EMBO/soda-nel huggingface dataset:
SGTA associates with nascent membrane protein precursors (2020)
Figure 6

Figure 6. SGTA binding is reduced in the presence of ER-derived membranes

A, B PPL-C99FL lacking a stop codon was in vitro translated in RRL in the presence of canine pancreatic microsomes or buffer control in reactions supplemented with ε-TDBA-Lys-tRNA analogue. Reactions were carried out in the presence (A) or absence (B) of 2 μM HisTrx-SGTA. Samples were subjected to UV light-induced photo-cross-linking and processed as described for Fig 5. "x SRP54" and "x HisTrx-SGTA" indicate cross-linking adducts between the translated nascent chain and endogenous SRP54 or recombinant HisTrx-SGTA, respectively, whilst "endo. SGTA" indicates cross-linking adducts with endogenous SGTA. Cross-linking products between stalled PPL-C99FL and SGTA are also indicated with arrowheads, whilst arrows indicate unmodified translation products.

C, D As for panel (A) but chemical cross-linking using DSS reagent was carried out.

E DSS-mediated HisTrx-SGTA cross-linking efficiency to PPL-C99FL in the absence or presence of ER-derived microsomes was quantified. For each repeat, intensity of the cross-linked SGTA adduct in the absence of microsomes was set as 100%. Shown is the mean with standard error of mean for n=3 biological replicates. Statistical significance was calculated using unpaired t test with Welch's correction.
SGTA associates with nascent membrane protein precursors (2020)
Figure 7

Figure 7. SGTA reduces the co-translational ubiquitination of nascent membrane proteins

A Indicated precursor proteins were translated in vitro using RRL supplemented with 20 μM HA-tagged ubiquitin and 2 μM of either HisTrx or HisTrx-SGTA. RNCs were isolated, ubiquitinated nascent chains recovered using anti-HA agarose, samples resolved by SDS-PAGE and analysed by phosphorimaging. Ubiquitinated precursor protein species were assigned based on SDS-PAGE electrophoretic mobility and are indicated in red. A schematic diagram of the proteins used is shown and the ~40 residues of the nascent chain located in the ribosomal exit tunnel are indicated. "ss" - signal sequence, "TMD" - transmembrane domain.

B Individual, clearly resolved ubiquitinated species of the indicated proteins (labelled 1 to 4) were quantified and their intensity in HisTrx-SGTA containing samples shown relative to samples supplemented with HisTrx control protein. Shown is the mean with standard error of mean for n=3 biological replicates. Statistical significance was calculated using unpaired t test with Welch's correction. ns - not significant.

C A schematic model for SGTA action during membrane protein biogenesis. A signal sequence or the first TMD of a nascent chain is bound by SRP as it leaves the ribosomal exit tunnel. If the elongation arrest is inefficient or the distance between the first and second hydrophobic signals short, SGTA is recruited to the ribosome by a downstream signal located inside the ribosomal exit tunnel (route i). As translation continues SGTA binds directly to the exposed hydrophobic signal and can reduce co-translational ubiquitination of the nascent chain. Upon arrival to the ER membrane both SRP and SGTA dissociate from the nascent chain, which is inserted into the ER lipid bilayer. In this model inefficient recruitment of SGTA (route ii) might result in increased co-translational ubiquitination of nascent chains and possibly their proteasomal degradation.

EXPANDED VIEW FIGURE LEGENDS
Compromised DNA repair is responsible for diabetes-associated fibrosis (2020)
Figure 1

Figure 1. Increased pO2, Mitochondrial Respiration, and Oxidative Stress lead to the Activation of Immediate DNA Damage Response after Parturition of Normal Live Pups

(A) Schematic representation of the oxygenic state of pre- and post-natal intrauterine life of mice pups.

(B) Quantitative PCR analysis of post-natal lungs for mitochondrial DNA (mtDNA). Actin was used as a normalization control. (mean ± SD, : p<0.05;: p<0.01; N=3).

(C) Quantitative analysis of oxidative stress in post-natal pups of indicated age (in days) using the peroxide linked oxidative stress marker DHR-123, as described in methods (mean ± SD, : p<0.01; N=8).

(D) Schematic representation of DNA damage (oxidative or DSBs) on naive DNA.

(E) Immunofluorescence analysis of oxidative DNA damage (left panel) and the DNA double-strand breaks sensor kinase marker pATM (right panel) in post-natal murine lungs. Here anti-8-oxo-7, 8-dihydroguanine was used for the detection of oxidative DNA modification (8-oxoG, left panel), and anti-pATM Serine 1981 was used for the analysis of the activation of the DDR (pATM, right panel). Green cytoplasmic staining represents α-Tubulin; blue DAPI staining represents nucleus (scale 10μm). No primary antibody control served as a negative control (P14) of the staining.

(F) Mean percentage of nuclei positive for oxidative DNA damage marker 8-oxoG in post-natal murine lungs, as determined by immunofluorescence analysis (mean ± SEM, : p<0.01; : p<0.001; N=8).

(G) Mean percentage of nuclei positive for DNA damage marker pATM in post-natal murine lungs, as determined by immunofluorescence analysis (mean ± SEM, : p<0.01; N=8).

(H) Quantitative analysis of the NAD+/NADH ratio in post-natal lungs after homogenization in extraction buffer (mean ± SD, : p<0.01; : p<0.001).

Data information: Statistical significance was assessed by unpaired two-sided student's t-test. As stated, data represented as mean ± SD or SEM, : p<0.05;: p<0.01; : p<0.001.
Compromised DNA repair is responsible for diabetes-associated fibrosis (2020)
Figure 2

Figure 2. Prolonged Exposure of High Concentration of Reducing Sugars Affect the Kinetics of DNA-DSBs Repair

(A) Immunofluorescence analysis of DSBs associated foci as marked by γH2AX in lung adenocarcinoma (A549) cells, cultured either in low glucose (5.5mM, left panel) or high glucose (30mM, right panel) for 5 days, then treated with etoposide (5µM for 60 minutes). The resolution of DNA-DSBs foci, as marked by γH2AX was monitored over 24 hours after drug treatment, CML (marked in green) served as induction control (scale 10µm).

(B) Mean percentage of DSBs positive nuclei as evidenced by γH2AX positivity after pre-treatment of different reducing carbohydrates at 0, 3 or 24 hours. More than 400 cells were analyzed for each bar (mean ± SD, : p<0.01;: p<0.001).

(C) Still images of A549 cells showing the live recruitment of hPARP-mCherry and hXRCC4-GFP, at the site of laser-induced DNA-DSBs from the cells pre-treated with the indicated reducing sugars as described in methods.

(D) Quantitative analysis of the efficiency of NHEJ-repair in a reporter cell line, as described in Methods. Cells were cultured in low glucose (5.5mM) or high glucose (30mM) for 5 days, fructose (30mM), ribose (20mM), for 3 days (graph bar 4). I-SceI, untransfected cells served as a negative control. Shown is the average from three independent experiments (mean ± SEM, : p<0.01 : p<0.001).

(E) Quantitative analysis of the NAD+/NADH ratio in A549 cells, cultured in either low (5.5mM, LG), or high glucose (30mM, HG) for 5 days, fructose (30mM, FR), ribose (20mM, RI), for 3 days (graph bar 4). Quantification was performed after homogenization in extraction buffer (Data represent mean ± SD, : p<0.01;: p<0.001; N=3).

(F) A549 cells were cultured in either low (5.5mM; lane 1) or high glucose (30mM; lane 2) for 5 days, fructose (30mM; lane 3), ribose (20mM; lane 4), for 3 days. The cell extracts were then for immunoprecipitation by using anti-PARP, or a non-specific species control antibody. The PARP, or its interacting partner DBC1 was then detected using PARP, or a DBC1 specific antibody.

Data information: Statistical significance was assessed by unpaired two-sided student's t-test. As stated, data represented as mean ± SD or SEM, : p<0.01; : p<0.001.
Compromised DNA repair is responsible for diabetes-associated fibrosis (2020)
Figure 3

Figure 3. Diabetes-Associated Persistent DNA Damage signaling is Associated with Cellular Senescence and SASP

(A) Representative images of lungs for nuclei positive for the DNA-DSBs marker γH2AX, versus DAPI, in age-matched 3-, or 6- months control, versus STZ induced diabetic mice, as determined by immunofluorescence analysis (scale 10μm).

(B) Mean percentage of nuclei positive for the DNA-DSBs marker γH2AX in lungs of age-matched control versus 6-month STZ induced diabetic mice, as determined by immunofluorescence analysis of lungs (mean ± SEM, :p<0.05;: p<0.01; : p<0.001; N=6).

(C) Representative images of lungs from age-matched control versus 3 or 6-month STZ mice, stained for the peroxide linked oxidative stress marker DHR-123(in red), as determined by mean fluorescence intensity. Blue nuclear staining represents DAPI (Scale 10µm).

(D) Quantitative analysis of respectively age-matched control versus 3 or 6-month STZ induced diabetic lungs for persistent DNA damage signaling associated inflammatory marker IL-6, as determined by mean fluorescence intensity of respective group lungs (mean ± SD; : p<0.01: p<0.001; N=6).

(E) Representative images of lungs from age-matched controls, versus 3 or 6-month STZ induced diabetic mice. Sections stained for cellular senescence-associated β-galactosidase [β-Gal] as described in Methods and visualized by bright field and polarized light, the senescent areas are recognized by its bluish-green staining (Scale 40µm).

Data information: Statistical significance was assessed by unpaired two-sided student's t-test. As stated, data represented as mean ± SD or SEM, : p<0.05;: p<0.01; : p<0.001.
Phospho-regulated Bim1/EB1 interactions trigger Dam1c ring assembly at the budding yeast outer kinetochore (2021)
Figure 1

Figure 1 Bim1 binds to Dam1c to form a stable complex in solution
A: Chromatogram and SDS-PAGE analysis of elution fractions from analytical size exclusion chromatography. Dam1c and Bim1 individually or in combination were subjected to gel filtration. Bim1 coelutes with Dam1c and forms a stoichiometric complex.
B: Lysine-lysine proximity map of Dam1c-Bim1 complex crosslinked by BS3. Each protein is represented as bar. Lines between the different proteins represent a pair of crosslinked lysine residues identified by mass spectrometry. Subunits of Dam1c that were crosslinked with Bim1 are shown in red, all other subunits in blue and Bim1 in green. The positions of an SxIP sequence in the C-terminus of Duo1 and Q205 in the C-terminus of Dam1 are marked by vertical lines.
C: Comparative structural analysis of representative 2D class averages of Dam1WTc (upper row) and Dam1WTc-Bim1 (lower row). Densities exclusive to Dam1WTc (arrow) indicate the topology of Bim1 (scale bar; 10 nm).
D: Three-dimensional structures of Dam1WTc (blue) and Dam1WTc-Bim1 (yellow) computed from the 2D class averages. The location of the protrusion domains of the Dam1WT complex (composed of Spc19/Spc34) is indicated (gray dashed ovals). Additional density in the Dam1WTc/Bim1 reconstruction (yellow dashed oval) indicates binding of Bim1 to the protrusion domains of Dam1c.
E, F: EM images of negatively stained Dam1c alone (E) and in the presence of Bim1 (F), at low salt conditions. The final concentrations of Dam1c and Bim1 were 0.3 µM and 0.1 µM, respectively. The scale bars are 100 nm, boxes mark representative complexes shown as magnified inserts.
Phospho-regulated Bim1/EB1 interactions trigger Dam1c ring assembly at the budding yeast outer kinetochore (2021)
Figure 2

Figure 2 An SxIP motif located in the C-terminus of Duo1 is required for binding of Bim1 to Dam1c
A: Scheme of the Duo1 subunit of the Dam1 complex. Predicted helical regions are highlighted in red, blue regions are unstructured (top). The position of an SxIP motif is marked by a vertical line. The multiple sequence alignment of Duo1 sequences from various fungal species highlights the conservation of the SxIP motif (black box) and adjacent amino acids across species. Amino acids are colored according to the Clustal color scheme.
B: Chromatogram and SDS-PAGE of elution fractions from gel filtration. Binding of Bim1 to Dam1∆SxIPc lacking the SxIP motif in the C-terminus of Duo1 was examined by analytical size exclusion chromatography. Elution fractions were analyzed by SDS-PAGE.
C: Solid phase binding assay to test for binding of different Dam1 complexes to Bim1. Recombinant GST-Bim1185-344 was immobilized on glutathione sepharose beads and incubated with either recombinant Dam1WTc or Dam1∆SxIPc. Input and pull down samples were analyzed by SDS-PAGE.
D: Pull down assay to compare binding of Dam1WTc and Dam1∆SxIPc to immobilized GST-Bim1185-344. Soluble cell lysates from yeast strains expressing the respective Duo1-6xFlag alleles were incubated with GST-Bim1185-344 immobilized on beads. Duo1-6xFlag bound to GST-Bim1185-344 was analyzed by western blot. An anti-Pgk1 antibody was used to confirm equal protein amounts in input samples.
Phospho-regulated Bim1/EB1 interactions trigger Dam1c ring assembly at the budding yeast outer kinetochore (2021)
Figure 3

Figure 3 Phenotypic analysis of the Duo1∆SxIP allele reveals impaired kinetochore localization of Dam1c and a delay in cell cycle progression
A: Serial dilution assay of yeast strains with different Duo1-6xFlag alleles in a wild type or Dad1-GFP strain background. Growth phenotypes of the respective yeast strains were analyzed by spotting serially diluted amounts of cells on YEPD plates and incubation at different temperatures.
B: FACS analysis of the DNA content of strains with Duo1WT-6xFlag or Duo1∆SxIP-6xFlag alleles with untagged Dad1. Samples were taken from log phase cultures grown at 30 °C. Cells with a 1C DNA content are in G1 phase, a 2C DNA content is characteristic of mitotic cells. Cells with an intermediate DNA content are in S phase.
C: Representative images from fluorescence live cell microscopy of Dad1-GFP strains with Duo1WT-6xFlag or Duo1∆SxIP-6xFlag. Displayed cells are in either metaphase (left) or anaphase (right). Scale bar: 2 μm.
D: Quantification of signal intensities of Dad1-GFP at metaphase and anaphase kinetochore clusters of Duo1WT-6xFlag and Duo1∆SxIP-6xFlag strains. Bars represent the mean +/- standard deviation. Each spot represents the value measured for a single kinetochore cluster. Values were normalized to the mean value of metaphase clusters of the Duo1WT-6xFlag strain. A one-way ANOVA test with Sidak's multiple comparisons test was used to calculate p values. n ≥ 76 kinetochore clusters.
E: Quantification of extra-large (XL) budded cells with short inter-kinetochore distance. Only cells with buds that were grown to more than half of the mother cell's size were counted. Mean +/- standard deviation of three independent experiments are displayed. p-value was calculated by an unpaired t-test.
F: Analysis of cell cycle progression of yeast strains with different Duo1 alleles. Accumulation and degradation of Pds1 after release from α factor arrest was analyzed by western blot (left). An antibody against Pgk1 was used to confirm equal protein amounts in samples. The DNA content of the cell population at indicated time points after release from α factor arrest was analyzed by FACS (right). Cells in G1 phase and mitosis are characterized by a 1C and 2C DNA content, respectively. An intermediate DNA content is detected for cells in S phase.
G: Pds1 signal intensities from western blots shown in F were quantified and corrected for Pgk1 signal of the corresponding sample. Finally, the highest signal intensity (45 minutes after release) was defined as 1.00 and values for other time points were normalized accordingly.
Phospho-regulated Bim1/EB1 interactions trigger Dam1c ring assembly at the budding yeast outer kinetochore (2021)
Figure 5

Figure 5 Bim1 recruits Bik1 to Dam1c and induces oligomerization of the complex in a concentration dependent manner
A, B: 5 µM, 7.5 µM or 10 µM Dam1WTc alone or in combination with respective concentrations of Bim1-Bik1 complex were analyzed by size exclusion chromatography. SDS-PAGE analysis of elution fractions is shown in B. High molecular weight species of Dam1WTc were eluted after 0.9 - 1.0 ml (see chromatogram in A). SDS-PAGE analysis of Dam1c in the absence of Bim1-Bik1 are shown in Figure EV4B.
C: Chromatogram of size exclusion chromatography of Dam1c with Bim1 and Bik1 and SDS-PAGE analysis of elution fractions. The elution buffer contained 100 mM NaCl in contrast to 200 mM in previous experiments. Please note that under these conditions the equilibrium is shifted towards the high oligomeric complex. Dashed boxes indicate elution fractions used for EM analysis shown in D.
D, E: EM images of negatively stained Dam1c in the presence of Bim1 and Bik1 at low salt conditions (10mM NaCl). The final concentrations of Dam1c, Bim1 and Bik1 were 0.3 µM, 0.1 µM, and 0.25 µM, respectively. The Dam1c-Bim1-Bik1 complex could be further purified by gel filtration in presence of 100 mM NaCl (20 mM HEPES, pH 7.4, 100 mM NaCl, 1 mM MgCl2, and 0.1 mM TCEP) prior to the final dilution to low salt conditions for EM (D). The scale bars are 100 nm, boxes mark representative complexes shown as magnified inserts.
Phospho-regulated Bim1/EB1 interactions trigger Dam1c ring assembly at the budding yeast outer kinetochore (2021)
Figure 6

Figure 6 Formation of Dam1c-Bim1 complex and Dam1c-Ndc80c complex are two consecutive steps during establishment of end-on attachments
A: Illustration of possible complex formation involving Dam1c, Bim1 and Ndc80c. After addition of Ndc80c to pre-formed Dam1c-Bim1 complex in solution two possible outcomes are expected. Either Dam1c and Ndc80c form a complex including Bim1 or Bim1 is replaced upon binding of Ndc80c.
B: Pull down assay for analysis of Dam1c-Bim1 binding to Ndc80c. Ndc80c immobilized on beads was incubated with either Dam1c or Bim1 alone or with pre-formed Dam1c-Bim1 complex with increasing amounts of Bim1. Input and elution samples were analyzed by SDS-PAGE. LC and HC denote the light and heavy chains of the M2 anti-Flag antibody used for immobilization of Ndc80c on beads. Protein names in blue, red and green refer to Dam1c, Ndc80c and Bim1, respectively.
C: Quantification of Dam1 and Spc34 signal intensities in pull down samples shown in B. Signal intensities from samples without Bim1 were normalized to the value of 1.0. Linear regression was calculated based on the data points.
D: Serial dilution assay of yeast strains with different Duo1 and Ndc80 alleles in an Ndc80-FRB-GFP background. Ndc80WT or Ndc80∆490-510 integration constructs were integrated at the LEU2 locus and combined with either the Duo1WT or Duo1∆SxIP allele (at DUO1 locus). Cells were spotted on YEPD medium without and with 1 µg/ml Rapamycin and incubated at the indicated temperatures.
Phospho-regulated Bim1/EB1 interactions trigger Dam1c ring assembly at the budding yeast outer kinetochore (2021)
Figure 7

Figure 7 The Duo1∆SxIP and Ask12A alleles cause a synthetic growth defect
A: Model of regulatory mechanisms of Dam1c oligomerization. Both binding of Bim1 to Dam1c and Cdk1-dependent phosphorylation of Ask1 promote oligomerization of the complex, while phosphorylation of Dam1 by Ipl1 prevents oligomerization.
B: Serial dilution assay of yeast strains with 6xFlag-tagged Duo1WT or Duo1∆SxIP and Ask1WT, Ask12A or Ask12D. Cells were grown at the indicated temperatures and images were taken two days (25 °C, 30 °C and 37 °C) or four days (16 °C) after spotting.
C: Mps1 localizes to unattached kinetochores and phosphorylates Dam1c on proximal microtubules (left). By this, Bim1 associates with Dam1c and the complex is enriched at microtubule plus ends which allows formation of end-on attachments (right). Since phosphorylation of Dam1c does not happen on distal microtubules, association with Bim1 and accumulation at plus ends is restricted to microtubules which are close to unattached kinetochores.

Tables
Table 1 List of plasmids used for protein expression and construction of yeast strains.

Table 2 List of yeast strains used in this study

Expanded View Figure legends
Figure EV1 Additional characterization of Dam1c interactions
A: Binding of Stu2 to Dam1c was analyzed by analytical size exclusion chromatography. Chromatograms and SDS-PAGE analysis of elution fractions are shown. The large peak at an elution volume of 2 ml is caused by the 3xFlag peptide used for elution of Stu2-Flag during purification.
B: Pull down assay with immobilized Stu2-Flag. Stu2-flag from Sf9 cells was immobilized on beads and incubated with either, buffer, Dam1c or Bik1. Empty beads were used as control for unspecific binding of proteins to beads. Input and pull down samples were analyzed by SDS-PAGE. HC and LC denote the heavy and light chain of the anti-Flag antibody, respectively. Subunits of Dam1c are labelled in blue. Bik1 and Stu2-Flag are labelled in green and orange, respectively.
C: Lysine-lysine proximity map of Dam1c crosslinked with BS3 in the absence of Bim1. Each subunit of the complex is represented by a blue bar. Blue and red lines indicate intermolecular and intramolecular crosslinks, respectively.
D, E: Representative TEM micrographs of negatively stained Dam1WTc (D) and Dam1WTc-Bim1 complex (E) (scale bar: 100nm). Subsequent 2D classification of Dam1WTc (D), revealed a mixture of Dam1c monomers (inset, upper row) and dimers (inset; lower row), whereas 2D classification of crosslinked Dam1WTc-Bim1 complex (E) revealed monodispersed particles of Dam1c-dimers+Bim1 (inset). Most particles adopt a side-view orientation on the EM grid. To allow for a direct structural comparison, the monomers were excluded from further analysis after this initial round of 2D classification.
Myopathy reversion in mice after restauration of mitochondrial complex I (2019)
Figure 1

Fig. 1. Creation and characterization of skeletal muscle specific Ndufs3 KO (smKO) mice. (A) Western blot analysis of NDUFS3 protein levels in homogenates from quadriceps muscle of 15-days old (upper panel) and 1-month old animals (lower panel). (B) Body weight comparison over time of Ndufs3 smKO male mice (filled blue squares; n=14) and age matched wild-type (WTFlx; n=15) males (empty blue squares). Ndufs3 smKO females (filled red circles) showed similar results (n=10 for smKO, and n=12 for WTFlx group). P values were calculated by Student's t test; (C) Representative image of a smKO male Ndfu3s3-smKO mouse and a WTFlx littermate of 6-month old showing decreased body weight. (D) Survival curve of Ndufs3 smKO female mice (red line) and males (blue line). P values were calculated using Log-rank (Mantel-Cox) Test; p=0.0010 for males (n=19), and p=0.0004 for females (n=10). (E) Nocturnal ambulatory activity of Ndufs3 smKO and aged matched wild-type littermates from 1 and 2 months old male mice. P values were calculated by Student's t test. (F) The number of falls in the treadmill of WTFlx and Ndufs3 smKO male mice, starting at 2 months (n=8). (G) Rotarod performed by Ndufs3 smKO and wild-type male mice. (n=8). Bars represent means ± standard error (SE). P values were calculated by Student's t test to determine the level of statistical difference. n.s.-not significant; *p<0.05, **p<0.01 and ***p<0.0001.