cellular fluorescent signal collapses into the one single aggregate, irrespective of whether cells contain little, high or very high amounts of GFP signal (groups indicated by three red circles on the quantification). M, mother; D, daughter. Bars, 4 µm.

E. Cells were shifted from galactose to glucose to stop expression of Q97-GFP. The quantified image shows mean values and SEM from 20 cells with aggregates and 15 cells without aggregate, indicating that soluble Q97-GFP is degraded whereas the aggregated protein is of much higher stability. Bars, 4 µm.

F. Schematic inheritance patterns of polyQ proteins in wild type and GAL-Mia40 cells. Compare also Fig. EV2 for more details.
Increased levels of mitochondrial import factor Mia40 prevent the aggregation of polyQ proteins in the cytosol (2021)
Figure 5

Figure 5. Overexpression of Mia40 leads to increased proteostatic robustness of the cytosol.

A. Workflow of the quantification of soluble proteins by mass spectrometry.

B. Q97-GFP was expressed for 4.5 h in wild type and GAL-Mia40 cells in two biological replicates each. Cells were lysed with NP-40 before soluble proteins were quantified. Mitochondrial proteins (mito.) and members of the mitochondrial carrier family (carrier) are visualized in a volcano plot. P-values were derived from a moderated t-test. Mitochondrial (mito.) and carrier proteins are indicated by yellow and red dots, respectively. Rnq1 is indicated in blue.

C. Transcript levels for RNQ1 were measured by qPCR in the strains indicated.

D. Antibodies against Rnq1 were used in Western blots to specifically detect the protein in whole cell extracts of yeast cells.

E, F. Cell extracts of the strains indicated were separated into soluble (S) and aggregated (P) fractions. Rnq1 was detected by Western blotting. Whole cell extracts (total, T) were loaded for control. The wild type and GAL-Mia40(N) strain shown in F were newly generated.

G. Drop dilution experiments of the strains indicated on galactose plates; ev, empty vector.

H. Growth curves of the indicated strains at 30°C.

I. Cells of the newly made strains were grown on galactose for 1 or 7 days, respectively. Cells were lysed before the distribution of Rnq1 in the soluble and pellet fractions was quantified. Shown are mean values and standard deviations of three independent replicates.

J. Mia40 overexpression reduces the toxicity of polyQ proteins. In newly made cells, Q97-GFP is still toxic (though less than in wild type) but after several generations, cells escape sensitivity by loss of Rnq1 nucleation seeds.
Increased levels of mitochondrial import factor Mia40 prevent the aggregation of polyQ proteins in the cytosol (2021)
Figure 6

Fig. 6. Cytosolic polyQ aggregates interfere with efficient protein import into mitochondria.

A. Wild type and GAL-Mia40 cells, and cells expressing a cytosolic version of Mia40 under GAL1 control were dropped on the indicated plates; ev, empty vector. Note, that the mitochondrial version of Mia40 suppressed polyQ toxicity, whereas the cytosolic version did not.

B. In wild type or GAL-Mia40 cells, Q25-GFP or Q97-GFP was expressed for 4.5 h. For control, wild type cells were used which expressed also for 4.5 h the 'clogger' protein cytochrome b2-DHFR, a strong competitive inhibitor of protein import. Samples were analyzed by Western blotting. The presence of precursor proteins (labeled as p) in addition to the mature forms (labeled as m) is indicative of a strong import defect.

C. ∆ura3 cells expressing the Oxa1-Ura3 reporter for the cytosolic accumulation of mitochondrial precursor proteins (Hansen et al., 2018) were transformed with plasmids expressing the proteins TDP-43, the TDP-43Q331K mutant, Q25-GFP, Q97-GFP, α-synuclein, FUS or GFP from a constitutive glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter. Note that the expression of all aggregation-prone proteins (indicated by red arrows) allows cells to grow on uracil-deficient plates indicating the cytosolic accumulation of the Oxa1-Ura3 fusion protein.

D, E. Wild type cells were transformed with plasmids for the expression of the indicated proteins. Q97-GFPnF is a non-fluorescent version of Q97-GFP. Close proximity of the two split GFP parts results in fluorescence which was quantified in a spectrometer and visualized by microscopy. Shown are mean values and standard deviations of three replicates. Bar, 5 µm.
Increased levels of mitochondrial import factor Mia40 prevent the aggregation of polyQ proteins in the cytosol (2021)
Figure 8

Figure 8. Overexpression of MIA40 reduces the number of human SH-SY5Y cells with polyQ aggregates.

A. Cell extracts of HEK293 cells expressing a C-terminally Strep-tagged version of MIA40 (MIA40-Strep) or a mock control were analyzed by Western blotting with antibodies against several IMS proteins as well as HSP70 which served as loading control. The enrichment factors shown in green are based on quantitative mass spectrometry data (Habich et al., 2019). P-values were derived from a Student's t-test. TCE, trichloroethanol protein stain.

B. SH-SY5Y cells co-expressing Q97-GFP with MIA40 (middle row), ∆N-MIA40 (lower row) or a mock-transfected control (upper row) were analyzed by immunocytochemistry and super-resolution microscopy. The magnification shows an enlarged section of the TOM20 staining. TOM20 and TRAP1 serve as markers for the mitochondrial outer membrane and the matrix, respectively. Bars, 20 µm. Magnifications: 40fold of a 63x objective field.

C. The percentage of cells with Q97-GFP aggregates were quantified in control (mock transfected) and MIA40-expressing cells. Data are displayed as mean ± standard deviations and were analyzed by two-tailed Student's t-test, n = 6.; * P ≤ 0.05.

D. SH-SY5Y cells co-expressing Q97-GFP with MIA40 (upper row) or ∆N-MIA40 (lower row) were analyzed by immunocytochemistry and super-resolution microscopy. The magnification shows an enlarged section of the TRAP1 staining used as a marker for the mitochondrial matrix, respectively. Bars, 20 µm. Magnifications: 40fold of a 63x objective field.

E. Model for competition of mitochondrial precursor proteins and aggregation-prone proteins for factors of the quality control (QC) machinery, such as chaperones and components of the proteasome system. Our observations indicate that mitochondrial membrane proteins pose a particular threat to cytosolic proteostasis. Overexpression of the indicated components of the import machinery suppress the toxicity of polyQ proteins for which the prion protein Rnq1 plays a critical role.

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CPEB2-dependent translation of long 3'-UTR Ucp1 mRNA promotes thermogenesis in brown adipose tissue (2018)
Figure 1

Figure 1. Alternative polyadenylation of mouse but not human Ucp1 mRNA generates 3'-UTRs with different translation efficiency.

The 3'-UTR sequence of mUcp1 RNA. Canonical CPEs (UUUUA1-2U in bold), the reported BRF1-binding site (UAUUUAU, gray box) and predicted IMP2-binding motifs (AYAYA and YA2YA, underlined) are denoted. Ucp1L and Ucp1S carrying long and short 3'-UTRs, respectively, are resulted from alternative use of two polyadenylation signals (Hex: AAUAAA, open box). ORF, open reading frame. The arrowhead marks the end of Ucp1S sequence. Northern blots of Ucp1 using 5 µg BAT total RNA and the denoted radiolabeled probes.

Schematic comparison of mUcp1L and hUcp1. Poly(A) signal (Hex, open box) and CPE (black box) are denoted. The RT-PCR results from human, WT (Ucp1+/+) and UCP1-KO (Ucp1-/-) BAT RNA samples.

Dual luciferase reporter assay and RT-qPCR. The reporter plasmids, Fluc-mUcp1S, -mUcp1L or -hUcp1L 3'-UTR, and Rluc were co-transfected into differentiated HIB1B adipocytes. The transfected cells were used for luciferase activity assay and RT-qPCR. Data are mean ± SD from 3 independent experiments. Translation efficiency was defined as normalized activity (Fluc/Rluc) divided by normalized RNA level (Fluc/Rluc). ***p < 0.001 compared with Fluc-mUcp1S, one-way ANOVA.
CPEB2-dependent translation of long 3'-UTR Ucp1 mRNA promotes thermogenesis in brown adipose tissue (2018)
Figure 2

Figure 2. Reduced UCP1 protein level, BAT temperature and adrenergic signaling-induced thermogenesis in Ucp1ΔL mice.

Two Ucp1ΔL mouse lines (#30 and #64) with a denoted deletion in the exon 6 of mUcp1 gene.

Northern blotting of Ucp1 and 18S rRNA and RT-PCR of Ucp1L and Ucp1S using total RNAs from the denoted BAT samples (2 mice per genotype, 2-3 mo old). The short PCR fragments were isolated for sequencing.

Western blot analysis using WT and Ucp1∆L BAT lysates. The protein level of UCP1 was normalized to GAPDH (4 mo old, n = 4 per group). **p < 0.01, Student's t test.

BAT temperature of WT (n = 5) and Ucp1∆L (n = 6) male mice measured during 12-h light time. *p < 0.05, Student's t test.

BAT temperature of WT (n = 5) and Ucp1∆L (n = 6) male mice measured before and after i.p. injection of CL316243. *p < 0.05, repeated-measures two-way ANOVA.

Hematoxylin and eosin sections of BAT from 5-mo-old WT and Ucp1∆L male mice. Scale bars, 50 µm.

Dual luciferase reporter assay. The reporter plasmids, Fluc-mUcp1L 3'-UTR, and Rluc were co-transfected with the plasmid expressing EGFP, or myc-tagged CPEB (immunoblots shown at the left) into differentiated HIB1B adipocytes (Data from 3 independent experiments). *p < 0.05 compared with EGFP, one-way ANOVA.

Western blot analysis using BAT lysates prepared from various CPEB-WT and KO male littermates (4 mo old, n = 4 per group). *p < 0.05, Student's t test.

Data information: Data are mean ± SEM in (D, E) and mean ± SD in (C, G, H).