EMBO/soda-nel huggingface dataset:
TMEM59 defines a novel ATG16L1‐binding motif that promotes local activation of LC3 (2013)
Figure 5

Alanine scanning approach to identify amino acids in the active subdomain of TMEM59 that are essential for LC3 activation. (A) Identification of critical amino acids for HALC3 conversion. 293 cells were transfected with the indicated CD16:7263281 mutants and HALC3A, subjected to anti-CD16 aggregation and lysed for western blotting against the indicated molecules. Asterisks mark mutations with reduced activity. Shown is one representative experiment of six repetitions. (B) Identification of critical amino acids for GFPLC3 activation and colocalization with the endocytosed chimera. JAR cells were transfected with the indicated CD16:7263281 mutants and GFPLC3A, aggregated and stained for the endocytosed chimera (red). Preparations were scored by blindly counting the number of red vesicles (chimera) and green vesicles (GFPLC3A) per cell, as well as the number of red vesicles colocalizing with green ones. The percentage of green vesicles colocalizing with red ones was close to 100% for all mutants, that is, virtually no GFPLC3A vesicles were unrelated to endocytosed chimera (not shown). At least 50 cells were scored per experimental point. The experiment was repeated three times. The graph shows the number of GFPLC3A vesicles per cell expressed as the percentage of the value obtained for the wild-type chimera (left axis), and the percentage of chimera vesicles labelled with GFPLC3A (right axis). Data are expressed as means ±s.d. of the triplicates. Asterisks indicate significant differences with respect to wild-type values (paired Student's t-test; P<0.01). (C) Representative confocal pictures of the phenotype produced by the indicated mutations. Procedures were as in B. (D) Simultaneous mutation of the four essential amino acids to alanine blocks HALC3 conversion induced by CD16:7263281. 293 cells were transfected with the indicated chimeras (4M, quadruple mutant) and HALC3A, aggregated and lysed for western blotting. (E) Simultaneous mutation of the four essential amino acids blocks GFPLC3 activation and colocalization with the endocytosed chimera. JAR cells were transfected with the indicated CD16:7263281 constructs and GFPLC3A, aggregated and stained for the endocytosed chimera (red). Representative confocal images are shown. (F) Quantification of the phenotype in E. Data gathering and expression were as in B.Source data for this figure is available on the online supplementary information page.
TMEM59 defines a novel ATG16L1‐binding motif that promotes local activation of LC3 (2013)
Figure 6

The minimal active subdomain of TMEM59 directly binds ATG16L1 through the identified functional motif. (A) Apposition events between GFPATG16L1 and endocytosed CD16:7263281. JAR cells were transfected with CD16:7263281 and GFPATG16L1 or GFPBECLIN (as indicated), aggregated for 4 h and stained for the endocytosed chimera (red). Representative confocal pictures are shown. Two different examples are provided for GFPATG16L1. (BD) 293T cells were transfected with the indicated constructs, lysed and subjected to GST immunoprecipitation using agarose beads coupled to glutathione (IP, immunoprecipitation; TL, total lysate). Shown are WBs against the indicated molecules. (B) AUATG16L1 co-precipitates with wild-type (WT) TMEM59GST but not with a mutated version where the four essential amino acids were mutated to alanine (TMEM594MGST). (C) Full-length TMEM59 and TMEM59?282 co-precipitate with GSTATG16L1, whereas the respective 4M versions do not. (D) AUATG16L1 co-precipitates with a fusion protein between GST and the minimal active peptide of TMEM59 (GST263281), but not with a 4M version (GST2632814M) or a GST fusion protein with the inactive portion of TMEM59ID (GST282323). (E) HAATG16L1 expressed in bacteria co-precipitates with a GST263281 recombinant protein purified from bacterial cultures, but not with a 4M version of the same construct or GST282323. The indicated GST partners were expressed in bacteria and purified using agarose beads coupled to glutathione. The loaded beads were then used for HAATG16L1 pull-down from crude bacterial lysates. Shown are WBs against the indicated molecules (PD, pull-down). A Coomasie staining of a protein gel with the purified GST fusion proteins is shown. The right panel compares the amount of HAATG16L1 pulled down by GST263281 with the signal provided by direct anti-HA immunoprecipitation. This result shows that about 2025% of the available HAATG16L1 protein is precipitated by GST263281. Asterisks indicate irrelevant bands in B and D.Source data for this figure is available on the online supplementary information page.
TMEM59 defines a novel ATG16L1‐binding motif that promotes local activation of LC3 (2013)
Figure 7

The ATG16L1-binding motif present in TMEM59 recognizes the WD-repeat domain of ATG16L1. (AF) 293T cells were transfected with the indicated constructs, lysed and subjected to GST immunoprecipitation with agarose beads coupled to glutathione. Shown are WBs against the indicated molecules. (A) A deleted version of ATG16L1 lacking the WD domain (HAATG16L1?WD) does not co-precipitate with TMEM59GST. (B) TMEM59 does not co-precipitate with ATG16L1?WD fused to GST. (C) The WD domain of ATG16L1 (HAATG16L1WD) suffices to co-precipitate with TMEM59GST. (D) TMEM59 co-precipitates with ATG16L1WD fused to GST. (E) HAATG16L1WD does not bind a 4M version of TMEM59GST. (F) TMEM594M does not co-precipitate with the ATG16L1WD fused to GST.Source data for this figure is available on the online supplementary information page.
TMEM59 defines a novel ATG16L1‐binding motif that promotes local activation of LC3 (2013)
Figure 8

The ATG16L1-binding motif is present with a similar ATG16L1-binding capacity in other molecules. (A) Alignment of the region that includes the motif in NOD2 with the same area in NOD1. The relevant amino-acid stretch is boxed. Individual amino-acid highlighting (black) in NOD2 indicates the residues identified by the Prosite algorithm as part of the motif. In NOD1, all residues that could be part of an eventual motif are highlighted to indicate that they form an incomplete motif. (B) Amino-acid region in TLR2 that includes the motif. Highlighting indicates residues identified by Prosite as part of the motif. (C, D, E, G, H) 293T cells were transfected with the indicated constructs, lysed and subjected to GST immunoprecipitation with agarose beads coupled to glutathione. Shown are WBs against the indicated molecules. (C) The N-terminal CARD of NOD2 (NOD2CARD1HA), but not the CARD of NOD1 (NOD1CARDHA), co-precipitates with GSTATG16L1. (D) The ID of TLR2 (HATLR2ID) co-precipitates with GSTATG16L1. (E) Mutated versions (MUT) of NOD2CARD1HA and HATLR2ID (as shown) do not co-precipitate with GSTATG16L1. (F) Amino-acid sequences of T3JAM and DEDD2 including the motif. Residues identified by the Prosite algorithm are highlighted. (G) Wild-type (WT) versions of HAT3JAM and HADEDD2 (as indicated) co-precipitate with GSTATG16L1. (H) Mutated versions (MUT) of HAT3JAM and HADEDD2 do not co-precipitate with GSTATG16L1. Asterisks indicate irrelevant bands in C, D and G.Source data for this figure is available on the online supplementary information page.
TMEM59 defines a novel ATG16L1‐binding motif that promotes local activation of LC3 (2013)
Figure 9

Endogenous TMEM59 mediates LC3 activation and interacts with ATG16L1 in response to S. aureus (SA) infection. (A) Depletion of TMEM59 inhibits LC3II generation by SA at early infection times. HeLa cells were transfected with the indicated siRNAs and, 48 h later, infected with the bacteria (SA) for the shown times before lysing them for western blotting against the indicated molecules. The right panel shows successful TMEM59 depletion (rabbit anti‐TMEM59 immunoprecipitation plus chicken anti‐TMEM59 WB). (B) Colocalization events between bacteria (GFP labelled), TMEM59–HA and Cherry–LC3A in infected cells. HeLa cells stably expressing TMEM59–HA and Cherry–LC3A were infected for the indicated times (moi=10), and processed for anti‐HA immunofluorescence. Representative confocal images are shown. The GFP, anti‐HA (Cy5) and DAPI signals were pseudocolored in blue, green and white, respectively. Asterisks indicate overlay images: (*), Cherry–LC3A/TMEM59; (**), Cherry–LC3A/TMEM59/bacteria; (***), Cherry–LC3A/TMEM59/bacteria/DAPI. (C) Colocalization between bacteria, TMEM59–HA and Cherry–ATG16L1. HeLa cells stably expressing TMEM59–HA and Cherry–ATG16L1 were infected with SA (2 h, moi=10), and processed for anti‐HA immunofluorescence. Shown are confocal pictures displaying two representative examples. Asterisks indicate overlays as in B. (D) SA promotes co‐precipitation of endogenous ATG16L1 with endogenous TMEM59. HeLa cells were infected with SA (2 h, moi=10), lysed and subjected to immunoprecipitation with anti‐TMEM59 antibodies or irrelevant protein G beads (as indicated). Immunoprecipitates were processed for western blotting. The right panel shows control WBs of the lysates used for immunoprecipitation. (E) Depletion of TMEM59 reduces recovery of SA from infected cells. HeLa cells were transfected with the indicated siRNAs, infected with SA 96 h later (5 h, moi=0.1) and lysed for CFU evaluation. The experiment was carried out three independent times. The graph displays average CFU counts obtained for the 10−1 dilution of the relevant extracts, ±s.d. of the three data sets. Asterisks indicate significant differences (P<0.01, paired Student's t‐test). The right panel shows successful TMEM59 depletion (anti‐TMEM59 immunoprecipitation plus anti‐TMEM59 WB).Source data for this figure is available on the online supplementary information page.
TDP-43 loss of function inhibits endosomal trafficking and alters trophic signaling in neurons (2016)
Figure 1

Figure 1. TDP-43 knockdown in primary rat and iPSC-derived human neurons inhibits trafficking of recycling endosome in dendrites

(A) Primary hippocampal neurons (DIV6+3) were transfected with shRNA targeting TDP-43 (shTDP) or control (shCtrl) together with GFP-RAB11 to visualize trafficking of recycling endosomes (RE). At least four dendrite segments per neuron were live imaged at 5 Hz for 1 minute to analyze recycling endosome transport. Representative dendrite segments and kymographs of GFP-RAB11 vesicle movement. Quantitative analysis of vesicle motility (B) and total number (C) from kymographs. (D) iPSC-derived human neurons were transduced with shRNA targeting human TDP-43 (shTDP) or control (shCtrl) seven days after thawing for three days. TDP-43 levels were analyzed by quantitative RT-PCR. Expression was normalized to the housekeeping gene YWHAZ and PGK1. (E) iPSC-derived human neurons (DIV7+3) were transduced with shTDP or shCtrl and GFP-RAB11 and at least three dendrite segments per neuron analyzed as in (a). Quantitative analysis of vesicle motility (F) and number (G) from kymographs. Mean +/- s.e.m., n = 3, unpaired two-tailed t-test: * denotes p < 0.05, ** denotes p < 0.01, *** denotes p < 0.001.
TDP-43 loss of function inhibits endosomal trafficking and alters trophic signaling in neurons (2016)
Figure 2

Figure 2. TDP-43 knockdown in primary neurons inhibits transferrin recycling

(A) Primary hippocampal neurons (DIV6+4) were transduced with shRNA targeting TDP-43 or a control together with tagRFP (not depicted). Representative images of neurons subjected to Alexa-transferrin (Alexa-Tf) for 20 min (pulse) and chased for 0, 20 or 60 min in complete media. Scale bar represents 100 µm. (B) Quantification of labeled cellular Alexa-Tf normalized to cell area (using tagRFP) after chase period. At least ten images per condition were analyzed per experiment in three independent experiments. (C) ELISA measurement of β2-transferrin in CSF of control subjects (n=65) and sporadic ALS patients (n=55), FTD-ALS patients (n=12) and behavioral variant FTD patients (bvFTD, n=16). (D) Primary hippocampal neurons (DIV6+3) were transfected shTDP or shCtrl and GFP as transfection marker. After 3 days neurons were subjected to Alexa-Tf for 20 min and intracellular Alexa-Tf movement was imaged as in Figure 1 afterwards. Quantitative analysis of Alexa-Tf movement (E) and vesicle number (F) from kymographs. n = 3. (F) Mean +/- s.e.m., unpaired, two-tailed t-test (E, F), Mann-Whitney-test with Bonferroni’s post-test (B) or one-way ANOVA with Dunnet’s post-test (C): * denotes p < 0.05, ** denotes p < 0.01, *** denotes p < 0.001.
TDP-43 loss of function inhibits endosomal trafficking and alters trophic signaling in neurons (2016)
Figure 3

Figure 3. Expression of nuclear but not cytoplasmic TDP-43 enhances recycling endosome motility

(A) Primary hippocampal neurons (DIV6+4) were transfected with myc-tagged TDP-43 wild-type or a mutant lacking the nuclear localization signal (ΔNLS) or an empty vector control. Immunostaining with the indicated antibodies and DAPI to label nuclei. Scale bar represents 50 µm. (B) Primary hippocampal neurons (DIV6+3) were transfected with either TDP-43 wild-type, TDP-43ΔNLS or an empty vector control together with GFP-RAB11 to visualize recycling endosomes and analyzed as in Figure 1. Quantitative analysis of recycling endosome movement (C) and vesicle number (D). Mean +/- s.e.m., n = 4, one-way ANOVA with Tukey’s post-test: * denotes P < 0.05, ** denotes P < 0.01.
TDP-43 loss of function inhibits endosomal trafficking and alters trophic signaling in neurons (2016)
Figure 4

Figure 4. VPS4B is increased upon TDP-43 knockdown

(A) Primary cortical neurons were transduced with shTDP or control (DIV2+7) and subjected to quantitative proteomic analysis. Volcano plot shows up and down regulated proteins from three biological replicates. Significant changes are colored. Compare Table EV1. (B) Selected significantly enriched annotation between TDP-43 knockdown and control in proteomic data (complete data in Table EV1) (C-E) Primary hippocampal neurons (DIV6+4) were transduced with lentivirus expressing shRNA targeting TDP-43 or a control. Immunoblots with the indicated antibodies (C). Quantification of VPS4B and TDP-43 protein level using densitometry (n = 4). Expression was normalized to the housekeeping gene β-actin (D). VPS4B mRNA level were analyzed by quantitative RT-PCR. Expression was normalized to the housekeeping genes YWHAZ and PGK1 (E). (F) Primary hippocampal neurons (DIV6+3) were transfected with either shTDP or shCtrl and iRFP670. Immunostaining with the indicated antibodies. Scale bar represents 50 µm. (G) iPSC-derived human neurons were transduced with shRNA targeting human TDP-43 (shTDP) or control (shCtrl) seven days after thawing for three days. VPS4B levels were analyzed by quantitative RT-PCR. Expression was normalized to the housekeeping gene YWHAZ and PGK1 (n = 3). mean +/- s.e.m., unpaired, two-tailed t-test with permutation based (A) or Benjamini-Hochberg (B) correction: * denotes p < 0.05, ** denotes p < 0.01, *** p denotes < 0.001.
TDP-43 loss of function inhibits endosomal trafficking and alters trophic signaling in neurons (2016)
Figure 5

Figure 5. TDP-43 represses VPS4B expression by binding to the promoter region

(A-B) Luciferase assay to analyze transcriptional regulation of VPS4B by TDP-43. (A) Reporter construct expressing renilla luciferase (R-Luc) driven by a VPS4B promoter fragment and firefly luciferase (FF) under the TK promotor. (B) HEK293 cells were transfected with shRNAs targeting human TDP-43, a control shRNA, TDP-43 wild-type or empty vector control together with the luciferase reporter containing the rat VPS4B promoter. VPS4B promoter-driven renilla luciferase activity was normalized to TK promoter-driven firefly luciferase. Quantification from six independent experiments. (C-D) ChIP assay to analyze binding of TDP-43 to VPS4B promoter region in rat cortical neurons, HEK293 cells and human brain tissue. PCR reaction from input, negative control and TDP-43 immunoprecipitates. Signal intensities from at least three independent experiments for each condition were quantified by densitometry and depicted as percent of input. (E-H) Analysis of VPS4B in tissue of seven patients with the neuropathological diagnosis of FTLD/ALS-TDP and five healthy controls. (E, G) Immunofluorescence with the indicated antibodies in the superior frontal gyrus or spinal cord. Scale bar represents 50 µm. (F, H) Quantification of cellular VPS4B levels. At least 50 cells per case were analyzed. Mean +/- s.e.m., unpaired, two-tailed t-test: * denotes p < 0.05, ** denotes p < 0.01.
TDP-43 loss of function inhibits endosomal trafficking and alters trophic signaling in neurons (2016)
Figure 6

Figure 6. VPS4B upregulation is necessary and sufficient for impaired recycling endosome trafficking upon TDP-43 knockdown

(A) Primary hippocampal neurons (DIV6+3) were transfected with either VPS4B or an empty vector control together with GFP-RAB11 to visualize recycling endosomes. Neurons were imaged as in Figure 1 to analyze recycling endosome transport. Quantitative analysis of vesicle motility (B) and number (C) from kymographs (n = 4). (D) Primary hippocampal neurons (DIV6+3) were transfected with the indicated combinations of shRNA targeting TDP-43, VPS4B and control together with GFP-RAB11 and imaged as in Figure 1A. Quantitative analysis of vesicle motility (E) and number (F) from kymographs (n=3). mean +/- s.e.m., unpaired, two-tailed t-test (B, C) or one-way ANOVA (Tukey’s post-test) (E, F): * denotes p < 0.05, *** denotes p < 0.001.
TDP-43 loss of function inhibits endosomal trafficking and alters trophic signaling in neurons (2016)
Figure 7

Figure 7. TDP-43 knockdown reduce surface expression and activity of ErbB4 and FGFR1

(A) Primary hippocampal neurons (DIV6+4) were transduced with shRNA targeting TDP-43 (shTDP) or control (shCtrl) and surface expression levels of glycoproteins was analyzed by metabolic labeling followed by surface biotinylation and subsequent streptavidin purification. Volcano plot depicts proteins that are significantly (p < 0.01, t-test, Benjamini-Hochberg, FDR: 0.05) decreased or increased on the cell surface in three independent experiments with two technical replicates. For full dataset see Table EV2. (B-E) Primary cortical neurons (DIV6+5) were transduced with shCtrl or shTDP. Neurons were treated with the ErbB4 ligand NRG1 (10 nM) or FGFR1 ligand FGF1 (10 ng/ml) for 10 min right before cell lysis. Immunoblots with the indicated antibodies (B, D). Quantification of total and phosho-ERBB4 (Y1056) and total and phosho-FGFR1 (Y654) using densitometry 3-7 biological replicates (C, E). (F) Primary hippocampal neurons (DIV6+4) were transduced with shTDP or shCtrl. Gene expression was analyzed by quantitative RT-PCR normalized to the housekeeping genes YWHAZ and GAPDH (n=3) (G) Hippocampal neurons were transfected with ErbB4 containing a luminal HA-tag (HA/T-ErbB4) and shCtrl or shTDP (DIV9+3). Surface HA/T-ErbB4 was stained in living cells and intracellular HA/T-ErbB4 was stained after fixation and permeabilization with HA-antibodies. (H) Quantification of HA/T-ErbB4 surface levels. At least 10 images per condition per experiment were analyzed in three independent experiments. Scale bar represents 50 µm. . mean +/- s.e.m., unpaired, two-tailed t-test: * denotes p < 0.05, ** denotes p < 0.01.
TDP-43 loss of function inhibits endosomal trafficking and alters trophic signaling in neurons (2016)
Figure 8

Figure 8. TDP-43 knockdown delays surface delivery of ErbB4

(A,B) Primary hippocampal neurons (DIV6+3) were transfected with shCtrl or shTDP and ErbB4 containing an N-terminal HA epitope tag and followed by thrombin cleavage site (HA/T-ErbB4). Surface HA/T-ErbB4 was stained in living cells and intracellular HA/T-ErbB4 was stained after fixation and permeabilization with two different HA-antibodies. After removing the HA-signal of surface ErbB4 (0 min) using thrombin cleavage (5min, 1U/ml) membrane insertion of HA/T-ErbB4 was measured at the indicated time points. At least 10 images per condition per experiment were analyzed in three independent experiments. Scale bar represents 50 µm. mean +/- s.e.m., unpaired, two-tailed t-test: * denotes p < 0.05, ** denotes p < 0.01.
TDP-43 loss of function inhibits endosomal trafficking and alters trophic signaling in neurons (2016)
Figure 9

Figure 9. TDP-43 knockdown blunts dendritic complexity and response to NRG1

Primary hippocampal neurons (DIV6+4) were cotransfected with shTDP, VPS4B or appropriate controls and GFP to visualize neuron morphology. Dendritic morphology from at least 23 neurons per condition per experiment was quantified by Sholl analysis and statistically evaluated using two-way ANOVA with Bonferroni’s (B, D) or Tukey’s (F, H) post-test. Scale bar represents 100 µm. (A, B) TDP-43 knockdown (DIV6+5) significantly reduces dendrite branching compared to control: at 25, 37.5 and 87.5 µm radius p < 0.05, at 50 µm p < 0.01 and from 62.5 to 75 µm p < 0.001. (C, D) VPS4B transfection (DIV6+4) significantly reduces dendrite complexity: at 25 µm radius p < 0.05, from 37.5 to 75 µm p < 0.001 and from 87.5 to 100 µm p < 0.01. (E, F) Two days after shRNA transfection neurons were treated with 1 nM NRG1-beta 1 (NRG1) or vehicle and analyzed at DIV6+5. shCtrl vs. shTDP: at 25 and 87.5 µm radius p < 0.05, from 37.5 to 75 µm p < 0.001. shCtrl vs. shCtrl + NRG1: from 37.5 to 62.5 µm p < 0.05. shTDP vs. shTDP+NRG1: no significant difference. (G, H) Transfection of the indicated combinations of shCtrl, shTDP, ErbB4 or vector control (DIV6+5). shCtrl + Ctrl vs. shTDP + Ctrl: from 25 to 75 µm radius p < 0.001, at 87 and 112.5 µm p < 0.01 and at 100 µm p < 0.05. shTDP + Ctrl vs. shTDP + ErbB4: from 25 to 100 µm radius p < 0.001, at 112.5 µm p < 0.01. shCtrl + Ctrl vs. shTDP + ErbB4: no significant differences.
ENTREP/FAM189A2 encodes a new ITCH ubiquitin ligase activator that is downregulated in breast cancer (2021)
Figure 1

Figure 1. The expression of FAM189A2/ENTREP.

A The mRNA expression of FAM189A2/ENTREP in the primary human mammary epithelium HMEC and human breast cancer cell lines. In qRT-PCR analyses, the expression was normalized to the immortalized normal human mammary epithelium HMEC4tertshp16. Relative expression ratios shown as mean + SD from three biological replicates.

B Relative expression of FAM189A2/ENTREP in the normal (white) and cancer (green) tissues of breast (Curtis Breast), lung (Okayama Lung), colorectal (TCGA Colorectal) and head and neck (Estilo Head-Neck). Data were downloaded from the Oncomine database (www.oncomine.org). P-values obtained by Student's t-tests and the number of cases are listed on the top and bottom of figures, respectively. P < 0.05 was considered as statistically significant. Box-whisker plot represents the interquartile range (25th and 75th percentiles) as a box and the median as a line. The maximn and minimun values within 1.5 x interquartile range are shown as whiskers. Outlier data are plotted as dots.

C Relative expression of FAM189A2/ENTREP in the primary (green) and the metastatic sites (blue) including lymph node, bone, liver, lung, soft tissues of prostatic cancer (Grasso Prostate) (left) and in the normal (white), primary (green) and metastatic sites (three cases of sentinel lymph node) (blue) of breast cancer (TCGA Breast) (right). Note that only three cases of the metastatic breast cancer were available in the dataset. Data were downloaded from the Oncomine database (www.oncomine.org). P-values obtained by Student's t-tests and the number of cases are listed on the top and bottom of figures, respectively. P < 0.05 was considered as statistically significant. Box-whisker plot represents the interquartile range (25th and 75th percentiles) as a box and the median as a line. The maximn and minimun values within 1.5 x interquartile range are shown as whiskers. Outlier data are plotted as dots.

D-F Relapse-free survival (RFS) of breast cancer patients. Data were downloaded from the Kaplan-Meier plotter (https://kmplot.com/analysis/). Statistical analysis is described in the Materials and Methods.