EMBO/soda-nel huggingface dataset:
TIP30 counteracts cardiac hypertrophy and failure by inhibiting translational elongation (2019)
Figure 5

Figure 5: A stable TIP30/eEF1A1 ratio is maintained in homeostasis and compensated growth conditions.

(A-J) Western blot analysis for TIP30, eEF1A1 and GAPDH in C57BL/6 WT mice 3 days (A, B), N=4 mice/group, 2 weeks (C, D), N=4 to 8 mice/group and 6 weeks (E, F), N=4 to 8 mice/group after TAC or sham surgery and their quantification. KO denotes TIP30 homozygous knock-out. (G, H) Western blot for TIP30, eEF1A1 and Actin in TIP30 Het mice 6 after TAC surgery and their quantification (N=4 mice/group). (I, J) Western blot for TIP30, eEF1A1 and Actin 2 weeks after sham or TAC surgery in AAV-Con or AAV-TIP30 treated C57BL/6 WT mice and their quantification (N=4 mice/group). A ratio of TIP30 and eEF1A1 expression was calculated for each condition. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. Two-sided Student's t -test.
TIP30 counteracts cardiac hypertrophy and failure by inhibiting translational elongation (2019)
Figure 6

Figure 6: The TIP30/eEF1A1 ratio decreases in human failing and hypertrophic cardiomyopathy hearts.

(A-C) Quantification of Tip30 and eEF1A1 mRNA transcript abundance in human failing hearts (A), N=6 to 8 hearts/group, in patients with Hypertrophic Cardiomyopathy (HCM; B, N=4 to 8 hearts/group) and 6 months old mdx mice or WT mice (C), N=4 mice/group. A ratio of Tip30 and eEF1A1 expression was calculated for each condition. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. Two-sided Student's t -test.

(D) Schematic representation of AAV-study design in mdx (MDX) mice.

(E) Echocardiographic diastolic left-ventricular wall-thickness in MDX mice at the age of 2 months at baseline, and 1-9 months (mo) after injection of AAV9-Con or AAV9-TIP30 (TIP; N=6 to 7 mice/group). *P<0.05, **P<0.01. One-way ANOVA with Sidak´s multiple comparisons test.

(F) Quantification of HW/TL ratio of 9mo old MDX mice, N=5 to 6 mice/group. *P<0.05. Two-sided Student's t -test.
TIP30 counteracts cardiac hypertrophy and failure by inhibiting translational elongation (2019)
Figure 7

Figure 7: TIP30 inhibits protein synthesis in cardiomyocytes.

(A) Western blot analysis of isolated neonatal rat cardiomyocytes (NRCM) after adenoviral transduction either with control adenovirus (Con) or Ad.TIP30 (TIP30) followed by stimulation with phenylephrine (PE, for 3 hours) and puromycin incorporation (for 30 min).

(B) Quantification of the Western blot shown in (A) (N=3 samples/group). *P<0.05, **P<0.01. One-way ANOVA with Sidak´s multiple comparisons test.

(C) Quantification of cell surface area of isolated neonatal mouse cardiomyocyte of Tip30 Het and WT mice treated with endothelin-1 (ET-1) and Narciclasine (Narci) or without stimulation as indicated (N=6 samples/group). *P<0.05, ***P<0.001. One-way ANOVA with Sidak´s multiple comparisons test.

(D) Schematic representation of Narciclasine study design.

(E-H) Quantification of HW/TL ratio (E), N=6 to 10 mice/group, echocardiographic wall thickness (F), N=6 to 10 mice/group, cardiomyocyte area (G), N=5 mice/group and fractional area change (FAC, H, N=6 to 10 mice/group) in Tip30 Het or WT mice 2 weeks after TAC. Animals were treated with Narciclasine daily for 14 days after TAC as indicated. *P<0.05, **P<0.01, ***P<0.001. One-way ANOVA with Sidak´s multiple comparisons test.

(I) Western blot analysis of puromycin incorporation in hearts of Tip30 Het and WT mice 3 days after TAC or sham surgery and daily Narciclasine (Narci) injection and their quantification (N=2 to 4 mice/group). Puromycin was injected 3 hours prior to sacrifice. *P<0.05, **P<0.01. One-way ANOVA with Sidak´s multiple comparisons test.

Expanded View Figure Legends
Partially inserted nascent chain unzips the lateral gate of the Sec translocon (2019)
Figure 1

Figure 1. Structure and dynamics of SecYEG translocon.

(A) Structure of quiescent SecYEG of Thermus thermophilus in the lipid cubic phase (PDB ID: 5AWW). TMHs 2b, 3, 7, and 8 of the lateral gate, as well as the proximate loop 6/7 involved in ribosome binding are indicated. The non-essential SecG subunit is omitted for clarity.

(B) Model of the SecY lateral gate opening upon inserting a nascent chain (red) in the lipid bilayer. The color-coding of SecYE TMHs is as in panel A. In the presence of the completely inserted and folded nascent chain, TMHs 2b and 3 of the N-terminal domain of SecY are displaced (arrows) thus opening a broad passage for the nascent TMH towards the lipid moiety.

(C) SDS-PAGE of SecYEG-ND sample after size-exclusion chromatography. Asterisks indicate translocon-enriched fractions used for forming the RNC FtsQ:SecYEG-ND complex. Lipid-loaded "empty" nanodiscs elute at larger volumes and so can be separated.

(D) Schematic drawing of a SecYEG-ND particle. Lateral dimensions of the nanodisc should be appropriate to accommodate a single SecYEG with surroundings lipids, thus mimicking the naturally occurring environment.
Partially inserted nascent chain unzips the lateral gate of the Sec translocon (2019)
Figure 2

Figure 2. Cryo-EM of the RNC FtsQ:SecYEG-ND complex.

(A) Representative cryo-EM micrograph of RNC FtsQ:SecYEG-ND. Exemplary individual ribosomes are encircled.

(B) Examples of two-dimensional classes of imaged particles. RNC:nanodisc assemblies can be seen at different view angles.

(C) Three-dimensional reconstruction of RNC FtsQ:SecYEG-ND complex. Primary structural elements of the ribosome and SecYEG-ND are indicated.

(D) Local resolution map of SecYEG-ND sub-particle. The cytoplasmic side of the translocon demonstrates higher resolution due to stabilization by the bound ribosome, while high-resolution at the periplasmic side is hindered by the SecYEG-ND dynamics within the complex. The associated ribosome is not shown for clarity.

(E) A planar slice through the SecYEG-ND core at different signal levels (blue/green/red) with indicated positions of SecYEG TMHs (SecY indicated in orange, SecE in purple, and SecG in green). A single helical turn could be fitted in a density in the area where SecG TMH 1 was expected (green asterisk).
Partially inserted nascent chain unzips the lateral gate of the Sec translocon (2019)
Figure 5

Figure 5. FtsQ nascent chain occupies the lateral gate of the translocon.

A, B Cryo-EM visualizes tRNA-bound nascent chain (red, NC) within the ribosomal tunnel (A) and a density between separated TMHs 2b and 7 of the lateral gate (B, shown in mesh). The proximate cysteine positions within SecY used for cross-linking are indicated and highlighted in red.

(C) Western-blot against the hemagglutinin tag within the nascent chain FtsQC40 reveals a cross-linking product of ~80 kDa in presence of nanodisc-reconstituted SecYC282EG and SecYC283EG, presumably assigned to FtsQxSecY adduct. The double band for FtsQC40 likely indicates the incomplete cleavage of the hexa-histidine tag by 3C protease.

(D) In-gel fluorescence imaging of SecYCF488A reveals a cross-linking product of ~80 kDa assigned to the FtsQC40xSecYC283 adduct ("SecYxFtsQ"). Bands for covalently cross-linked SecY dimer ("SecYxSecY"), and adducts of SecY and ribosomal proteins ("SecYxRiboP") are indicated. The nascent chain-specific adduct "SecYxFtsQ" does not depend on the presence of PE lipids.

(E) FtsQ:SecY cross-linking is position specific. FtsQ TMH C-terminal residues 40-42 could be cross-linked to the lateral gate (SecY residue 283), while no adduct could be detected at the upstream position 35.

(F) A refined scheme of the nascent chain insertion via the translocon. In the early intermediate state, the ribosome binding to the loop 6/7 and the emerging nascent chain cause displacements of SecY TMHs 2b and 7 at the cytoplasmic interface and "unzip" the lateral gate. The rigid-body tilt of TMH 7 leads to a close contact between TMHs 3 and 7 at the periplasmic side, so the V-shaped crevice is formed. The flexible nascent chain reaches the core of the lateral gate and gradually egresses the lipid moiety, where the helical fold is stabilized by hydrophobic interactions. Upon further nascent chain elongation and movements of SecY TMHs 2b, 3, 7 and 8, the widely open "inserting" state of the translocon is formed, with the complete nascent TMH at the lateral gate prior the release into the membrane.

Expanded View Figure Legends
Structural mechanism of CRL4-instructed STAT2 degradation via a novel cytomegaloviral DCAF receptor (2022)
Figure 1

Figure 1. Host protein regulation during RCMV-E infection and the role of E27

(A) Mass spectrometric analyses of protein abundance changes upon RCMV-E infection, compared to mock-infected samples. Host and virus proteins are indicated as grey and black dots, respectively. Host proteins involved in the type I IFN response and the STAT signalling pathway are highlighted in blue and red.

(B) Replication analysis of WT and ΔE27-RCMV-E in REF. Symbols indicate mean values (pooled data of two biological replicates each titrated in triplicates).

(C) Western blot analysis of STAT2 protein levels in mock-, WT-RCMV-E-, ΔE27-RCMV-E- or UV-inactivated RCMV-E-infected REF. 24 h p.i., cells were either left untreated or treated for 5.5 h with DMSO, 2.5 µM MLN4924 for blocking CRL activity or 10 µM MG132 for proteasome inhibition. IE1 and β-tubulin served as infection and loading controls, respectively.
Structural mechanism of CRL4-instructed STAT2 degradation via a novel cytomegaloviral DCAF receptor (2022)
Figure 2

Figure 2. In vitro reconstitution and XL-MS analysis of DDB1ΔBPB(/DDA1)/E27/STAT2 protein complexes

(A, B) GF analysis of in vitro reconstitutions containing the indicated protein combinations. Coomassie blue-stained SDS-PAGE analyses of fractions collected during the GF runs are shown next to the chromatograms. The red * indicates a contaminant, which we identified by cryo-EM analysis as E. coli ArnA, a notorious contaminant in E. coli Ni-NTA protein purifications (Andersen et al., 2013) (Appendix Fig S2, S3). Note that ArnA is present in GF fraction 6 (Ve ~ 10.2 ml) of all runs containing E27, and migrates similarly to E27 on the SDS-PAGE. * - additional contaminant from E27 preparation; ** - contaminant from STAT2 preparation.

(C) Schematic of proteins used for XL-MS analysis with domains annotated. Heteromeric cross-links obtained from XL-MS involving DDA1 are shown as light grey lines, otherwise as dark grey lines. ND - N-terminal domain, CCD - coiled-coil domain, DBD - DNA-binding domain, LD - linker domain, SH2 - Src homology 2, TAD - transactivation domain, BP - β-propeller, CTD - C-terminal domain.
Structural mechanism of CRL4-instructed STAT2 degradation via a novel cytomegaloviral DCAF receptor (2022)
Figure 4

Figure 4. E27 replaces endogenous CRL4 substrate receptors

(A) Comparison of the DDB1/E27-binding mode with endogenous and viral CRL4 substrate receptors. Structures of DDB1 (blue) in complex with E27 (green), DCAF1 (grey, PDB 6zue (Banchenko et al., 2021)), SV5 V (orange, PDB 2hye (Angers et al., 2006)), and HBx (wheat, AlphaFold2 prediction, Appendix Fig S6) are shown as cartoons. Regions of the AlphaFold2 HBx model with very low confidence have been removed. The insets display enlarged views of the boxed interaction areas, with DDB1 shown as semi-transparent blue surface. Zinc ions are shown as grey spheres. The position of the HBx zinc ion was modelled based on information from (Ramakrishnan et al., 2019).

(B) "Molecular footprint" of substrate receptors from A on the DDB1 surface. DDB1 is shown in the same representation as in A, but slightly rotated to allow an unobstructed view of the receptor-binding cleft. The receptors themselves have been removed for clarity. Parts of the DDB1 surface in contact with the respective substrate receptor are coloured according to the scheme in A.

(C) Detailed comparison of a helical DDB1-binding motif in E27 (green) and the DCAF1 "H-box" (grey). DDB1 is shown as semi-transparent blue surface, E27 and DCAF1 as semi-transparent cartoon. Selected amino acid residues involved in intermolecular contacts are shown as sticks.

(D) Amino acid sequence alignment of the "H-box" DDB1-binding motif in endogenous substrate receptors and viral factors hijacking DDB1. Black boxes: >80% type-conserved; dark grey: >60%; light grey: >40%. >60% type-conserved residues are marked by red dots, and underlined in C.

(E) Pre-formed DDB1/DCAF1 protein complex was incubated with indicated amounts of E27 and analysed by GF. SDS-PAGE analyses of GF fractions are shown next to the chromatogram. The red * indicates the ArnA contaminant (see Fig 2). * - additional contaminant from E27 preparation.
Structural mechanism of CRL4-instructed STAT2 degradation via a novel cytomegaloviral DCAF receptor (2022)
Figure 7

Figure 7. E27 structure reveals a conserved zinc-binding motif of functional importance in vivo

(A) Detailed view of the zinc-binding motif. E27 is shown as green cartoon, residues involved in zinc-binding as sticks. Zinc coordination bonds are indicated as dashed lines. Cryo-EM density, contoured at 6σ, is shown as grey mesh.

(B) 293T cells were transfected with expression plasmids encoding E27-HA or indicated variants. 24 h after transfection, cells were lysed and E27-HA was precipitated using an HA-specific antibody. Precipitates were separated by SDS-PAGE, blotted, and probed using the indicated antibodies.

(C) Amino acid sequence alignment of E27 and homologues from other CMV species. Residues involved in zinc coordination are marked by red dots.

(D) Superposition of the E27 zinc-binding site (green, structure from this work) with the one from M27 (grey, AlphaFold2 prediction, see Appendix Fig S8).

(E) 293T cells were transfected with expression plasmids encoding M27-FLAG or the indicated M27-FLAG variants. 24 h after transfection, cells were lysed and M27-FLAG was precipitated using a FLAG-specific antibody. Precipitates were separated by SDS-PAGE, blotted and probed using the indicated antibodies.

(F) Mouse fibroblasts were infected with MCMV or the indicated MCMV mutants. At the indicated time points, cells were lysed, proteins were separated by SDS-PAGE and blotted. Blots were probed using the indicated antibodies. pIE1 and GAPDH were used as infection and loading controls.

(G) NIH3T3 reporter cells stably expressing ISRE-Luc were infected with the indicated MCMV strains, treated with IFNβ or IFNλ, and luciferase activity was measured. The error bars represent the standard deviation, N=4 biological replicates, *** - P ≤ 0.001 (ANOVA).

(H) BALB/c mice were infected i.p. with WT-MCMV and indicated MCMV mutants. At 3 and 21 d p.i., salivary glands were collected and frozen. Virus titres were determined from organ homogenates by plaque titration. Titrations were performed in quadruplicates. Bars depict the geometric mean, dots show titres of individual mice (N=5 biological replicates). DL - detection limit.

Expanded View Figure legends
COMPREHENSIVE EVALUATION OF CODING REGION POINT MUTATIONS IN MICROSATELLITE UNSTABLE COLORECTAL CANCER (2018)
Figure 4

Figure 4. Molecular and cellular landscape of SMARCB1 wild type and R377C mutant.

Immunofluorescence microscopy analysis reveals highly similar and mostly nuclear localization of the SMARCB1 wild type and the R377C mutant, visualized by anti-HA staining (green). Phalloidin and DAPI staining was used to visualize the actin cytoskeleton and the nucleus, respectively. A novel MS-microscopy approach was used to further define the molecular context of the proteins. This analysis identified possible chromosomal, endosomal and membrane localization of the proteins. The possible endosomal localization is in agreement with the anti-HA immunofluorescence microscopy results. (key: the scale bar for immunofluorescence images is 10 µm, and the color gradient on the MS-microscopy indicates the localization scores calculated by the MS-microscopy tool.)

The physical (AP-MS, green) and functional (BioID, red) interactions of SMARCB1 wild type and the R377C mutant (key: lower right corner). The majority of the physical interactions remained highly similar with the R377C mutant, whereas, several functional interactions decreased. The interactions that decreased (<0.6 fold) with the R377C mutant are shown with green node color, and the interactions that increased (>2 fold) are shown in blue. The interaction map of the proteins is grouped based on participation in known protein complexes (CORUM) or on Gene Ontology Biological Processes of the proteins.

The cell proliferation assay in HCT116 CRC cells shows growth advantage conferred by the SMARCB1 R377C mutation compared with the wild type SMARCB1 or the vector control. The error bars designate the standard deviation (SD).
COMPREHENSIVE EVALUATION OF CODING REGION POINT MUTATIONS IN MICROSATELLITE UNSTABLE COLORECTAL CANCER (2018)
Figure 5

Figure 5. Molecular and cellular landscape of STK38L wild type and R105W mutant.

Immunofluorescence microscopy analysis of the STK38L wild type and the R105W mutant display no clear difference in localization. Both the wild type and the R105W mutant display nuclear, cytoplasmic and plasma membrane localization. However, the MS-microscopy analysis suggests a more prominent endosomal localization with the R105W mutant. (key: the scale bar for the immunofluorescence images is 10 µm, and the color gradient on the MS-microscopy indicates the localization scores calculated by the MS-Microscopy tool.)

The physical (AP-MS, green) and functional (BioID, red) interactions of the STK38L wild type and the R105W mutant (key: lower right corner). The R105W mutant displays increased interaction with 11 HCIPs, whereas five interactions decreased. The interactions that decreased (<0.6 fold) with the R105W mutant are indicated with green node color and the interactions that increased (>2 fold) are in blue.

The homology modelling of the STK38L shows a shift from the positively charged arginine to a non-polar and bulky tryptophan pointing inside towards the ATP-binding site, thereby possibly inhibiting the ATP-binding and kinase activity of STK38L.

The cell proliferation assay in HCT116 CRC cells shows growth advantage conferred by the expression of the wild type STK38L, whereas the R105W mutant only slightly increased the number of colonies. The error bars designate the standard deviation (SD).
Involvement of mitochondrial dynamics in the segregation of mitochondrial matrix proteins during stationary phase mitophagy (2013)
f1

(a) WT and dnm1Δ cells expressing Vph1-RFP and Idp1-GFP were grown in minimal synthetic dextrose medium and transferred to lactate medium at an initial density of OD600 0.08. The cells were then sampled daily and analysed by fluorescence microscopy (× 100 objective) during stationary phase mitophagy in lactate medium. Scale bar, 5 μm. (b) WT, dnm1Δ and dnm1Δ mgm1Δ mutants were grown in SL medium as described in Methods, and samples (10 OD600 units) were taken at each time point. Protein extracts were prepared and equal protein amounts (20 μg) were subjected to SDS–polyacrylamide gel electrophoresis and immunoblotting with anti-GFP antibody. (c) Quantification of the percent free GFP at the day 4 time point in each of the three strains analysed in b, using four independent experiments for each strain. Error bars indicate s.d. analysis of variance, P0.001; N=4.
Involvement of mitochondrial dynamics in the segregation of mitochondrial matrix proteins during stationary phase mitophagy (2013)
f4

A comparison of the SILAC-derived abundance as a function of time, for several proteins that either show differential behaviour between WT and the mutants (Idh2, Idp1 and Aco2) and proteins that behave similarly between all the genetic backgrounds tested (Hsp78). Shown are mean protein ratios (two replicates for mutants, three replicates for WT), error bars indicating peptide-based relative s.d.
Involvement of mitochondrial dynamics in the segregation of mitochondrial matrix proteins during stationary phase mitophagy (2013)
f5

WT (a), dnm1Δ (b) and atg1Δ cells (c) and atg32Δ cells (d) expressing chromosomally tagged GFP chimeras of Aco2, Idh2, Idp1 and Hsp78 were incubated in SL medium for 7 days, and 10 OD600 units were sampled at day 1 and day 7 and processed for immunoblotting with anti-GFP antibodies as described in the Methods section. Positive control used in c and d was from dnm1Δ cells expressing Idp1-GFP from a plasmid at 4-day incubation (Fig. 1b, lane 4). Release of free GFP in WT cells is reproducibly variable between these reporters, and varies between 0 (Idh2) and 60% (Aco2). (e) Densitometric quantification of the release of free GFP (as % of total signal) in four independent experiments, comparing the results between WT and dnm1Δ cells. Bars denote s.d. (n=4).
Involvement of mitochondrial dynamics in the segregation of mitochondrial matrix proteins during stationary phase mitophagy (2013)
f6

(a) Cells expressing chromosomally tagged GFP chimeras of Aco2, Idh2, Idp1 and Hsp78 as well as a plasmid-borne (pDJB12), mitochondrially targeted RFP (mtRFP) were incubated in SL-leucine medium for 3 days and imaged daily. Aco2 and Idp1 show clear mitophagic targeting (white arrows point at specific examples) by day 3, whereas Idh2 and Hsp78 show weak or no mitophagy. In addition, Idh2 and Hsp78 show different degrees of segregation relative to the mtRFP marker, whereas Idp1-GFP and Aco2-GFP show a near-complete co-localization with mtRFP. Scale bar, 5 μm. (b) Quantification of the frequency of mitophagy from a; the percentage of cells showing mitophagic profiles in the red (magenta bars; plasmid-borne mtRFP) or green (turquoise bars; integrated C-terminal GFP chimerae) was calculated for each GFP chimera. Idp-GFP, red N=76, green N=82; Aco2-GFP, red N=79, green N=82; Idh2-GFP, red N=72, green N=72; Hsp78-GFP, red N=70, green N=70.
Involvement of mitochondrial dynamics in the segregation of mitochondrial matrix proteins during stationary phase mitophagy (2013)
f7

WT and dnm1Δ cells expressing integrated Hsp78-GFP and plasmid-borne mtRFP were grown on lactate medium for 3 days. Cells were imaged daily and the images were analysed using ImageJ as described in Methods. Graphs illustrate the distribution of pixels associated with specific values of RFP and GFP channel intensities, respectively. Diagonal concentrations of dots indicate spatial correlation between the signals. The coloured photographic panels at the bottom illustrate the increased intensity correlation of the two channels at the 3-day time point in the dnm1Δ mutant, relative to the WT cells. Scale bar, 5 μm.