EMBO/soda-nel huggingface dataset:
Infection-driven activation of transglutaminase 2 boosts glucose uptake and hexosamine biosynthesis in epithelial cells (2020)
Figure 4

Figure 4. GFPT is a substrate of TG2 transamidase activity.

A - HeLa cells were infected with C. trachomatis (MOI = 1) and 40 µM CP4d was added or not 2 hpi. After 24 h 0.5 mM BP was added and cells were lysed at 48 hpi. Lysates were precipitated with streptavidin-coated beads. After separation with SDS-PAGE, proteins were transferred to a membrane and blotted with anti-GFPT antibody followed with HRP-conjugated secondary antibody.

B - In vitro assay testing the ability of purified TG2 to crosslink purified rhGFPT1 with BP. Samples were incubated for 3 h at 37°C before separation by SDS-PAGE. Proteins were transferred to a membrane and BP was revealed using HRP-conjugated streptavidin. rhGFPT1 is 77.5 kDa.

C -GFPT1 sequence: glutamine residues identified by mass spectrometry as cross-linked to BP are in bold letter .

D - In vitro assay was performed as described in B using wild type rhGFPT1 (WT), rhGFPT1 Q58N, rhGFPT1 Q328N or rhGFPT1 Q555N as substrates. The reaction was performed at 37 °C for 30 min. After probing with HRP-Streptavidin the membrane was washed and probed with anti-GFPT antibodies followed with HRP-conjugated secondary antibodies. The ratio of modified protein (streptavidin signal) to the total GFPT is shown, normalized to its value with WT rhGFPT1. The mean ± SD of five independent experiments is shown, the p-value of the Student's ratio-paired t-test is indicated when <0.05.

E - Lysates of cells treated or not for 6 h with ionomycin were incubated at 37 °C for 45 min with fructose-6-P and glutamine. The production of glucosamine-6-P was measured using HPAEC-PAD. Results of three independent experiments are shown, with mean ± SD, and p-value of the Student's paired t-test is indicated (*P < 0.05).
Infection-driven activation of transglutaminase 2 boosts glucose uptake and hexosamine biosynthesis in epithelial cells (2020)
Figure 5

Figure 5. TG2 activation results in increased UDP-GlcNAc production.

A - Schematic view of the hexosamine biosynthesis pathway. Production of glucosamine-6-P by GFPT is the first and rate-limiting step of the pathway that produces UDP-GlcNAc. HK: hexokinase; G6PI: glucose-6-P isomerase; GFPT: glutamine:fructose-6-P amidotransferase; GNA: glucosamine-6-P N-acetyltransferase; PGM3: phosphoglucomutase 3; UAP: UDP-N-acetylglucosamine pyrophosphorylase; OGT: O-GlcNAc transferase OGA: O-GlcNAcase; GlcNAc: N-acetylglucosamine.

B - Endocervical epithelial cells were pre-treated or not with 40 μM CP4d for 2 h before addition of the indicated concentration of ionomycin (or an equivalent volume of DMSO) and 0.5 µM BP. Six hours later whole cell lysates were analyzed by western blot. The membrane was first blotted with HRP-conjugated streptavidin to detect TG2 activity, then extensively washed and probed with anti-O-GlcNAcylation antibody followed with HRP-conjugated secondary antibodies. Last the membrane was probed with anti-actin as a loading control.

C - The same experimental procedure as described in (B) was applied to HeLa cells treated for 48 h prior to ionomycin treatment (8 µM) with siRNA control or directed against TG2.
Infection-driven activation of transglutaminase 2 boosts glucose uptake and hexosamine biosynthesis in epithelial cells (2020)
Figure 6

Figure 6. Optimal bacterial growth requires GFPT and prevents UDP-GlcNAc accumulation.

A - HeLa cells were infected or not (NI) with C. trachomatis (MOI = 1), then lysed 24 or 48 hpi. After separation with SDS-PAGE, proteins were transferred to a membrane, probed with anti-O-GlcNAcylation antibody followed with HRP-conjugated secondary antibodies. After extensive washes the membrane was blotted again with anti-GFPT and anti-actin antibodies before revelation with HRP-conjugated secondary antibodies.

B - HeLa cells were infected or not with C. trachomatis (MOI = 1). Twenty-four hours later, 8 µM ionomycin (or DMSO alone) and 0.5 mM BP were added. After 6 h of treatment, cells were lysed and proteins revealed as in (A).

C - HeLa cells treated for 72 h with siRNA targeting GFPT1 or GFPT2 were lysed. After separation with SDS-PAGE, proteins were transferred to a membrane, probed with anti-GFPT and anti-actin antibody before revelation with HRP-conjugated secondary antibodies.

D - HeLa cells were transfected with control siRNA or siRNAs against GFPT or TG2. Two days later the cells were infected for the indicated times (MOI=0.3) before fixation, rupture of the cells and measurement of bacterial diameter. The mean diameter ± SD and p-values of Student's paired t-test on 4 independent experiments are shown.

E - HeLa cells treated for 48 h with siRNA targeting GFPT1 or not (siCTRL) were infected with C. trachomatis (MOI = 0.15). Thirty hours later cells were fixed and analyzed by flow cytometry. The percentage of infected cells (top) and the mean fluorescence of the infected population (middle) ) ± SD are shown for at least four independent experiments. Duplicate wells were lysed and used to re-infect fresh HeLa cells to determine the bacterial titer (bottom). P-values of Student's ratio-paired t-test are indicated.

F - Schematic view of the outcome of TG2 activation in infection. The increase in TG2 expression and activity in cells infected with C. trachomatis results in the up-regulation of the expression of glucose transporters. Increasing quantities of glucose are thus imported in the host cytoplasm and redirected to the vacuole, where they fuel bacterial growth. Parallel to this transcriptional outcome, the transamidating activity of TG2 targets the host enzyme GFPT, thereby boosting the hexosamine biosynthesis pathway. The bacteria consume the resulting UDP-GlcNAc, or an intermediate along this pathway, in particular to sustain bacterial division.

Expanded View Figure Legends
Non-canonical proline-tyrosine interactions with multiple host proteins regulate Ebola virus infection (2021)
Figure 1

Figure 1. VP30 interacts with multiple host proteins that contain PPxPxY motifs.

(A) A model for RBBP6 and NP interaction with VP30 via PPxPxY motifs to regulate viral transcription. Both EBOV NP and human RBBP6 share a common PPxPxY motif for binding to EBOV transcription factor VP30. Interaction between NP and VP30 modulates viral RNA synthesis because RBBP6 outcompetes NP for VP30 binding.

(B) An EBOV VP30-human protein-protein interaction network. Purple and green prey nodes indicate that the protein was identified as a VP30 interactor in HEK293T or Huh7 cells, respectively; purple-green bifurcation indicates that the interaction was identified in both cell types. Grey lines correspond to human-human protein-protein interactions curated in the publicly available CORUM database. Host proteins containing PPxPxY motif(s) are circled in red.

(C) Co-immunoprecipitation between VP30 and the indicated PPxPxY containing host factors. Empty expression plasmid (Vector) or FLAG-tagged host protein expression plasmids were co-transfected with an HA-VP30 expression plasmid in HEK293T cells. An anti-FLAG immunoprecipitation (IP: Anti-FLAG) was performed. Western blots of IP and whole cell lysates (WCL) are shown. Anti-β-tubulin was used as a loading control for the WCLs.

(D) Cells were transfected with empty vector or HA-VP30 expression plasmid and IPs were performed with anti-HA antibody (IP: Anti-HA). Western blotting with anti-HA tag or antibodies to the indicated host proteins was performed on IP and on whole cell lysates (WCL).
Non-canonical proline-tyrosine interactions with multiple host proteins regulate Ebola virus infection (2021)
Figure 2

Figure 2. PPxPxY containing peptides derived from host proteins interact with VP30 and modulate EBOV RNA synthesis.

(A) Schematic representation of the arrangement and number of PPxPxY motifs present in the indicated host proteins (left panel). Multiple sequences aligned using ClustalW, of peptides containing PPxPxY motif derived from cellular proteins and from the EBOV NP protein (right panel).

(B) Co-immunoprecipitation between HA-VP30 and either GFP or GFP-fused to peptides derived from the host proteins was performed. Western blots of the co-immunoprecipitation and whole-cell lysate (WCL) are shown.

(C) Equilibrium dissociation curves of FITC-RBBP6 from eVP30130-272 in the presence of increasing concentrations (0.13-500 μM) of the PPxPxY containing peptides. Fluorescence polarization was determined with constant concentrations of FITC-RBBP6 and eVP30130-272, at 0.50 μM and 3.8 μM respectively. Experiments were performed in two independent replicates. Error bars represent standard deviation.
Non-canonical proline-tyrosine interactions with multiple host proteins regulate Ebola virus infection (2021)
Figure 3

Figure 3. HDX-MS analysis of VP30 and host factor peptide interactions.

(A) HDX kinetics data for VP30 alone and for the indicated host peptides incubated with VP30 are shown. VP30 peptide amino acid residue positions and sequences are indicated at the top of each curve. The error bars indicate mean ± S.D. (N=3).

(B) Deuterium uptake differences were mapped onto the VP30 structure (PDB 6E5X), revealing similar binding regions for the peptides. Residue-level uptake difference was achieved by overlapping peptides.
Non-canonical proline-tyrosine interactions with multiple host proteins regulate Ebola virus infection (2021)
Figure 4

Figure 4. PPxPxY containing host proteins target the NP binding cleft on VP30.

(A) Co-immunoprecipitation experiment demonstrating that NP interferes with VP30-PPxPxY binding. VP30 was co-expressed with the indicated HA-tagged PPxPxY proteins and either empty vector (-) or increasing amounts of FLAG-tagged NP expression plasmid. IPs were performed with anti-HA antibodies followed by Western blots of IPs or WCL by using anti-HA, anti-FLAG and anti-VP30 antibodies.

(B) Representative immunoblots of co-immunoprecipitations between hnRNP L and the indicated VP30 mutants which were previously described to exhibit varying degrees of NP binding activity.

(C) Co-immunoprecipitations between VP30 and the indicated host proteins upon titration of RBBP6. VP30 was co-expressed with FLAG-tagged PPxPxY containing host proteins and either a 5-fold or 10-fold excess of RBBP6 plasmid. IP was performed using anti-FLAG beads followed by Western blotting using the indicated antibodies.

(D) Densitometric analysis of the Western blots shown in panel C. The graph represents the relative amount of VP30 present in the eluates compared to the input. The data represent the mean ± S.D. from two independent experiments (n=2). Statistical significance was calculated using ANOVA with Tukey's multiple comparisons test. **p<0.005.
Non-canonical proline-tyrosine interactions with multiple host proteins regulate Ebola virus infection (2021)
Figure 5

Figure 5. Defining the contribution of PPxPxY residues to VP30 interaction.

(A) Co-immunoprecipitation between HA-VP30 and either GFP, or GFP-fused to WT or mutant RBBP6 peptides. Each proline or tyrosine residue in the P1xP2P3P4P5xY motif was mutated to alanine and the indicated mutant peptides were expressed as GFP fusions. Co-immunoprecipitations of HA-VP30 with GFP or GFP-peptide fusions were performed using anti-GFP magnetic beads (IP: Anti-GFP). Whole cell lysates (WCL) were blotted with anti-GFP, anti-HA and anti-β-tubulin antibodies.

(B) Equilibrium dissociation curves of FITC-RBBP6 from eVP30130-272 as it is outcompeted

by increasing concentrations (0.13-500 μM) of RBBP6 alanine mutants. Fluorescence polarization was determined with constant concentrations of FITC-RBBP6 and eVP30130-272 at 0.50 µM and 3.8 µM respectively. Experiments were performed in two independent replicates. Error bars represent the standard deviation.

(C) Co-immunoprecipitation between HA-VP30 and wildtype (WT) or mutant RBBP6 peptides fused to GFP. GFP alone served as a negative control. IP was performed for GFP and blots were probed with anti-GFP and anti-HA antibodies. Anti-β-tubulin served as a loading control for the whole cell lysates.

(D) Minigenome (MG) activity upon titration of plasmids encoding GFP fused to wildtype or single point mutant-RBBP6 peptides. GFP alone or GFP-RBBP6 peptide plasmids were transfected at 12.5 and 25 ng amounts along with the plasmids of the MG system. The data represent the mean ± S.D. from one representative experiment in which each transfection condition was performed in triplicate (n=3). The experiment was reproduced in two additional, independent experiments (see Appendix Figure S2). Statistical significance was calculated using ANOVA with Tukey's multiple comparisons test. ****p<0.00005; ***p<0.0005
Non-canonical proline-tyrosine interactions with multiple host proteins regulate Ebola virus infection (2021)
Figure 6

Figure 6. Functional interaction between VP30 and multiple host proteins that contain PPxPxY motifs.

(A) The effect of over-expression of the indicated host proteins on EBOV RNA synthesis was assessed using an EBOV MG assay. HEK293T cells were transfected with plasmids expressing components of the MG assay and either 12.5 or 125 ng of expression plasmids for the indicated host factors. Empty expression plasmid (vector) served as a control. Luciferase activity was measured 48h post-transfection and fold MG activity was calculated relative to a no VP30 control.

(B) MG activity upon transient expression of GFP or GFP-fused PPxPxY-containing peptides. Plasmids expressing GFP fused peptides were transfected at 12.5 and 125 ng amounts. Statistical significance was calculated relative to the GFP control for each concentration.

(C) MG activity upon knockdown of host genes. HEK293T cells were transfected with scrambled siRNA or siRNA targeting RBBP6, hnRNP L, hnRNPUL1 or PEG10. Twenty-four hours post-transfection, cells were transfected with MG assay plasmids including VP30 at 12.5 and 25ng doses.

(D) MG activity was assessed as in panel C upon single or double knockdown of RBBP6 and hnRNP L.

Data information: For each panel (A-D), the data represent the mean ± S.D. from one representative experiment in which each transfection condition was performed in triplicate (n=3). The experiment was reproduced in two additional, independent experiments (see Appendix Figures S3, S4, S8 and S9). Statistical significance was calculated using ANOVA with Tukey's multiple comparisons test. ****p<0.00005; ***p<0.0005, **p<0.005, *p<0.05.
Non-canonical proline-tyrosine interactions with multiple host proteins regulate Ebola virus infection (2021)
Figure 7

Figure 7. PPxPxY containing host proteins modulate EBOV infectivity, viral transcription and VP30 phosphorylation.

(A) HeLa cells were transfected with scrambled siRNA (SCR si) or siRNAs targeting hnRNP L, hnRNPUL1 or PEG10, 48 h post-transfection, cells were challenged with EBOV-EGFP at an MOI of 0.5. 24 h post-infection, cells were fixed, stained for nuclei with Hoechst dye and imaged. Scale bar is 200 µm.The graph (right) depicts relative number of infected cells as determined by calculating the ratio of number of infected cells to nuclei and shown relative to SCR siRNA. The data shows a representative result of two independent experiments with the mean ± S.D. calculated from three independently treated wells. Statistical significance was calculated relative to the values obtained in SCR siRNA-treated cells using ANOVA with Dunnett's multiple comparisons test. ***p<0.0001, ***p<0.001, **p<0.01

(B) Levels of transcription and replication in the MG assay upon over-expression of the indicated host factors. MG assays were performed with 50 and 500 ng of host protein expression plasmid. RNA was isolated and analyzed by qRT-PCR to measure the levels of viral mRNA/cRNA and vRNA. The sample with vector control was set to 100% and mean ± S.D. values from two independent experiments are shown (each experiment was performed in duplicate, n=2). **p<0.005, *p<0.05. Statistical significance was calculated relative to vector control using ANOVA with Tukey's multiple comparisons test.

(C) Immunoblots to assess VP30 and pVP30 levels in lysates from MG assay upon knockdown of RBBP6, hnRNP L or hnRNPUL1.

(D) Immunoblots to assess the levels of VP30, pVP30 and NP in EBOV infected lysates upon knockdown of NPC1 or RBBP6. HeLa cells were mock transfected or transfected with siRNA targeting NPC1 or RBBP6. 48 h post-transfection cells were infected with EBOV at MOI=0.1. At 24 h post-infection, cells were lysed in TRIzol. Protein was extracted from the TRIzol reagent and analyzed by Western blotting to determine levels of VP30, pSer29 VP30 and NP.

(E) Western blots detecting pVP30, VP30 and NP levels in EBOV infected HeLa cells upon hnRNP L, hnRNPUL1 or PEG10 knockdown.

(F) Proposed model for inhibition of EBOV replication by host factors containing PxPPPPxY motifs. Host proteins present at varying endogenous levels disrupt VP30-NP interaction thereby preventing VP30 dephosphorylation by NP-recruited host phosphatase PP2A-B56, and therefore inhibit virus transcription.

Expanded View figures
Active poly-GA vaccination prevents microglia activation and motor deficits in a C9orf72 mouse model (2019)
Figure 1

Figure 1. Vaccination of carrier coupled poly-GA peptides induces high-titer anti-GA antibodies. (A) Experimental paradigm for vaccination of GA-CFP mice and wild-type littermates. (B) Anti-GA response in vaccinated mice measured by ELISA using GST-(GA)15 antigen. Tukey-style box plot shows 25th, 50th and 75th percentile and whiskers extend to ±1.5 interquartile range. Outliers depicted as dots. Two-way repeated-measure ANOVA (group x time) revealed a significant main effect of treatment (F(5, 57) = 478.2, P <0.0001) and time (F(6,342) = 296.1, P <0.0001) and a significant interaction between factors (F(30,342) = 119.5, P <0.0001), followed by Tukey's post hoc test. p values are indicated in Table S1. (C) Immunohistochemistry of sections from a C9orf72 patient and a healthy control. Monoclonal anti-GA and antiserum from Ova-GA vaccinated mice detect neuronal cytoplasmic inclusions (arrows) in the molecular layer of the cerebellum. Note that crude antiserum shows higher background than the monoclonal antibody. Scale bar indicates 20 μm. (D) Quantitative analysis for GA-positive inclusions per 100 neurons in (C). One-way ANOVA, Tukey's post hoc test, F(3,15)=2.676, p=0.0846, C9: Anti-GA 1A12 vs C9: TG-Ova-(GA)10 serum p=0.9483. (E) Immunohistochemistry of sections from occipital cortex of a C9orf72 patient and a healthy control with monoclonal anti-GA and antiserum preincubated with 0.1 mg/ml recombinant GST-(GA)15 or GST. Scale bar indicates 20 μm.
Active poly-GA vaccination prevents microglia activation and motor deficits in a C9orf72 mouse model (2019)
Figure 2

Figure 2. Ova-GA immunization prevents motor deficits and reduces poly-GA aggregation. (A) Analysis of motor function in vaccinated GA-CFP mice and wild-type littermates in a beam walk assay. Average time to cross the beam from duplicate repeat measurements in consecutive weeks. Box plots as in Fig 1B. Pairwise Wilcoxon rank sum test with Benjamini-Hochberg correction. All comparisons with WT-PBS and the TG-Ova-(GA)10 mice vs. TG-PBS comparison are depicted with * p<0.05, **, p<0.01. Number of mice per group as in Fig. 1A, exact p-values are indicated in Table S2. (B) Average number of drop-downs per mouse from all aggregated runs. Kruskal-Wallis test with Dunn's multiple comparisons. * p<0.05. TG-Ova-(GA)10 vs. TG-PBS p=0.0188. (C) Representative image of poly-GA aggregates in the anterior horn in spinal cord using immunohistochemistry. (D) Quantitative analysis of poly-GA aggregates in the spinal cord using immunohistochemistry. Images from complete spinal cord sections at 1 mm intervals. One-way ANOVA, Tukey's post hoc test, F(5,12)=1.625, p=0.2270. *** p<0.001, ns not significant. TG-Ova-(GA)10 vs. TG-PBS p<0.0001; TG-(GA)15 vs. TG-PBS p=0.2460. Scale bar indicates 100 μm. (E) Immunoassay of poly-GA from the insoluble fraction. One-way ANOVA, Tukey's post hoc test, F(5,24)=3.098, p=0.0269. ** p<0.001, ns not significant. TG-Ova-(GA)10 vs. TG-PBS p<0.0001; TG-(GA)15 vs. TG-PBS p=0.1975.
Active poly-GA vaccination prevents microglia activation and motor deficits in a C9orf72 mouse model (2019)
Figure 3

Figure 3. Ova-GA immunization prevents microglia/macrophage activation, TDP-43 mislocalization and neuroaxonal damage. (A) Gene ontology analysis of differentially expressed genes in TG-PBS mice comparing genes significantly rescued by Ova-(GA)10 immunization and non-rescued genes (absolute log2 fold-change > 0.585, compare Dataset EV1-EV4). The dot size and color represent the fraction of the differentially expressed genes in each category and adjusted p-values, respectively. Mouse number as indicated in Fig. 3F. (B) Network of the genes dysregulated in TG-PBS and significantly rescued in TG-Ova-(GA)10. (C, D) Analysis of microglia/macrophage activation using Iba1 immunohistochemistry from complete spinal cord sections at 1 mm interval. One-way ANOVA, Tukey's post hoc test. ** p<0.01, * p<0.05, ns not significant. Scale bar indicates 100 μm. F(5,12)=0.6974, p=0.6357; TG-Ova-(GA)10 vs. TG-PBS p= 0.0131; TG-(GA)15 vs. TG-PBS p=0.7650. TG-PBS vs. WT-PBS p=0.0018. (E) The percentage of cells with partial cytoplasmic mislocalization of TDP-43 (compare EV5E) was quantified from six images from spinal cord sections at 1 mm intervals were analyzed. One-way ANOVA, Tukey's post hoc test. * p<0.05, ns not significant. F(5,12)=0.6533, p=0.6650; TG-Ova-(GA)10 vs. TG-PBS p= 0.0096; TG-(GA)15 vs. TG-PBS p=0.4215. (F) Immunoassay of NFL level in cerebrospinal fluid. One-way ANOVA, Tukey's post hoc test, F(3,11)=1.911, p=0.1862. ** p<0.01. TG-Ova-(GA)10 vs. TG-PBS p=0.0081.