EMBO/soda-nel huggingface dataset:
The RAS-related GTPase RHOB confers resistance to EGFR-tyrosine kinase inhibitors in non-small cell lung cancer via an AKT-dependent mechanism (2016)
Figure 5

Figure 5. AKT inhibition sensitizes RHOB-expressing cells to erlotinib.

A. HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing (AdRHOB) adenoviruses and treated for four hours with erlotinib (100 nM), G594 (100 nM), or a combination of both drugs. The phosphorylation status of GSK3β (Ser9), ERK1/2 and EGFR (Tyr1173) was assessed by western blotting and normalized according to the total protein levels. RHOB overexpression was also monitored by western blotting. B. HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing (AdRHOB) adenoviruses and treated for 72 h with erlotinib alone (black and red curves) or in combination with the AKT inhibitor G594 at 100 nM (green and blue curves). The surviving cell fraction was determined by an MTS assay. C. HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing adenoviruses (AdRHOB) and treated for 72 h with increasing concentrations of erlotinib in the absence or presence of increasing doses of G594. The surviving cell fraction was determined by an MTS assay, and erlotinib IC50 values were determined for each condition. (**: p<0.001 vs. AdCont cells; ***: p<0.0001 vs. AdCont cells). HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing (AdRHOB) adenoviruses and treated for 48 h with erlotinib (100 nM), G594 (100 nM), or a combination of both drugs. Apoptosis was then determined by either quantification of the subG1 cell population (D) or by detection of cleaved PARP and caspase-3 (E). (***: p<0.0001 vs. untreated cells; ###: p<0.0001 vs. G594 treated cells; †††: p<0.0001 vs. erlotinib-treated AdCont cells). In vitro data are representative of at least three independent experiments.
The RAS-related GTPase RHOB confers resistance to EGFR-tyrosine kinase inhibitors in non-small cell lung cancer via an AKT-dependent mechanism (2016)
Figure 6

Figure 6. The AKT inhibitor G594 resensitizes EGFRL858R mouse tumors to erlotinib.

A. Representative H&E staining of lung tumors from EGFRL858R/Rhob-/-, EGFRL858R/Rhob+/- and EGFRL858R/Rhob+/+ mice treated or not during four days with erlotinib (12.5 mg/kg/day), with the AKT inhibitor G594 (25 mg/kg/day), or in combination with both drugs. Scale bar: 500 µm. B. Quantification of the tumor/lung ratio of mice treated or not with the individual drugs or with a combination of both. C. Representative Ki67 immunostaining of lung tumors from EGFRL858R/Rhob-/-, EGFRL858R/Rhob+/- and EGFRL858R/Rhob+/+ mice treated or not with erlotinib alone (12.5 mg/kg/day) or in combination with the AKT inhibitor G594 (25 mg/kg/day) for four days (scale bar: 50 µm), and the corresponding quantification (D). (**: p<0.001; ***: p<0.0001). EGFRL858R/Rhob+/+ (placebo: n=6; G594: n=5; erlotinib: n=7; G594+erlotinib: n=8); EGFRL858R/Rhob+/- (placebo: n=7; G594: n=6; erlotinib: n=8; G594+erlotinib: n=7); EGFRL858R/Rhob-/- (placebo: n=6; G594: n=6; erlotinib: n=6; G594+erlotinib: n=6). Three independent zones/mice lung were used for Ki67 quantification.
ᵳ8;-secretase cleavage of the Alzheimer risk factor TREM2 is determined by its intrinsic structural dynamics (2020)
Figure 1

Figure 1 - Structural analysis of TREM2 transmembrane helix (TMH) in dodecyl-phosphocholine (DPC) micelles.

(a) Proteolytic processing­­ of TREM2. First, ADAM10 or 17 cleave off the ectodomain followed by sequential cleavage of the CTF by γ-secreta­­­se creating a p3-like extracellular peptide and the intracellular domain (ICD). Only an intact CTF can bind to DAP12. CTF: C-terminal fragment.

(b) 2D-[1H;15N]-TROSY spectrum of 400 µM 2H,13C,15N-labeled TREM2-TMH (residues R161-H206) in DPC micelles at 37 °C. Assigned resonances are labeled.

(c) Secondary chemical shifts plotted against the residue number indicates an α-helical region between residues 173 and 198. The used TREM2-TMH construct along with the predicted transmembrane helix content (aa 175-195) is also indicated.

(d) Circular dichroism (CD) spectrum of TREM2-TMH in DPC micelles indicates an α-helical secondary structure content of ~50%.

(e) 3D-15N-edited NOESY spectra show that the transmembrane helix is interrupted at residues 190-192. Dashed red lines indicate sequential amide NOE contacts that are characteristic for an α-helical secondary structure.

(f) Final NMR-derived structural bundle of the 20 lowest-energy structures of TREM2 shows that the C-terminal end of the transmembrane helix can adopt various positions relative to the well-defined TMH. r.m.s.d.: root mean squared deviation.
ᵳ8;-secretase cleavage of the Alzheimer risk factor TREM2 is determined by its intrinsic structural dynamics (2020)
Figure 2

Figure 2 - Interaction of TREM2-TMH with the signaling partner DAP12.

(a, b) Overlay of 2D-[1H,15N]-TROSY spectra of 2H,15N-labeled TREM2-TMH in DPC micelles (black) and in complex with (a) unlabeled DAP12 (red) or (b) TREM2-TMH lacking its positively charged lysine residue (K186A, red).

(c) Chemical shift perturbations (CSPs) between the two spectra in panels (a) or (b). The α-helical secondary structure as determined for wild-type TREM2-TMH is indicated above and the position of the K186A mutation is labeled in green.

(d) NOE-derived lowest-energy structure of TREM2-TMH in complex with disulfide-bridged dimeric DAP12 shows one continuous α-helix.

(e) The experimental structure of TREM2-TMH was docked to a previously determined structure of DAP12 in complex with NKG2C (Call et al., 2010). The positive charge in K186 is engaged in a salt bridge with D50 of DAP12. This charge complementarity together with hydrophobic interactions stabilize the complex and lead to a straight α-helical conformation in TREM2-TMH. D50 of the second DAP12 monomer is not in direct contact with TREM2, similar to the DAP12-NKG2C complex (Call et al., 2010).

(f) For TREM2-K186A-TMH a ~7% increase in α-helical secondary structure can be detected by CD spectroscopy.

(g) A continuous α-helical secondary structure in the kink region (aa 189-192) was probed by sequential amide NOE contacts in TREM2-K186A-TMH. Red dots indicate the chemical shift of intra-residual (i) amide resonance and red broken lines the inter-residual connectivity (i±1).

(h) Removal of the positive charge (K186A) leads to a very similar structure of the TREM2-TMH as in its complex with DAP12.

Data information: The C­­SP values color coded onto the NOE-derived structures in each case (e, h) show similar patterns (color coding according to the dotted lines in panel (c), i.e. lower line: mean CSP value, upper line: mean + standard deviation). The location of the K186A mutation is indicated by a green sphere. Spheres shown in panels (e and h) represent residues with CSP values above the mean value plus one standard deviation.
ᵳ8;-secretase cleavage of the Alzheimer risk factor TREM2 is determined by its intrinsic structural dynamics (2020)
Figure 3

Figure 3 - Dynamics of TREM2-TMH wild-type and the K186A variant.

(a,b) Generalized order parameter S2 for backbone amide resonances derived from NMR relaxation experiments with wild-type TREM2-TMH (a) and the K186A variant (b), indicative of motions in the nanosecond to picosecond time scale. S2 can adopt values between 1 (rigid) and 0 (unrestricted motion). Error bars have been obtained by a Monte-Carlo-simulation-based error estimation and motional model selection procedure implemented in the software Relax (Bieri et al., 2011) and published previously (Mandel et al, 1995).

(c,d) Molecular dynamics simulations of TREM2-TMH wt (c) and K186A (d) in a POPC/POPG (3:1) bilayer provide information on the stability of helix insertion into a lipid bilayer membrane. Since K186 is initially located in the hydrophobic membrane, its positively charged side chain is pulled towards the negatively charged phosphates in the lipid head group region leading to a pronounced tilt in helix orientation in the nanosecond time scale and on a µ-second to milli-second time scale to a kink in the C-terminal half of the α-helix, as probed by NMR line broadening. In contrast, in the K186A variant the transmembrane helix is stably inserted within the bilayer.
ᵳ8;-secretase cleavage of the Alzheimer risk factor TREM2 is determined by its intrinsic structural dynamics (2020)
Figure 4

Figure 4 - Membrane position of TREM2-TMH and the K186A variant.

(a) Paramagnetic relaxation enhancement (PRE) with a free-radical-modified stearic fatty acid in DPC micelles. Low values indicate location inside whereas values close to one suggest a location outside the detergent micelle. Orange bars: TREM2-TMH wt, green bars: K186A variant. The N-terminal residues of TREM2-TMH-wt are located deeper inside the micelles as in the K186A case, whereas the C-terminal end is positioned at the outside for wild-type and inside for the K186A variant. Negative bars indicate missing assignments.

(b) NOE-cross peaks between backbone amides of TREM2-TMH wt (top) or the K186A variant (bottom) and selected regions of DPC (see color coding of the structure of the DPC molecule on the right). The K186A variant shows strong contacts to the hydrophobic aliphatic regions of the detergent throughout the transmembrane helix whereas no such contacts can be seen at the kink site of the wild-type helix, indicating a location outside of the micelle for this part.

(c) Model of the position of TREM2-TMH wt and K186A within the membrane. The amino acids in the kink region are exposed to the solvent in the wild-type case but buried in the membrane for the K186A variant.
ᵳ8;-secretase cleavage of the Alzheimer risk factor TREM2 is determined by its intrinsic structural dynamics (2020)
Figure 5

Figure 5 - Identification of γ-secretase cleavage sites in TREM2 wild-type and selected K186 variants.

(a) Schematic overview of the in vitro γ-secretase assay to determine the ε-cleavage site(s) of TREM2.

(b) Immunoblot detection of ICD accumulation of TREM2 wildtype, K186A, K186P, and K186L variants mediated by γ-secretase cleavage upon overnight incubation of membrane preparations at 37 °C. ICD formation is blocked by 1 µM of the transition state analogue L-685,458. Note that inhibition of ICD formation from wt TREM2 was less efficient. Due to the charge within the TMH wt TREM2 is less efficiently transported to the cell surface (Lanier et al, 1998) and therefore retained within the endoplasmic reticulum, where other proteases not related to γ-secretase and therefore not inhabitable by L-685,458 may cleave. In vitro generation of the ICD of APP (AICD) was used as a positive control. Immunoblot detection of mature nicastrin and the presenilin 1 NTF, which results from presenilin endoproteolysis necessary to activate γ-secretase (Thinakaran et al, 1996), proves comparable amounts of γ-secretase in the individual assays.

(c) MALDI MS analysis of the ε-cleavage sites of wt and mutant TREM2 C-terminal fragments. Mass spectrometry reveals ε-cleavage C-terminal of A192 for the wt CTF. MS determination of the ε-cleavage site of TREM2 CTF K186A reveals major and minor ε-cleavage sites C-terminal of A195 and L193, respectively. The exact same cleavage pattern is observed for the K186L mutant. MS analysis of the cleavage pattern of the K186P mutant reveals major and minor ε-cleavage sites C-terminal of L193 and S191. Minor peaks that could not be assigned are indicated by an asterisk.

(d) Schematic representation of the ε-cleavage sites of CTF wildtype and selected K186 mutants. Residue 186 is indicated in bold. The location of helices is indicated by boxes.

(e) Table listing the calculated and observed masses of both wildtype and mutant ICDs as determined by MALDI MS. Note that molecular weights are increased by 58 Da due to alkylation of C213. As a positive control, we ran immunoblots confirming reduction of APP ICD levels upon γ-secretase inhibition (Appendix Fig. S2).
ᵳ8;-secretase cleavage of the Alzheimer risk factor TREM2 is determined by its intrinsic structural dynamics (2020)
Figure 6

Figure 6 - Cleavage specificity of TREM2-TMH by γ-secretase is governed by its local helix stability.

(a) Structural model of the complex of TREM2-TMH wild-type and γ-secretase obtained by docking guided by the cryo-EM structure of the γ-secretase-NOTCH complex (Yang et al., 2019). Protein regions in γ-secretase that occlude the substrate binding site have been removed for clarity.

(b, c) TREM2-TMH wild-type in the active site of γ-secretase in a register that would lead to cleavage between residues A192 and L193 (marked by spheres in red and purple), as detected by mass spectrometry (see Fig. 5). The K186 residue in TREM2 is also shown as well as the catalytically relevant residues in γ-secretase (D257 and D385). In TREM2-TMH K186A, the register of the helix moves upwards leading to positioning of A195 and A196 in the active site (c). The cleavage site needs to be unwound by γ-secretase, thus, the intrinsic stability of the helical substrate is a critical determinant of the cleavage position.

(d, e) Chemically induced unfolding of TREM2-TMH wild-type (d) and the K186A variant (e) demonstrates that the less dynamic variant is more stable than the wild-type protein.

(f) Water exchange peak as observed in a 3D-15N-edited-[1H,1H]-NOESY experiment which is only absent if the amide moiety is present in a stable hydrogen bond.

(g) Amino acid sequence comparison of TREM2-TMH wild-type and K186A with Notch1 and APP. α-helical secondary structure elements in the free form of each protein are labeled by grey boxes. Positively charged residues are marked by blue and polar residues close to the cleavage site (indicated by the comma mark) by yellow boxes. Red boxes indicate the unfolded sequence stretches in the bound state preceding the cleavage site. The secondary structure of the substrate helices (or derived from the structural model for TREM2-TMH) in complex with γ-secretase is shown above.

Expanded View Figure Legends
TNF stimulation primarily modulates transcriptional burst size of NF-κB-regulated genes (2021)
Figure 1

Figure 1. Basal mean and transcriptional noise of NF-κB targets varies systematically with chromatin environment at the promoter.

A NF-κB can recruit a variety of binding partners to target promoters, including the chromatin modifying enzyme p300, the elongation complex P-TEFb, and components of transcriptional machinery. NF-κB target genes with a variety of functions were chosen for this study.

B Induction of NF-κB targets in Jurkat T cells in response to 20 ng/ml TNF treatment for 1, 2, and 4 hours as measured by RT-qPCR. Target values were normalized to GAPDH and are reported as fold change relative to basal expression. Data are presented as mean ± standard deviation (SD) of 3 biological replicates.

C Enrichment of RelA before and 30 and 60 minutes after treatment with 20 ng/mL TNF as measured by ChIP-qPCR and shown as % input (non-IP control). Data are presented as mean ± SD of three biological replicates.

D Maximum intensity projections of smFISH fluorescence microscopy z-stacks of basal Jurkat T cells stained for the indicated genes. Nfkbia, Tnfaip3, Tnf, and Il6 were labeled with Quasar 670, and Il8 and Csf2 were labeled with fluorescein. All images were filtered as described in Materials and Methods. Brightness and contrast were enhanced for visualization. Scale bars: 10 μm.

E Histograms of transcripts per cell for target genes (blue) overlaid with probability density plots (red) generated from smFISH data. Cells were combined from three replicates (Nfkbia, Tnfaip3, Tnf, and Il6) or one replicate (Il8, Csf2).

F-G Bar graphs of mean (F) and CV (G) of smFISH distributions for the indicated genes. Error bars indicate bootstrapped 95% confidence intervals (CIs) for the samples in (E). Significant differences indicated by non-overlapping CIs.

H Ratio of enrichment of total histone H3 to acetylated H3 (AcH3) in Jurkat T cells at the indicated target promoters quantified by ChIP-qPCR. Data are presented as mean of % input (non-IP control) ± SD of three biological replicates.

I Graph of log10(mean) versus log10(CV2) of basal mRNA distributions measured in Jurkat T cells (black), HeLa cells (red) or murine bone marrow derived macrophages (green) for endogenous genes and four latent HIV LTR integrations. Gray shading indicates 95% CI of the linear regression for the basal trendline. Poisson trendline indicated by dashed line. HeLa data from Lee et al. (2014) and HIV LTR data from Wong et al. (2018).
TNF stimulation primarily modulates transcriptional burst size of NF-κB-regulated genes (2021)
Figure 2

Figure 2. TNF induces gene-specific changes in transcript distributions at NF-κB targets.

A Maximum intensity projections of smFISH fluorescence microscopy z-stacks of Jurkat T cells stained for the indicated genes after 1-hour (Nfkbia, Tnfaip3, Tnf, Il8) or 4-hour (Il6, Csf2) treatment with 20 ng/mL TNF. Nfkbia, Tnfaip3, Tnf, and Il6 were labeled with Quasar 670, and Il8 and Csf2 were labeled with fluorescein. All images were filtered as described in Materials and Methods. Brightness and contrast were enhanced for visualization. Scale bars: 10 μm.

B Bar graphs of mean of smFISH distributions before and after TNF treatment for the indicated genes. Cells were combined from three replicates (Nfkbia 1h; Tnfaip3 1h; Tnf 1h, 2h; and Il6 2h, 4h) two replicates (Nfkbia 2h) or one replicate (Tnfaip3 2h; Il8 1h, 2h; Csf2 2h, 4h). Basal data are same as in Fig. 1E. Error bars indicate bootstrapped 95% CIs for the samples in (C). Significant differences indicated by non-overlapping CIs.

C Probability density plots of single-cell mRNA distributions from smFISH as described in (B) before and after treatment with 20 ng/mL TNF for the indicated time points.

D-E Graph of log10(mean) versus log10(CV2) for endogenous gene targets that maintain CV2 (D) or decrease CV2 (E) after treatment with 20ng/mL TNF in Jurkat T cells (black), HeLa cells (red) or murine bone marrow derived macrophages (green). Gray shading indicates 95% CI of basal trendline. Poisson trendline indicated by dashed line. HeLa data from Lee et al. (2014).
TNF stimulation primarily modulates transcriptional burst size of NF-κB-regulated genes (2021)
Figure 3

Figure 3. Inferred fits from two-state promoter model shows that TNF treatment increases transcriptional burst size at most targets.

A Schematic of a two-state promoter model for transcriptional bursting. Burst frequency (ka) and burst size (b = kt/ki) were fit to combined transcript distributions measured by smFISH.

B-C Burst size (B) and burst frequency (C) parameter fits from the two-state model in the basal state and after treatment with 20 ng/mL TNF for 1, 2 or 4 hours. Error bars indicate bootstrapped 95% CIs. Significant differences indicated by non-overlapping CIs. The fit for Nfkbia (1h TNF) was unstable and thus is not reported (see Materials and Methods). Sample sizes are displayed in Figs. 1E and 2C.
TNF stimulation primarily modulates transcriptional burst size of NF-κB-regulated genes (2021)
Figure 4

Figure 4. RNAPII pausing is associated with increases in transcriptional bursting upon TNF treatment.

A Enrichment of histone H3, AcH3, total RNPII, ser5-p RNPII, ser2-p RNPII, and NELF-E in the basal state (0 hours) and after treatment (2 and 4 hours) with 20 ng/ml TNF quantified using ChIP and shown as % input (non-IP control). Data are presented as mean ± standard error of the mean (s.e.m.) of three biological replicates. Significance calculated by Dunnett's multiple comparison test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

B Hierarchical clustering of ChIP data before and after TNF treatment separates promoters with TNF-mediated increases in burst frequency or burst size. For each protein target, color bar indicates the % maximum ChIP value measured across all genes and time points.
TNF stimulation primarily modulates transcriptional burst size of NF-κB-regulated genes (2021)
Figure 5

Figure 5. Small molecule inhibitors of H3 acetylation and RNAPII pause release alter TNF-mediated changes in transcriptional bursting.

A Schematic of A-485 inhibition of the histone acetyl transferase p300/CBP, which is recruited by NF-κB, and of JQ1 inhibition of BET bromodomains, which recruit the positive transcription elongation factor (P-TEFb).

B Change in enrichment of histone H3 and AcH3 after treatment with 300 nM A-485 for 4 hours measured by ChIP-qPCR and shown as % input (non-IP control) normalized to the uninhibited control for each gene. Data are presented as mean ± SD for two or four biological replicates.

C Bar graphs of mean mRNA level for basal condition, 1h TNF, 1h TNF + 4h pretreatment with 300nM A-485, and 1h TNF+62.5nM JQ1 cotreatment measured by smFISH for Tnfaip3 (left) and Tnf (right). Cells were combined from two replicates (Tnfaip3 A-485, JQ1; Tnf A-485) or one replicate (Tnf JQ1). Error bars indicated bootstrapped 95% CIs. Samples with non-overlapping CIs are significant.

D Change in enrichment of total and Serine-5-phosphorylated RNAPII after 20ng/mL TNF treatment for 1 hour with 62.5nM JQ1 measured by ChIP-qPCR and shown as % input (non-IP control) normalized to the uninhibited control for each gene. Data are presented as mean for two biological replicates.

E-F Burst size (E) and burst frequency (F) parameter fits from the two-state model for the same conditions in (C) measured by smFISH for Tnfaip3 (left) and Tnf (right) for the data described in (C). Error bars indicated bootstrapped 95% CIs. Samples with non-overlapping CIs are significant.

G-H Probability density of mRNA distributions measured by smFISH for 20 ng/ml TNF for 1h (blue) versus 1h TNF + 4h pretreatment with 300nM A-485 (green) (G) and 1h TNF (blue) versus 1h TNF+62.5nM JQ1 cotreatment (yellow) (H) for Tnfaip3 (left) and Tnf (right).
Cancer-secreted miRNAs regulate amino-acid-induced mTORC1 signaling and fibroblast protein synthesis (2020)
Figure 4

Figure 4 - Cancer-derived EVs suppress protein synthesis through downregulating RAGC.

A. Western blot analysis of WI-38 and NIH3T3 cells treated with EVs or PBS for 48 h.

B. Western blot analysis of NIH3T3 cells transfected with indicated siRNA for 48 h.

C. Western blot analysis of NIH3T3 cells that had been transfected with indicated siRNA and labeled with Puro in the presence or absence of CHX.

D. Western blot analysis of NIH3T3 cells that had been transfected with a pRK5-RAGC-HA-GST expression plasmid or control vector and treated with EVs or PBS for 48 h, and then labeled with Puro.

E. Western blot analysis of CAF that had been treated with PBS or EVs from indicated cells for 48 h.
Cancer-secreted miRNAs regulate amino-acid-induced mTORC1 signaling and fibroblast protein synthesis (2020)
Figure 5

Figure 5 - miR-105 and miR-204 in cancer-secreted EVs target RRAGC.

A. Western blot analysis of CAF and NIH3T3 cells transfected with indicated anti-miRNAs or control for 24 h and then treated with EVs or PBS for 48 h.

B. Sequences of the predicted binding sites for miR-105 and miR-204 in the 3'UTR of human RRAGC.

C. Dual luciferase assay of the psiCHECK reporters containing the 3'UTR of RRAGC that was wild-type, with all three miR-105 sites mutated, or with the miR-204 site mutated. NIH3T3 cells were co-transfected with the reporter and indicated miRNA mimic (n=3 biological replicates for each group).

D. Dual luciferase assay of the indicated psiCHECK reporters in NIH3T3 cells treated with EVs or PBS for 48 h (n=3 biological replicates for each group).

Data information: In (C,D), data are presented as mean ± SD. **P<0.01 (one-way ANOVA).