EMBO/soda-nel huggingface dataset:
A protein conjugation system essential for autophagy (1998)
figf5

a, Amino-acid sequence of Apg7. b, Homology between Apg7 and Uba1, S. cerevisiae E1 enzyme. Black circles indicate a putative ATP-binding site (GxGxxG). The putative active-site Cys residue of Apg7 is indicated. c, In vitro conjugation of Apg5 and Apg12. A cell lysate of Δapg12 carrying HA-APG5 (lane 1) was incubated with an equal amount of lysate from Δapg5 carrying HA–APG12 (2 µ plasmid) (lane 2) at 30 °C with (lane 4–6) or without (lane 7) 5 mM ATP. Samples were mixed with SDS-sample buffer at the times indicated.
A protein conjugation system essential for autophagy (1998)
figf6

Spheroplasts were generated from cells expressing either HA-Apg12 or HA-Apg5. Their lysates were mixed and layered on top of a 10-step (18–54 % w/w) sucrose gradient, and centrifuged at 174,000g for 2.5 h (ref. 29). Fifteen fractions were collected and the positions of free Apg5, free Apg12 and Apg5/Apg12 conjugate were examined by western blotting. The peak fractions of alcohol dehydrogenase (ADH)(cytosol), ALP (vacuole), Kex2 (Golgi) and Sec12 (endoplasmic reticulum) are indicated by arrows.
Signal Peptide-Peptidase-Like 2c (SPPL2c) impairs vesicular transport and cleavage of SNARE proteins (2018)
Figure 1

Figure 1: Identification of SPPL2c substrates in HEK293 cells.

A) Volcano plot of proteome analysis. Plotted is the negative log10 of the p-value (y-axis) versus the log2 LFQ ratio of SPPL2c overexpressing and control cells (x-axis) of a given identified protein. A permutation based FDR correction for multiple hypotheses was applied (p = 0.05; s0 = 0.1; dashed line). Open grey circles indicate proteins not-significantly altered in SPPL2c expressing cells, red circles significantly altered proteins after FDR correction. Filled red circles indicate type IV membrane proteins and filled blue circles with red line and blue names type II proteins altered in SPPL2c expressing cells, n=6 biological repl. per cell line.

B) Pathway analysis of SPPL2c candidate substrates. All type II and type IV membrane proteins significantly decreased in the proteomic analysis following overexpression of SPPL2c (see Table 1) were uploaded on the online STRING 10.5 software (https://string-db.org/). Interaction networks of the candidate substrates are depicted and the thickness of the lines indicates the degree of confidence in the prediction of the interaction.

C) Western Blot of selected type IV SPPL2c candidate substrates. Endogenous protein levels of VAPA, VAPB, VAMP8, Syntaxin 8, 18 (Stx8 & Stx18) and Membrin (encoded by the gene Golgi SNAP Receptor Complex Member 2, GOSR2) were analyzed in HEK293 cells ectopically expressing wild type SPPL2c (SPPL2c WT) or a catalytically inactive SPPL2c (SPPL2c D/A). Non-transfected cells (-) or stably transfected but non-induced SPPL2c wt cells (n/i) served as controls. VAPA, VAPB, VAMP8, Stx8 and Membrin protein levels were decreased in SPPL2c expressing cells but not in control or SPPL2c D/A expressing cells. For Stx18 a cleavage product was detected in SPPL2c WT expressing cells. Expression levels of SPPL2c are depicted and Calnexin serves as loading control.
Signal Peptide-Peptidase-Like 2c (SPPL2c) impairs vesicular transport and cleavage of SNARE proteins (2018)
Figure 2

Figure 2: SPPL2c downregulates SPPL3 and affects trafficking of membrane proteins in HEK293 cells.

A) SPPL2c overexpression causes a decrease on SPPL3 levels. Endogenous levels of SPPL3 were assessed in cells ectopically expressing wild type SPPL2c (SPPL2c WT) or catalytically inactive SPPL2c (SPPL2c D/A) by Western Blot. Non-transfected cells (-) or stably transfected but non-induced SPPL2c wt cells (n/i) served as controls. Note that SPPL3 expression is reduced in cells expressing SPPL2c wt.

B) SPPL2c impairs secretion and maturation of glycan-modifying enzymes. Protein levels of endogenous EXTL3, B4GALT1, OGFOD3 and GnT-V were monitored in lysates of cells treated with control siRNA (siCtr) or with siRNA targeting SPPL3 (siSPPL3) and in cells ectopically expressing either SPPL2c or SPPL3 by Western Blot. In addition, the secreted forms (sEXTL3, sB4GALT1, sOGFOD3, sGnTV,) were analyzed in the corresponding conditioned media using Western Blot. Note that SPPL2c reduces secretion of the glycosyltransferases similar to SPPL3 knock down, but additionally induces a decrease of the mature glycosyltransferases in the cell lysate similar to ectopic expression of SPPL3.

C) Processing of ectopically expressed GnT-V with an N-terminal Flag- and C-terminal V5-tag (Flag-GnTV-V5) was analyzed in lysates and conditioned media (sup.) of either SPPL2c wt or SPPL2c D/A expressing cells by Western Blot. Non-transfected cells (-) or stably transfected but non-induced SPPL2c wt cells (n/i) served as controls. In addition, maturation of the glycoproteins Nicastrin (NCT), Lamp2 and SPPL2a was monitored by detection of the respective mature (mat) and immature (im) version. Note that secretion of GnT-V is impaired in SPPL2c wt expressing cells and immature glycoproteins accumulate.

D) SPPL2c induces mislocalization of GnT-V. Ectopically expressed GnT-V was visualized by immunofluorescence staining using the anti-V5 antibody either in control cells (Ctr) or in cells co-expressing either SPPL2c wt or SPPL2c D/A. The ER was stained using the anti-BiP antibody and the Golgi with the anti-TGN46 antibody. While in control cells GnT-V mainly localizes to the Golgi, its localization shifts to more ER-like structures in SPPL2c expressing cells. Scale bar 5 µm.

Data information: Expression levels of the ectopically expressed SPPL proteases are shown and Calnexin serves as loading control in (A-C).
Signal Peptide-Peptidase-Like 2c (SPPL2c) impairs vesicular transport and cleavage of SNARE proteins (2018)
Figure 3

Figure 3: Impact of the validated SPPL2c substrates on GnT-V trafficking and glycoprotein maturation in HEK293 cells.

A-F Protein levels of the validated SPPL2c substrates VAPA (A), VAPB (B), VAMP8 (C), Syntaxin 8 (Stx8) (D), Syntaxin 18 (Stx18) (E) and Membrin (F) were reduced using specific siRNAs. Non-targeting control siRNA (Ctr) served as negative control and overexpression of SPPL2c WT was used as a positive control. Non-transfected cells (-) or stably transfected but non-induced SPPL2c wt cells (n/i) served as additional controls for antibody specificity. Processing and maturation of ectopically expressed GnT-V and maturation of endogenous SPPL2a were monitored by Western Blotting. None of the substrates' knock down fully recapitulated the effect on GnT-V or on endogenous glycoproteins observed in SPPL2c overexpressing cells. Calnexin served as loading control and ectopic expression of SPPL2c WT and SPPL2c D/A was confirmed using a SPPL2c specific antibody.
Signal Peptide-Peptidase-Like 2c (SPPL2c) impairs vesicular transport and cleavage of SNARE proteins (2018)
Figure 4

Figure 4: SPPL2c mediated cleavage of Syntaxin 5 impairs GnT-V trafficking in HEK293 cells.

A) Additional candidate SPPL2c substrates associated with vesicular trafficking were identified using the function "More" of the STRING 10.5 software. The type IV membrane proteins identified as candidate substrates in the proteomic analysis served as basis for the analysis.

B) Processing of endogenous Syntaxin 5 (Stx5) and Syntaxin 6 (Stx6) by SPPL2c wt was assessed as described in Fig. 1C by Western Blot. Non-transfected cells (-), stably transfected but non-induced SPPL2c wt cells (n/i) and induced cells stably expressing SPPL2c D/A served as controls. Results in Figure 1C and 4B are derived from one experiment, thus expression (SPPL2c) and loading (Calnexin) control are the same. Note, that Syntaxin 5 is cleaved by SPPL2c while Syntaxin 6 is not.

C) Syntaxin 5 protein levels were down regulated as described in Fig. 3 and processing and maturation of ectopically expressed GnT-V, as well maturation of the endogenous glycoproteins Nicastrin (NCT) and SPPL2a was analyzed in Western Blot. Calnexin served as loading control and ectopic expression of SPPL2c WT was confirmed using a SPPL2c specific antibody. While GnT-V and Nicastrin maturation were similarly affected by Syntaxin 5 knock down and SPPL2c overexpression, SPPL2a maturation was hardly impaired in Syntaxin 5 knock down cells.

D) Syntaxin 5 localization is impaired by overexpression of SPPL2c. Endogenous Syntaxin 5 was visualized in control cells without SPPL2c expression (Ctr) and in cells overexpressing SPPL2c wt or SPPL2c D/A using immunofluorescence. BiP was used as a marker protein of the ER and TGN46 as Golgi marker. White arrow indicating cells with disturbed Golgi and Syntaxin 5 staining. Scale bar 5 µm.

E) Syntaxin 5 knock down induces mislocalization of GnT-V similar to SPPL2c overexpression (Fig. 2). Cells ectopically expressing GnT-V were treated either with non-specific siRNA (siCtr) or with siRNA targeting Syntaxin 5 (siStx5). GnT-V was visualized in immunofluorescence using anti-V5 antibody targeting its C-terminal tag and either BIP was co-stained as ER marker protein or TGN46 as Golgi marker protein. Scale bar 5 µm.
Signal Peptide-Peptidase-Like 2c (SPPL2c) impairs vesicular transport and cleavage of SNARE proteins (2018)
Figure 5

Figure 5: SPPL2c impacts on the integrity of subcellular compartments in HEK293 cells.

After seeding cells were cultured for 72h and expression of catalytically active (wt) or non-active (D/A) SPPL2c was induced by addition of doxycycline for either 24h or 48h as indicated. Non-transfected cells (Ctr) or non-induced SPPL2c cells (n/i) served as controls. The ER was stained with the anti-BiP antibody, the cis/medial-Golgi with the anti-Giantin, the trans-Golgi Network with the anti-TGN46 antibody and all Golgi subcompartments with the CAB45-specific antibody in immunofluorescence. Note that only upon expression of catalytically active SPPL2c the morphologies of ER and cis-Golgi change compared to controls. Scale bar 5 µm.
Signal Peptide-Peptidase-Like 2c (SPPL2c) impairs vesicular transport and cleavage of SNARE proteins (2018)
Figure 6

Figure 6: SPPL2c cleaves SNARE proteins in testis and is involved in acrosome formation.

A) Western Blot analysis of SPPL2c in membrane preparations from HEK293 inducibly overexpressing SPPL2c, human testis and mouse testis tissue. For human and mouse tissue, 5 μg protein of each total testis preparation is loaded. The membrane fraction obtained from a confluent 6 cm dish of SPPL2c expressing HEK293 cells is diluted 1:50 (0.1 μg total protein).

B) Western Blot analysis of selected glycoproteins in mouse tissues from two wildtype C57BL/6J (Black6) mice. Endogenous Nicastrin (NCT) and SPPL2a were analysed in membrane preparations from testis, kidney, brain, spleen and liver in littermate male mice. Note that immature proteins (im) accumulate in testis compared to other tissues. SPPL2c protein is only detected in testis tissue.

C) Analysis of Syntaxin 8 (Stx8) in SPPL2c-/- mice compared to wildtype (wt) littermates. Membrane isolates from whole testis were analyzed for Syntaxin 8 (Stx8) and SPPL2c expression. TMEM192 serves as loading control.

D) Syntaxin 8 protein levels were quantified from the respective Western blots by densitometric analysis and normalized to those of TMEM192. Bars represent mean values of n=3 animals ± SD normalized to mean Syntaxin 8 levels of wildtype mice. Statistical significance was calculated applying an unpaired, two-sided Student's t-test. * p≤0.05. Note that Syntaxin 8 is accumulating in SPPL2c-/- mice.

E) Real-time qPCR analysis of Syntaxin 8 (Stx8) mRNA levels in testis of SPPL2c-/- and the wildtype mice normalized to that of Tuba1a. The diagram depicts mean values of Stx8 mRNA levels normalized to that of wildtype animals of n=6 individuals from two independent experiments ± SD. For statistical analysis an unpaired, two-sided Student's t-test was performed. ns = not significant.

F) Immunohistochemical stainings of cross-sectioned seminiferous tubules of SPPL2c+/+ and SPPL2c-/- mice using anti-Cab45 antibody. Stages VII/VIII are depicted. Higher magnifications are indicated with dashed lines and black arrows indicate pre-acrosomal structures. Scale bar 50 µm.

G) Volcano plot depicting the binding affinities of lectins on sperm lysates from SPPL2c-/- mice compared to SPPL2c+/+ (wt). Each circle represents one lectin. The lectins above the dashed line (light blue) have a significant p-value<0.05. Lectins on the left side of the blot demonstrate reduced binding while UEA-I on the right side has increased binding. n=4 animals per genotype, two-sided, unpaired Student's t-Test.
Loss of CHD1 causes DNA repair defects and enhances prostate cancer therapeutic responsiveness (2016)
Figure 1

Figure 1. CHD1 accumulates at the DNA damage sites in proximity to γH2AX.

(A) Frequency of CHD1 gene mutation (green) deep deletion (blue) or amplification (red) in prostate cancer patients. (B) CHD1 is recruited to an I-SceI-induced DSB site and is co-localized with γH2AX. Immunofluorescence studies using U2OS19 ptight13 GFP-LacR cells containing a stably integrated I-SceI cleavage site flanked by 256 copies of lac operator (lacO) on one side and 96 copies of the tetracycline response element on the other side (tetO). The localization of the GFP-lac repressor protein (GFP-LacR) at the lac-operator DNA sequences in the nucleus before (− I-SceI) and 16 h after I-SceI-induced (+ I-SceI) DSB. After 16 h of doxy treatment, CHD1 and γH2AX co-localized at I-SceI cleavage site, along with DNA-bound GFP-LacR but not in uninduced cells (−I-SceI). Scale bar 10 µm. (C) Quantification of co-localization of CHD1 with the lac array, mean values of three independent experiments counting at least 100 cells are represented in the graph as ± SD. (D) PC3 cells were treated with NCS (100 ng/ml) for 2 h and with EdU for 45 min. Cells were stained with EdU first and then proximity ligation assay (PLA) was performed using γH2AX and CHD1 antibodies. Scale bar 10 µm. (E) Quantification of PLA signal from (D) in EdU-positive and -negative cells using ImageJ. For quantification more than 100 cells were analyzed for each condition and represented as mean value (n=3, ± SD). (F) CHD1 is recruited to the chromatin upon DNA double strand break induction. PC3 cells with stable control (shCont) or CHD1 shRNA (shCHD1) expression were treated with NCS for the indicated time points and chromatin fractions were immunoblotted with CHD1 and γH2AX antibodies. H2B was used as a loading control. See also Figure EV1A-EV1F.
Loss of CHD1 causes DNA repair defects and enhances prostate cancer therapeutic responsiveness (2016)
Figure 2

Figure 2. Loss of CHD1 leads to DNA damage response defects.

(A, B) BHP1 or PC3 cells with stable expression of either control (shCont) or CHD1 shRNA (shCHD1) were treated with γ-radiation (3 Gy) and after 1 h and 24 h cells were immunostained for γH2AX (A) and the number of γH2AX foci per cell were determined for each time point, more than 50 cells were counted in each condition (B). Scale bar 5 µm. The mean values of three independent experiments (± SD) are shown. p-values (0.0008 and 0.006, **p ≤ 0.01, ***p ≤ 0.001) were calculated using ANOVA. (C) CHD1 depleted cells show prolonged γH2AX accumulation. PC3 cells with shCont or shCHD1 were treated with γ-radiation as in (A) and total protein was analyzed by Western blot. (D) CHD1-depleted cells show increased sensitivity to γ-radiation. For colony formation assay, both BHP1 and PC3 cells with shCont or shCHD1 were treated with the indicated doses of γ-radiation and surviving fractions were measured by counting colonies after 3 weeks, mean values are represented in the plot (n=3, ± SD). Data were normalized to the plating efficiency. p-values (0.0009 and 0.002, **p ≤ 0.01, ***p ≤ 0.001) were calculated using ANOVA. See also Figure EV2A-EV2B.
Loss of CHD1 causes DNA repair defects and enhances prostate cancer therapeutic responsiveness (2016)
Figure 3

Figure 3. CHD1 is required for homologous-recombination (HR) repair.

(A, B) HeLa cells harboring single copies of HR (pGC) or NHEJ (pEJ) repair substrates were transfected with either negative control (siCont) or siRNAs targeting CHD1, RAD51 or XRCC4. After 24 h of transfection, DSB was induced by transfecting cells with I-Sce-I-expressing vector (pCMV-I-SceI-3xNLS). After 48 h of transfection, GFP-positive cells were measured by flow cytometry. HR (A) or NHEJ (B) efficiency was calculated based on the fraction of GFP-positive cells and represented as mean values from three independent experiments as ± SD, 50,000 cells were counted for each condition. (C) CHD1-depleted cells show decreased RAD51 foci after DNA damage induction. The shCont or shCHD1 PC3 cells were irradiated and co-immunostained with γH2AX and RAD51 antibodies after the indicated time points. Scale bar 5 µm. (D) The number of RAD51 foci co-localized with γH2AX per cell (from C) were counted (n=50) and represented in a graph. The data is represented in mean value of three independent experiments as ± SD, more than 50 cells were counted for each condition. See also Figure EV3A-EV3F.
Loss of CHD1 causes DNA repair defects and enhances prostate cancer therapeutic responsiveness (2016)
Figure 4

Figure 4. Depletion of CHD1 affects end resection by decreasing binding of CtIP, RPA1 and RAD51 to chromatin in response to DNA DSB induction.

(A) PLA assay with CtIP and CHD1 in PC3 cells after 2 h of NCS treatment. Scale bar 5 µm. (B) shCont and shCHD1 PC3 cells were treated with NCS for the indicated times and chromatin fractions were immunoblotted for CtIP, RPA1 and RAD51. H2B is shown as a loading control. CHD1 depletion leads to decreased (C) CtIP and (D) RPA1 recruitment to I-SceI-induced DSB in U2OS19 ptight13 GFP-LacR cells. CHD1 was depleted in U2OS19 ptight13 GFP-LacR cells by siRNA and 48 h after transfection cells were treated with doxy for 16 h and co-immunostained for γH2AX and CtIP or RPA1. The number of cells with CtIP or RPA1 foci from (C) and (D) were counted and represented the mean in the graph as percentage of foci positive cells as ± SD (n=3), more than 50 cells were counted. Scale bar 5 µm. (E) PC3 cells were transfected either with mock or siCHD1 followed by empty vector, wt mChd1 (Wt) or ATPase-mutant (Mt) mChd1. After 48 h of transfection cells were treated with of 4-OH tamoxifen (4-OHT) for 24 h and processed for PLA with γH2AX and CtIP antibodies. Cells demonstrating positive focal interactions indicative of DNA repair hubs (punctate staining) are present only in control and (Wt) CHD1-rescued cells but not following knockdown or reconstitution of an ATPase-mutated CHD1 (Mt). Scale bar 20 µm. (F) PC3 cells which stably express HA-mChd1-ERT2, were transfected with either mock or siCHD1. After 24 h of transfection cells were treated with 4-OHT for 24 h to induce HA-mChd1-ERT2 nuclear translocation. Western blot analysis of chromatin fractions for CHD1, ERT2 (mChd1), CtIP and RAD51 shown in Figure EV4O were analyzed by densitometry using ImageJ. The relative quantification for the indicated proteins is shown in the graph. See also Figure EV4A-EV4P.
Loss of CHD1 causes DNA repair defects and enhances prostate cancer therapeutic responsiveness (2016)
Figure 5

Figure 5. CHD1 facilitates chromatin opening at HR DSB sites.

(A) CHD1 recruitment is dependent on MRE11 activity. PC3 cells were transfected with mock or siMRE11 (SmartPool) and 48 h after transfection cells were treated with NCS and chromatin fractions were isolated and analyzed by Western blot. (B) CHD1 is upstream of CtIP. PLA with γH2AX and CHD1 antibodies in mock or CtIP depleted cells after 2 h of NCS. Scale bar is 50 µm. Verification of knockdown efficiency is shown by Western blot on the right. HSC70 is shown as a loading control. Scale bar 20 µm. (C) Quantification of PLA signal from (B) represented as mean values from three independent experiments as ± SD, more than 100 cells were counted per condition. (D) qPCR analyses for chromatin accessibility at two HR-repaired sites (DSB-I and DSB-II) was analyzed by FAIRE in AsiSI–ER-U20S cells transfected with either mock or CHD1 siRNA. After 48 h of transfection cells were treated with 4-OHT for the indicated time points and processed for FAIRE. The data are represented as mean ± SD (n=3) p-values were calculated using ANOVA (0.02 and 0.03, *p ≤ 0.1). (E) Native BrdU staining of PC3 cells transfected either with mock or siCHD1 (SmartPool) were grown for 48 h and then treated with NCS for 2 h prior to staining with anti-BrdU and γH2AX antibodies. Scale bar 5 µm. (F) Quantification of Brdu positive cells from (E). The data are represented as mean ± SD from three independent experiments, more than 50 cells were counted for each condition. p-values (0.03, *p ≤ 0.1) were calculated using ANOVA. See also Figure EV5A-EV4J.
Loss of CHD1 causes DNA repair defects and enhances prostate cancer therapeutic responsiveness (2016)
Figure 6

Figure 6. CHD1-depleted cells show hypersensitivity to Mitomycin C, Irinotecan and PARP inhibition.

(A) CHD1-depleted cells show hypersensitivity to Mitomycin (MMC) treatment. For colony formation assay shCont and shCHD1 PC3 cells were treated with the indicated MMC concentrations for 4 h and surviving fractions were measured by counting colonies after 3 weeks and mean values were represented (n=3, ± SD, p-value=0.0002, ANOVA, ***p ≤ 0.001). (B) CHD1-depleted cells show increased hypersensitivity to Irinotecan. For cell proliferation analysis, shCont and shCHD1 cells were treated with 1 µM Irinotecan and proliferation was measured by Celigo and the relative confluency (mean) was plotted in the graph (n=3, ± SD, p-value=0.0004, ANOVA, ***p ≤ 0.001). (C) Loss of CHD1 leads to increased sensitivity to PARP inhibition. Control or CHD1-depleted BPH1 cells were treated with the indicated concentrations of the PARP inhibitor olaparib and surviving fractions were measured by counting colonies after 3 weeks. Data represented as mean values (n=3, ± SD, p-value=0.0003, ANOVA, ***p ≤ 0.001). (D) CHD1 depletion render cells sensitive to PARP inhibition in combination with irradiation. shCont- or shCHD1-expressing BHP1 cells were treated with 1 µM of PARP inhibitor olaparib for 2 h before irradiation with indicated doses of X-rays and surviving fractions (normalized to the unirradiated condition) were measured by counting colonies after 3 weeks. Data is represented as mean ± SD (n=3, p-value=0.07, ANOVA, *p ≤ 0.1). (E) Frequency of CHD1 gene alterations in comparison with DSB repair genes in prostate cancer patients. Data obtained from cBioPortal for Cancer Genomics. (F) Frequency of CHD1 gene alterations from indicated data sets. See also Figure EV5A-EV5B.
Dephospho-CoA kinase, a nuclear-encoded apicoplast protein, remains active and essential after Plasmodium falciparum apicoplast disruption (2021)
Figure 2

Figure 2 Colocalization of DPCK-mCherry with the apicoplast marker api-SFG

A.) Genotyping PCR confirming the insertion of a 40bp attB element in the p230p gene, with amplification demonstrating integration at the Δ5' and Δ3' loci and lack of wild type parasites (as indicated by the failure to amplify at the wild type 5' and 3' loci) as compared to the parental control. A schematic describing these genotyping PCR reactions is shown in Figure EV1.

B.) Genotyping PCR confirming the insertion of the PfDPCK-mCherry construct using attP/attB integration. PCR reactions demonstrate integration at the Δ5' and Δ3' integration sites and lack of parental parasites (P) as compared to the parental control (P230p-attB). A schematic describing these genotyping PCR reactions is shown in Figure EV2a.

C.) Live epifluorescence microscopy of PfMev parasites that are expressing api-SFG (green) and the P. falciparum DPCK-mCherry protein (red) with DAPI marking the nuclei (blue). Colocalization statistics are shown in Table 1. The borders of the red blood cell and parasite are marked with dotted blue and white lines, respectively. Microscopy images represent fields that are 10µm long by 10µm wide.