EMBO/soda-nel huggingface dataset:
Listeria monocytogenes ActA-mediated escape from autophagic recognition (2009)
figf2

(a) Schematic diagram of the domain of ActA involved in actin nucleation. The signal peptide, the membrane anchor, the proline-rich repeats, the putative actin-binding motif and the Arp2/3 complex-recruiting motif are shown in violet, brown, yellow, pink and light-blue, respectively. The deleted amino acid regions of ΔactA3, 5, 9, 13, 16 and 17 are 129–153, 265–389, 142–153, 90–109, 154–262 and 390–584, respectively. ΔactA14 has R148S substituted for a single amino-acid. ΔactA21 harbours deletions of amino–acids 93–98, 265–389 and the substitution R148S. Right, semiquantitative assessment of actA mutants for bacterial motility, recruitment of the Arp2/3 complex or VASP, and entrapment by autophagy. +, positive; ++, very strong. (b, c) Quantification of the number of GFP–LC3-positive bacteria (b) or actin-accumulating bacteria (c). Data are mean ± s.e.m., at least 500 bacteria were counted in each experiment (n = 3 experiments). (d) Colocalization of ubiquitin with ΔactA2. MDCK/pGFP–LC3 cells infected with ΔactA2 bacteria were stained with an anti-Listeria antibody (blue) and anti-ubiquitin antibody (red). Scale bars, 10 μm. Arrowheads indicate GFP–LC3-positive or ubiquitin-positive bacteria. (e) High magnification of framed image in d, and the graph shows the intensity of the fluorescence signal along the arrow. (f) Quantification of the number of GFP–LC3-positive, ubiquitin-positive or GFP–LC3- and ubiquitin-positive bacteria. Data are mean ± s.e.m., at least 500 bacteria were counted in each experiment (n = 3 experiments). (g) Immunogold electron micrograph of MDCK/pGFP–LC3 cells infected with ΔactA2. Gold particles of 5 nm (arrowheads) indicate GFP–LC3, and gold particles of 10 nm (arrows) indicate ubiquitin. Scale bars, 0.5 μm. (h) Kinetics of the generation of GFP–LC3-positive or ubiquitin-positive bacteria. Data are mean ± s.e.m., at least 500 bacteria were counted in each experiment (n = 3 experiments). *P 0.001. (i) Quantification of the number of ubiquitin-positive or GFP–LC3-positive bacteria. MDCK/pGFP–LC3 cells infected with wild-type or ΔactA2 bacteria were untreated or treated with 3-MA (10 mM). Data are mean ± s.e.m., at least 500 bacteria were counted in each experiment (n ≥ 3 experiments). Ub, ubiquitin. WT, wild type.
Listeria monocytogenes ActA-mediated escape from autophagic recognition (2009)
figf3

(a) Quantification of the number of ubiquitin-positive, p62-positive or GFP–LC3-positive bacteria. MDCK/pGFP–LC3/pp62-3×Myc cells were infected with a wild-type or ΔactA2 strain of L. monocytogenes for 2 h. Data are mean ± s.e.m., at least 500 bacteria were counted in each experiment (n = 6 experiments). (b) Colocalization of p62 with ΔactA2. MDCK/pGFP–LC3/pp62-3×Myc cells were stained with an anti-ubiquitin antibody (light blue), anti-Myc antibody (red), DAPI (white) and anti-GFP–LC3 (green). Scale bar, 10 μm. Arrowheads indicate GFP–LC3-, ubiquitin- or p62-positive bacteria. (c) Quantification of the number of ubiquitin-positive, p62-positive or GFP–LC3-positive bacteria. p62−/−/p62 cells were infected with ΔactA2 bacteria for 2 h. Data are mean ± s.e.m., at least 500 bacteria were counted in each experiment (n = 6 and 3 experiments for p62−/− and p62−/−/p62, respectively). (d, e) Intracellular survival of wild-type or ΔactA2 bacteria in p62−/− cells (d) or p62−/−/p62 cells (e; colony-forming units, CFU, relative to CFU 30 min after infection). Data are presented as mean ± s.e.m. (n ≥ 4 experiments). (f) Schematic representation of p62 mutants. The deleted amino-acid regions of ΔLIR and ΔUBA are 321–342 and 394–431, respectively. ΔLIR-ΔUBA has deleted 321–342 and 394–431 amino-acid regions. K7AD69A has two amino–acid substitutions. (g) Generation of p62-3×Myc mutants in p62−/− cells. (h) Quantification of the number of p62-positive or GFP–LC3-positive bacteria. p62−/−/p62 (wild type, ΔLIR, ΔUBA, ΔLIR-ΔUBA or K7AD69A) cells were infected with ΔactA2 bacteria and stained with anti-Listeria and anti-Myc antibodies (Supplementary Information, Fig. S4e). Data are mean ± s.e.m., at least 500 bacteria were counted in each experiment (n = 3 experiments). P 0.001 versus p62-positive bacteria in p62−/−/p62 wild-type cells, P 0.001 versus GFP–LC3-positive bacteria in p62−/−/p62 wild-type cells. Ub, ubiquitin. WT, wild type. A full scan of g is shown in Supplementary Information, Fig. S7.
Listeria monocytogenes ActA-mediated escape from autophagic recognition (2009)
figf4

(a) Schematic representation of the GFP–ActA-Q79C series of constructs. FL, N and C comprise ActA residues 30–612, 30–389 and 390–612, respectively. (b–e) The series of pEGFP–ActA–Q79C constructs was transfected into COS-7 cells. Cells were fixed with 4% Paraformaldehyde after 18 h and stained with an anti-Arp2 antibody (b), an anti-VASP antibody (c), an anti-ubiquitin antibody (d) or an anti-p62 antibody (e; red). Scale bars, 10 μm. Arrowheads indicate Arp2-, VASP-, ubiquitin- or p62-positive Q79C. Ub, ubiquitin.
Listeria monocytogenes ActA-mediated escape from autophagic recognition (2009)
figf5

(a) Schematic representation of the GFP–ActA–170 series of constructs. FL, N and C comprise ActA residues 30–612, 30–389 and 390–612, respectively. (b–e) The series of pEGFP–ActA–170 constructs was transfected into COS-7 cells. Cells were fixed with 4% Paraformaldehyde after 14 h and stained with an anti-Arp2 antibody (b), an anti-VASP antibody (c), an anti-ubiquitin antibody (d) or an anti-p62 antibody (e; red). Scale bars, 10 μm. Arrowheads indicate Arp2-, VASP-, ubiquitin- or p62-positive 170. Ub, ubiquitin.
PTP-MEG2 regulates quantal size and fusion pore opening through two distinct structural bases and substrates (2021)
Figure 1

Figure 1. Inhibition of PTP-MEG2 reduced the spike amplitude and 'foot' probability of catecholamine secretion.

(A-D). The epinephrine (A, B) or norepinephrine (C, D) secreted from the adrenal medulla was measured by ELISA method after stimulation with high KCl (70 mM) (A,C) or Angiotensin II (AngII, 100 nM) (B,D) for 1 min, with or without pre-incubation with a specific PTP-MEG2 inhibitor (400 nM) for 1 hours.

(E, F). Amperometric spikes of primary mouse chromaffin cells induced by AngII (100nM) were determined by electrochemical experiments after incubation with PTP-MEG2 inhibitor at different concentrations.

(G). The distribution of the quantal size of AngII (100 nM)-induced amperometric spikes by primary chromaffin cells after incubation with different concentrations of PTP-MEG2 inhibitor. Histograms show the number of amperometric spikes of different quantal sizes.

(H, I). Secretory vesicles of primary chromaffin cells were examined by transmission electron microscopy after 100 nM AngII stimulation, with or without pre-incubation with 400 nM Compound 7. Scale bars: (H, I), 150 nm. Red arrows indicate morphologically docked LDCVs and green arrows stands for undocked LDCVs. PM represents plasma membrane.

(J). Vesicle numbers according to different distances from the plasma membrane were calculated in the presence or absence of 100 nM AngII or 400 nM Compound 7. PM represents plasma membrane.

(K). The percentage of vesicles with different diameters was measured after 100 nM AngII stimulation or incubation with 400 nM Compound 7. Compound 7 significantly decreased vesicle size under AngII stimulation.

(L). The percentage of pre-spike foot (PSF) (left panel) and stand-alone foot (SAF) (right panel) were calculated after incubation with the indicated concentrations of Compound 7.

Data information: Data were analyzed using one-way ANOVA and displayed as the mean ± s.e.m. (A-D, J-L) *, P<0.05; **, P<0.01; ***, P<0.001: cells stimulated with KCl or AngII compared with un-stimulated cells. #, P<0.05; ##, P<0.01; ###, P<0.001: cells pre-incubated with PTP-MEG2 inhibitors compared with control vehicles. N.S. means no significant difference. Data were from 6 (A-D, J-K) or 8 (L) independent experiments. # indicates P<0.05 and ### indicates P<0.001 compared with control group.
PTP-MEG2 regulates quantal size and fusion pore opening through two distinct structural bases and substrates (2021)
Figure 2

Figure 2 Interaction of PTP-MEG2 and tyrosine-phosphorylated NSF in the medulla and the crystal structure of the PTP-MEG2/phospho-NSF-segment complex.

(A). NSF was phosphorylated after stimulation with AngII or KCl. Adrenal medulla cells were stimulated with 100 nM AngII or 70 mM KCl for 5 min and lysed. NSF was immunoprecipitated with a specific NSF antibody coated with Protein A/G beads. A pan-phospho-tyrosine antibody pY20 was used in Western blot to detect the tyrosine-phosphorylated NSF in the adrenal medulla under different conditions.

(B). NSF was co-localized with PTP-MEG2 in the adrenal medulla. After stimulation with 100 nM AngII, NSF and PTP-MEG2 in the adrenal medulla were visualized with immunofluorescence. White arrow stands for co-localization of the PTP-MEG2 and NSF.

(C). Analysis of PTP-MEG2 and NSF fluorescence intensities by Pearson's correlation analysis. The Pearson's correlation coefficient was 0.7.

(D). Phosphorylated NSF interacted with PTP-MEG2. PC12 cells were transfected with FLAG-NSF. After stimulation of the cells with 100nM AngII in the presence or absence of 100μM H2O2 for 5 minutes (H2O2 was exploited to increase the overall tyrosine phosphorylation of the cellular proteins), the potential PTP-MEG2 substrate in cell lysates was pulled down with GST-PTP-MEG2-D470A or GST control.

(E). The overall structure of PTP-MEG2-C515A/D470A in complex with the NSF-pY83 phospho-segment.

(F). The 2Fo-Fc annealing omit map (contoured at 1.0σ) around the NSF-pY83 phospho-segment. The P-loop was highlighted in blue.

(G). Plot of distance RMSDs of individual residues between the crystal structures of the PTP-MEG2/NSF-pY83 phospho-peptide complex (PDB: 6KZQ) and the PTP-MEG2 native protein (PDB: 2PA5).

(H). Superposition of the PTP-MEG2/NSF-pY83 phospho-peptide complex structure (red) on the PTP-MEG2 native protein structure (PDB: 2PA5, blue). The structural rearrangement of the WPD loop and β3-loop-β4 is highlighted.

(I, J). The closure of the WPD loop and corresponding conformational changes in the inactive state (I) and the active state (J) of PTP-MEG2. The rotation of R521 leads to the movement of W468 and a corresponding 7 Å movement of the WPD loop.

(K, L). The structural rearrangement of β3-loop-β4 of PTP-MEG2 in the active state (L) compared with the inactive state (K). The disruption of the salt bridge between E406 and R521 contributed to the new conformational state of the N-terminal of β3-loop-β4.

(M, N). The conformational change of the P-loop of the inactive state (M) and the active state of PTP-MEG2 in response to NSF-pY83 segment binding (N). The disruption of the charge interaction between R409 and D335 resulted in the movement of the main chain from G408 to K411.
PTP-MEG2 regulates quantal size and fusion pore opening through two distinct structural bases and substrates (2021)
Figure 3

Figure 3. Molecular determinants of PTP-MEG2 interaction with the NSF-pY83 site.

(A). Schematic representation of interactions between PTP-MEG2 and the NSF-pY83 site.

(B). Relative values of the decreased phosphatase activities of different PTP-MEG2 mutants towards pNPP (p-Nitrophenyl phosphate) compared with wild-type PTP-MEG2.

(C). Fold-change decreases in the phosphatase activities of different PTP-MEG2 mutants towards the NSF-pY83 phospho-segment compared with wild-type PTP-MEG2.

(D). Sequence alignment of PTP-MEG2 from different species and with other PTP members. Important structural motifs participating in phosphatase catalysis or the recognition of the NSF-pY83 site are shown, and key residues contributing to the substrate specificity are highlighted.

Data information: *** P<0.001: PTP-MEG2 mutants compared with the control group. Data were obtained from 3 independent experiments. N.S. means no significant difference. All the data were analyzed using one-way ANOVA, and displayed as the mean ± s.e.m. In (B, C) the red column indicates that the mutation sites have greater effects on the enzyme activities toward phospho-NSF segement than the pNPP. The green column indicates that the mutation sites only have significant effects on the enzyme activity toward NSF phospho-segement but not pNPP. The white column stands for the mutants that have similar effects on pNPP and NSF phospho-segement.
PTP-MEG2 regulates quantal size and fusion pore opening through two distinct structural bases and substrates (2021)
Figure 5

Figure 5. Effects of different PTP-MEG2 mutants on properties of catecholamine secretion from primary chromaffin cells.

(A). Primary mouse chromaffin cells were transduced with a lentivirus containing the gene for wild-type PTP-MEG2 or different mutants with a GFP tag at the C-terminus. Positive transfected cells were confirmed with green fluorescence and selected for electrochemical analysis. Typical amperometric current traces evoked by AngII (100 nM for 10 seconds) in the control (transduced with control vector) (top left panel), WT (top right panel), G334R (bottom left panel), and D335A (bottom right panel) are shown.

(B). The schematic diagram shows key residues of PTP-MEG2 defining the substrate specificity adjacent to the pY83 site of NSF.

(C). The distribution of the quantal size of chromaffin cells transduced with lentivirus containing the genes encoding different PTP-MEG2 mutants.

(D). Statistical diagram of the quantal size in Fig. 5A and Appendix Figure S6A-D. The secretion amount of each group was standardized with respect to the control group.

(E-G). Calculated parameters of secretory dynamics, including the spike amplitude (E), PSF frequency (F), and SAF frequency (G).

Data information: (B, D-G) Green color represents the amino acids which are found to modulate foot probability based on structure analysis, and red color means amino acids which does not regulate foot probability. (D-G), * indicates PTP-MEG2 mutants overexpression group compared with the WT overexpression group. # indicates the overexpression group compared with the control group. **, P<0.01; *** P<0.001 and #, P<0.05; ##, P<0.01; ### P<0.001. N.S. means no significant difference. Data were from 6 independent experiments. All the data were analyzed using one-way ANOVA and showed as the mean ± s.e.m.
PTP-MEG2 regulates quantal size and fusion pore opening through two distinct structural bases and substrates (2021)
Figure 6

Figure 6. Identification of potential candidate substrates of PTP-MEG2 that participate in fusion pore initiation and expansion.

(A). Flowchart for the workflow to predict the candidate substrates of PTP-MEG2 during fusion pore initiation and expansion. A total of 51 proteins were enriched with the functional protein association network STRING and the text mining tool PubTator by searching the keywords "fusion pore", "secretory vesicle" and "tyrosine phosphorylation". These proteins were filtered with UniProt by selecting proteins located only in the membrane or vesicle, which resulted in 28 candidates. The Human Protein Atlas database was then applied to exclude proteins with no expression in the adrenal gland. Finally, we used the post-translational-motif database PhosphoSitePlus to screen candidate proteins with potential phospho-sites that matched our sequence motif prediction at the pY+1 or pY+2 positions.

(B). After the bioinformatics analysis, a total of 12 candidate PTP-MEG2 substrates that may participate in fusion pore initiation and expansion and their potential phospho-sites were displayed.

(C). The GST pull-down assay suggested that PACSIN1, MUNC18-1, VAMP7, SNAP25, DYNAMIN1 and DYNAMIN2 directly interact with PTP-MEG2. PC12 cells were transfected with plasmids of candidate substrates, including SYN1, MUNC18-3, PACSIN1, SCAMP1, MUNC18-1, PPP3CA, STX17, VAMP7, SYT7, SYT11, SNAP25, DYNAMIN1, and DYNAMIN2 stimulated with 100 nM AngII. The tyrosine phosphorylation of these proteins was verified by specific anti-pY antibodies (Appendix Figure S8A-E). The potential substrates of PTP-MEG2 in cell lysates were pulled down with a GST-PTP-MEG2-D470A trapping mutant and then detected by Western blot. White arrow stands for Western blot band consistent with the predicted molecular weight of the potential PTP-MEG2 substrate.

(D-F). Co-immunostaining assays of PTP-MEG2 with potential substrates in the adrenal medulla. MUNC18-1, VAMP7 and DYNAMIN2 all showed strong co-localization with PTP-MEG2 after 100 nM AngII stimulation in the adrenal medulla. White arrow stands for co-localization of PTP-MEG2 with MUNC18-1, VAMP7 or DYNAMIN2. The Pearson's correlation coefficients for (D, E and F) were 0.61, 0.65 and 0.79 respectively. The co-immunostaining results of PTP-MEG2 with other potential substrates are shown in Appendix Figure S7.
PTP-MEG2 regulates quantal size and fusion pore opening through two distinct structural bases and substrates (2021)
Figure 8

Figure 8. The structural details of the PTP-MEG2-MUNC18-1-Y145 interaction and catalytic bias of PTP-MEG2 towards NSF-pY83 and MUNC18-1-pY145

(A). The 2Fo-Fc annealing omit map (contoured at 1.0σ) around MUNC18-1-pY145 phospho-segment.

(B). Comparison of residues of PTP-MEG2 interacting with NSF and MUNC18-1. Amino acid residues of NSF and MUNC18-1 are coloured as follows: green, residues interacting with both NSF and MUNC18-1; red, residues specifically contributing to NSF recognition; blue, residues selectively contributing to MUNC18-1 interaction.

(C). The structural alteration of the interactions surrounding Y471 of PTP-MEG2 with the MUNC18-1-pY145 site.

(D). The structural alteration of the interactions surrounding I519 of PTP-MEG2 with the MUNC18-1-pY145 site.

(E). Relative phosphatase activities of different PTP-MEG2 mutants towards the MUNC18-1-pY145 phospho-segment compared with wild-type PTP-MEG2.

(F). Relative phosphatase activities of different PTP-MEG2 mutants towards the DYNAMIN 2-pY125 phospho-segment compared with wild-type PTP-MEG2.

(G).The percentages of PSF and SAF for PTP-MEG2-R409A or R410A were calculated.

(H). Schematic illustration of the PTP-MEG2-regulated processes of vesicle fusion and secretion in chromaffin cells via the dephosphorylation of different substrates with distinct structural basis. PTP-MEG2 regulates vesicle fusion and vesicle size during the catecholamine secretion of adrenal gland by modulating the phosphorylation state of the pY83 site of NSF, which relies on the key residues G334, D335 (pY loop), Y471 (WPD loop), I519 (P-loop) and Q559 (Q loop). PTP-MEG2 regulates the fusion pore initiation and expansion procedures of catecholamine secretion by the adrenal gland (also designated as foot probability) by modulating the newly identified substrate MUNC18-1 at its pY145 site and DYNAMIN2 at its pY125, through distinct structural basis from that of its regulation of NSF phosphorylation.

Data information: (E, F) Residues are coloured according to Figure 8B. (E, F), * P<0.05, ** P<0.01, *** P<0.001: PTP-MEG2 mutants compared with the control. N.S. means no significant difference. Data were obtained from 3 independent experiments. (G), * indicates PTP-MEG2-R409A or R410A mutants overexpression group compared with the WT overexpression group. # indicates the PTP-MEG2 overexpression group compared with the control group. **, P<0.01; *** P<0.001 and ## P<0.01; ### P<0.001. N.S. means no significant difference. Data were obtained from 10 cells in each group of 6 independent experiments. . All the data were analyzed using one-way ANOVA and showed as the mean ± s.e.m.

.
Negative regulation of the NEMO signaling by the ubiquitin E3 ligase MARCH2 (2020)
Figure 1

Figure 1. Deficiency of MARCH2 strengthens antimicrobial immunity in vivo.

A, B MARCH2+/+ (n = 8) or MARCH2-/- (n = 8) mice were intravenously infected with VSV-GFP (2 × 108 PFU/mouse), and serum samples were collected at 12 hpi (hours post-infection). (A) Virus titer was analyzed in a plaque assay. (B) Secretion of IFN-β, IL-6, IL-12, and TNFα was measured in specific ELISAs.

C MARCH2+/+ (n = 6) or MARCH2-/- (n = 6) mice were intravenously injected with poly(I:C) (200 μg/mouse). Serum samples were collected at 2 hpi and secretion of IFN-β, IL-6, IL-12, and TNFα was measured in specific ELISAs.

D-J MARCH2+/+ (n = 8) or MARCH2-/- (n = 8) mice were intraperitoneally challenged with 1 × 106 CFU of Listeria monocytogenes. (D) The percentage of surviving mice (D, log-rank test, **P < 0.01) and body weight changes for each group (E) are shown. (F, G) At 3 dpc, the bacterial load in the spleen (F) and liver (G) was examined. (H-J) Amount of IL-6, IL-12, CCL5, and CXCL10 in serum (H), spleen (I), and liver (J) of mice in each group at 24 hpc.

Data information: *P < 0.05, **P < 0.01 (two-tailed Student's t-test). Data are expressed as the mean ± SEM.

Source data are available online for this figure.
Negative regulation of the NEMO signaling by the ubiquitin E3 ligase MARCH2 (2020)
Figure 2

Figure 2. Deficiency of MARCH2 induces susceptibility to LPS-induced septic shock.

A-B MARCH2+/+ (n = 8) or MARCH2-/- (n = 8) mice were challenged intraperitoneally with LPS (24 mg/kg). (A, B) Percentage of surviving mice (A, log-rank test, **P < 0.01) and body weight changes (B) in each groups.

C MARCH2+/+ (n = 4) or MARCH2-/- (n = 4) mice were challenged intraperitoneally with LPS (24 mg/kg). Representing slides of H&E staining of spleen sections from each group. Scale bar, 6mm.

D MARCH2+/+ (n = 6) or MARCH2-/- (n = 6) mice were challenged intraperitoneally with LPS (24 mg/kg). Levels of IL-6, TNF-α, CXCL-10, CCL-5 in serum from mice in each groups were measured at12 hpc by ELISA.

E, F MARCH2+/+ (n = 5) or MARCH2-/- (n = 5) mice were challenged intraperitoneally with LPS (24 mg/kg). cDNA was prepared from total RNA extracted from spleen and liver of mice. Expression of mRNA encoding IL-6, TNF-α, CXCL10, IL-1β in spleen (E) and liver (F) from mice in each group was examined at 6 hpc by qPCR.

Data information: *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed Student's t-test). Data are expressed as the mean ± SEM.

Source data are available online for this figure.