EMBO/SourceData huggingface dataset:
Figure 4A Effects of the HDAC6 inhibitor treatment on IL-1β secretion after NLRP3 inflammasome activation. ELISA assays show IL-1β secretion and caspase-1 processing (p10) in BMDMs upon NLRP3 inflammasome activation following pretreatment with tubacin (left to right: 10 and 20 mM) or rocilinostat (left to right: 30 and 60 mM).Co-immunoprecipitation assay showing the interaction between Lamtor1 and HDAC6. Immunoblot analysis of Lamtor1-Flag co-immunoprecipitated with HDAC6-myc from lysates of HEK293T cells transfected with the indicated plasmidsCo-immunoprecipitation assay showing the interaction between Lamtor1 and HDAC6. Immunoblot analysis of Lamtor1-Flag co-immunoprecipitated with HDAC6-myc from lysates of HEK293T cells transfected with the indicated plasmidsCo-immunoprecipitation assay showing the interaction between Lamtor1 and HDAC6. Lamtor1-Flag co-immunoprecipitated with endogenous HDAC6 from the lysate of Lamtor1-Flag knocked-in THP-1 cells. (D)Endogenous Lamtor1 co-immunoprecipitated with endogenous HDAC6 from lysates of WT THP-1 cells Effects of NLRP3 inflammasome activation on the interactions between Lamtor1 and HDAC6. Co-immunoprecipitation assay shows interactions between Lamtor1 and HDAC6.Endogenous Lamtor1 co-immunoprecipitated with endogenous HDAC6 from lysates of WT THP-1 cells Effects of NLRP3 inflammasome activation on the interactions between Lamtor1 and HDAC6. Co-immunoprecipitation assay shows interactions between Lamtor1 and HDAC6.G PLA to confirm endogenous binding between Lamtor1 and HDAC6. Lamtor1-HDAC6 complexes are shown in red, and nuclei are depicted in blue. In situ PLA signals were quantified over nine images in each experiment.H NanoBRET assay to confirm endogenous binding between Lamtor1 and HDAC6 in living cells. HEK293T cells were transiently transfected with NanoLuc-fused Lamtor1 and Halo-tag-fused HDAC6 using Lipofectamine 2000. Luminescence was measured 48 h after transfection following the addition of Nano-Glo Luciferase Assay Substrate (Promega).I Immunoblot analysis of the truncated form of Flag-tagged Lamtor1 mutant (∆145-161, Met1-144; ∆95-161, Met1-Val94) co-immunoprecipitated with HDAC6-myc from lysates of HEK293T cells transfected with the indicated plasmids.Effect of truncated Lamtor1 mutant on NLRP3 inflammasome activation in THP-1 cells. ELISA assay showing IL-1β secretion in supernatants from WT cells, Lamtor1 KO cells, Lamtor1 KO cells reconstituted with full-length Lamtor1, and Met1-Val94 Lamtor1 cells; all cell types were treated with nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight).
Figure 5A Yeast two hybrid (Y2H) assays for Lamtor1 interactions with NLRP3. Positive Y2H interaction between Lamtor1 as prey, and NLRP3 as bait. Lamtor2 as prey was used as a positive control and the empty bait vector as a negative control.B PLA to confirm endogenous binding between Lamtor1 and NLRP3. Lamtor1-NLRP3 complexes are shown in red, and nuclei are depicted in blue. In situ PLA signals were quantified over 10 images in each experiment.C, D Immunoblot analysis of Lamtor1-V5 co-immunoprecipitated with NLRP3-Flag from lysates of HEK293T cells transfected with the indicated plasmids.Immunoblot analysis of the truncated form of V5-tagged Lamtor1 mutant (∆145-161, Met1-Ser144) co-immunoprecipitated with NLRP3-flag from lysates of HEK293T cells transfected with the indicated plasmids.Immunoblot analysis of the truncated form of V5-tagged Lamtor1 mutant (∆145-161, Met1-Ser144) co-immunoprecipitated with NLRP3-flag from lysates of HEK293T cells transfected with the indicated plasmids.G Effect of the HDAC6 knockdown on NLRP3-Lamtor1 interactions. Immunoblot analysis of Lamtor1-V5 co-immunoprecipitated with NLRP3-Flag from lysates of HEK293T cells transfected with the indicated plasmids.
Figure 6A, B Representative images of Lamtor1 and ASC. PMA-treated THP-1 cells stably expressing GFP-tagged ASC and td-Tomato-tagged Lamtor1 were primed with LPS (200 ng/mL, 2 h) and then stimulated with nigericin for the indicated duration. Time shows after nigericin stimulation (min:s).C Effects of Lamtor1 deficiency on ASC speck formation after NLRP3 inflammasome activation. Representative images of PMA-primed THP-1 macrophages stably expressing GFP-tagged ASC treated with or without LPS (200 ng/mL, 2 h) and nigericin (15 μM, 1 h). Scale bars = 10 μm.
Figure 7Effects of DL-all-rac-α-tocopherol on the interactions between Lamtor1 and HDAC6. Immunoblot analysis of Lamtor1-Flag co-immunoprecipitated with HDAC6-myc from lysates of HEK293T cells transfected with the indicated plasmids after pretreatments with the DL-all-rac-α-tocopherol (B)IL-1β secretion upon NLRP3 inflammasome activation after overnight pretreatment with DL-all-rac-α-tocopherol (C) at the indicated concentrations. ELISA assay showing IL-1β secretion in supernatants from WT THP-1 cells treated with LPS (200 ng/mL, 2 h) and nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight).D) IL-6 secretion after overnight pretreatment with DL-all-rac-α-tocopherol. ELISA assay showing IL-6 secretion in supernatants from WT BMDMs treated with LPS (200 ng/mL, overnight) and DL-all-rac-α-tocopherol at the indicated concentrations (overnight).Effects of α-tocopherol on the interactions between Lamtor1 and HDAC6. Immunoblot analysis of Lamtor1-Flag co-immunoprecipitated with HDAC6-myc from lysates of HEK293T cells transfected with the indicated plasmids after pretreatments with the D -α-tocopherol (E)IL-1β secretion upon NLRP3 inflammasome activation after overnight pretreatment with D -α-tocopherol (F) at the indicated concentrations. ELISA assay showing IL-1β secretion in supernatants from WT THP-1 cells treated with LPS (200 ng/mL, 2 h) and nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight).G Effects of DL-all-rac-α-tocopherol in an acute gouty arthritis model. MSU crystals (0.5 mg) suspended in 20 μL endotoxin-free PBS or PBS control were injected intra-articularly into the tibia-tarsal joint (ankle) of C57BL/6 WT mice with or without DL-all-rac-α-tocopherol. Ankle joint swelling at 24 h was assessed with electronic calipers and with photomicrographs and hematoxylin and eosin-stained sections of ankle joints.H Effects of DL-all-rac-α-tocopherol in an MSU-induced peritonitis model. Twelve hours after DL-all-rac-α-tocopherol pretreatment, 100 μL MSU solution (10 mg/mL) was intraperitoneally injected for 4 h. Peritoneal IL-1β production was then measured by ELISA, and CD11b+ Ly6G+ neutrophil recruitment was measured by FACS.
Figure 1B. Western blot analysis of purified autophagic vesicles. Isolations were performed using antibodies directed against LC3B or all GABARAP isoforms, respectively, and are represented with total lysate (TL). Depicted are representative blots of 14 independent approaches.
Figure 2A. Differential interference contrast microscopy images of purified autophagic vesicles at high (I) or low (II) dilution. Images are representative of 3 independent approaches. B. Negative stain electron microscopy images of isolated vesicles. C. Size evaluation of isolated vesicles. The diameters of approx. 60 individual vesicles were determined using EM images.D. Western blot analysis of isolated autophagic vesicles upon proteinase K digestion. Mechanically opened vesicles served as positive control. For negative control, isolates were incubated with BSA instead of proteinase K. Depicted are two different blots that are representative for 5 independent experiments
Figure 3E. Phospholipids identified in autophagic vesicles isolated upon different conditions. Relative amounts were calculated based on total levels of detected phospholipids. Abbreviations are depicted in A.
Figure 4A. Volcano plot showing the differential appearance of proteins in autophagic vesicles of EBSS treated cells in comparison to vesicles isolated under basal autophagy. Log2-transformed fold changes. For proteins that were excluded from autophagic vesicles upon EBSS treatment or which exclusively appeared within these vesicles, no fold changes could be calculated and they are indicated as not determinable (nd). B. Volcano plot showing the differential appearance of proteins in autophagic vesicles of MG132 treated cells in comparison to vesicles isolated upon basal autophagy. Log2-transformed fold changes. For proteins that were excluded from autophagic vesicles upon MG132 treatment or which exclusively appeared within these vesicles, no fold changes could be calculated and they are indicated as not determinable (nd).
Figure 1(A) Classical, (B) Rolled, and (C) Rotated subpopulations of the 80S*HCVIRES complex. Left column shows an overview of the final cryo-EM reconstructions with the HCVIRES (pink), 40S subunit (yellow), 60S subunit (blue) and tRNA (green). Middle and right column shows mesh representation of the maps and docked models of the 60S and 40S parts, respectively. 28S-5S-5.8S rRNA (blue), 60S proteins (gold), 18S rRNA (yellow), 40S proteins (dark gray), HCVIRES (pink), tRNA (green).(D) Subpopulations of the 80S*HCVIRES complex containing P-site tRNA. The 80S is in the classical configuration and the head tilt is not present. Left column shows an overview of the final cryo-EM reconstructions with the HCVIRES (pink),40S subunit (yellow),60S subunit (blue) and tRNA (green). Middle and right column shows mesh representation of the maps and docked models of the60S and40S parts, respectively.28S-5S-5.8S rRNA (blue), 60S proteins (gold), 18S rRNA (yellow), 40S proteins (dark gray),HCVIRES (pink), tRNA (green).(E) The cryo-EM map was filtered according to resolution. 40S*HCV IRES. Left column shows an overview of the final cryo-EM reconstructions with the HCV IRES (pink), 40S subunit (yellow), 60S subunit (blue) and tRNA (green). Middle and right column shows mesh representation of the maps and docked models of the 60S and 40S parts, respectively. 28S-5S-5.8S rRNA (blue), 60S proteins (gold), 18S rRNA (yellow), 40S proteins (dark gray), HCV IRES (pink), tRNA (green).
Figure 2(A) Comparison of 40S subunit positions in Rolled, Rotated, Classical/no head tilt (orange) and Classical 80S*HCVIRES complex (yellow) in common 60S alignment. The cryo-EM map of the Classical 80S*HCVIRES was filtered to 6 Å. Arrows indicate the direction of movement during transition between two different states. The distance changes in the 40S subunit positions resulting from the rigid body transformation are color-coded in Å units.(B) Comparison of the HCVIRES in Classical 80S*HCVIRES complex (gray) and upon P-tRNA binding (Classical, no head tilt; purple) in a common 40S body alignment. The distance of the movement of domain II was indicated with an arrow and Å units
Figure 3(A) Adopted secondary structure of the IRES representing the tertiary fold.(B) Atomic model of the HCV IRES RNA. IRES domains are presented as coloured ribbons, overlaid with experimental IRES density (gray, semi-transparent).(C). Molecular model of the HCV IRES (pink) with ribosomal interaction sites (coloured proteins and helices).
Figure 4(A) The secondary structure of the HCVIRES domain III (hIII1, hIII2, IIIe and IIId) and helix 26 (h26) from the 18S rRNA. Colors of HCVIRES Domains are the same as in Fig 3. Interaction between the HCVIRES and 18S rRNA are indicated as red lines and a tertiary IRES interaction is indicated as black line.(B) The interaction core between IIId, IIIe, hIII1 of HCVIRES (colored as in (A)) and Expansion segment 7 (ES7) of h26 (dark gray).(C) The rearrangement of h26 (red) upon binding of HCVIRES. The 80S*HCVIRES complex (yellow) is compared to the elongating POST state (gray; PDB 5Aj0) (Behrmann et al, 2015). The loop closing the helix is highlighted in red and dark gray.(E) Close up on the interactions between GGG266-268 of IIId (cyan) and CCC1116-1118 of ES7 (gray).(F) Close up on the interactions between G295, A296 and U297 of IIIe (green), A136 and A288 of hIII1 (yellow) and U1114, U1115 of ES7 (gray).
Figure 5(A) Comparison of the 40S subunit from Classical 80S*HCVIRES complex (yellow) and canonical POST state (gray; EMD 2875) (Behrmann et al, 2015) in common 40S body alignment. The arrow indicates the direction of the head tilt movement. Ribosomal proteins of the 40S head (uS7, eS25 and eS28) that interact with the HCVIRES domains II and IV (pink) on the 40S head are illuminated with blue, green, and purple, respectively. The positions of the proteins in the canonical POST state are illuminated in lighter color. The numbers indicate interaction site of HCVIRES and the 40S subunit.(B) Close up view on the interaction site of HCVIRES and the 40S head region. Numbers correspond to red labels in (A). The rearrangement of the uS7 loop (blue) compared to the Classical 80S*HCVIRES complex (gray) is indicated. The arrow indicates the direction of the rearrangement of the uS7 loop. (B-1 and B-4) inter subunit view from 40S subunit. (B-2 and -3) inter subunit view from 60S subunit.
Figure 6(A, B) Comparative solvent side views of the small subunit with the bacterial SD-chamber on the 30S from the 70S POST state (PDB 2HGR) (Yusupova et al, 2006) and the corresponding region of the eukaryotic 40S in the classical 80S*HCV IRES complex. Analogous proteins and rRNA helices are highlighted in the same colours, respectively. (A) Crystal structure of the 30S subunit with mRNA. The SD-helix is established between the mRNA (orange) and the 3' end of the 16S rRNA (blue).(B) Atomic model of the 40S subunit with the HCV IRES model (pink). The mRNA-part and S2 of the IRES are shown in orange and cyan, the 3'-end of the 18S rRNA is shown in blue.(C) Overview and zoomed view of Classical 80S*HCVIRES. The cryo-EM map of the Classical 80S*HCVIRES was filtered according to resolution.(D and E) mRNA exit channel from in Classical 80S*HCVIRES and POST state (PDB 5AJ0) (Behrmann et al, 2015). The two structures are aligned at the 40S body. We note eS28 as part of the 40S head is differently located because of the head tilt. The surface representation was created using the pdb models. Shown are the 18S rRNA (yellow), ribosomal proteins (gray), eS26 (cyan) eS28 (purple), HCVIRES (pink).
Figure 7(A) Comparison of HCV IRES from 80S*HCV IRES Classical (pink) and tRNAs from POST state (gray)(PDB 5AJ0) (Behrmann et al, 2015) in a common 40S body alignment of the cryo-EM maps.(B) Same as above but in a common 40S head alignment.(C) Comparison of the 40S subunit and bound ligands of the classical 80S*HCVIRES complex (orange) and 80S*HCVIRES*Met-tRNA*eIF5B*GMPPNP complex (POST-like state) (yellow)(EMD 2683) (Yamamoto et al, 2014) in common 60S alignment.(D) The models of the 80S*HCVIRES Classical complex (gray), 80S*HCVIRES*Met-tRNA*eIF5B*GMPPNP complex (POST-like state) (green)(PDB 4UPZ) (Yamamoto et al, 2014) and 80S*HCVIRES Classical/no head tilt complex (pink) are rigid body fitted into the corresponding densities in a common 40S body alignment. The Helix 68 from the 28S rRNA is depicted as well (blue).
Figure 8(A and B) Comparison of the 80S*HCV IRES Classical complex and Classical/no head tilt complex (40S yellow, 60S blue, IRES pink, P-tRNA green) at intersubunit view. Inter subunit bridge 1b/c and 1a are indicated as B1b/c and B1a, respectively. The cryo-EM map of classical 80S*HCV IRES classical complex was filtered to 6 Å.(C and D) Close up of the mRNA tunnel. The distance between helix 29 and 44 of the 18S rRNA are indicated with an arrow and Å units
Figure 5(C) Benign breast epithelial MCF10A and breast carcinoma BT-474 cells were embedded in 3D collagen as single cells or as spheroids, respectively, and the growth was followed for 5 days. Light micrographs show filamentous actin (phalloidin) and nuclei (Hoechst) in representative cell colonies. Quantitative assessment of the nuclei counts per colony show the induced proliferation in MCF10A cells after ECHCD1 sgRNA knockout. At 72 hours, MCF10A mock versus ECHDC1_sgRNA_1 and ECHDC1_sgRNA_2 P < 0.05; at 96 hours mock versus ECHDC1_sgRNA_1, ECHDC1_sgRNA_2 and ECHDC1_sgRNA_3 P < 0.001; at 120 hours mock versus ECHDC1_sgRNA_1, ECHDC1_sgRNA_2 and ECHDC1_sgRNA_3 P < 0.0001. Nuclei count relative to mock 0 hours. Error bars indicate mean ± SEM; n ≥ 10 colonies. Statistical significance was assessed with one-way ANOVA with Tukey's multiple comparison test. Scale bar 50 µm.(E) Measured metabolite levels of intermediates in propanoate metabolism in select breast cancer cell lines with or without the ECHDC1 rCCS status (n=7 in both groups). Boxes represent the interquartile range, whiskers represent the range of the values and solid line within the box correspond to the median value. Outlier points indicates values not included between the whiskers. Statistical significance was assessed with Wilcoxon test.
Figure 6(H) Survival analysis based on mRNA expression levels of DDX27 in patients with endometrial cancer in the TCGA dataset. Expression levels were divided into 2 classes, high (n=203) and low (n=322), based on mean expression level of DDX27 (logFPKM=18.27). Patients in the high class showed lower survival probability than those in the low class (P=.4.2×10-4; log-rank test).(I) mRNA expression levels of DDX27 in PTEN mutated (Mut, n=302) and PTEN wild type (WT, n=224) endometrial patient tumors in the TCGA dataset. Triangles correspond to the median value. P-value was calculated with Wilcoxon test.
Figure 2A FBN2 mRNA levels in multiple human tissues. Human tissue cDNAs were used as templates for PCR of FBN2, followed by agarose gel analyses. GAPDH levels served as loading controls (biological replicates, n=2).B FBN2 mRNA levels in mouse tissues based on quantitative RT-PCR. Data were normalized based on levels in adipose tissues.C Comparison of FBN2 transcript levels between human and mouse placentas. Common primers for FBN2 and GAPDH in both species were used for RT-PCR analyses (biological replicates, n=3) .D Immuno-histochemical detection of placensin in human placental villi obtained from women at 9 and 38 weeks of pregnancy. During early pregnancy, placensin immunostaining was found in cytotrophoblasts (c), whereas syncytiotrophoblasts (s) showed weak staining. At 38 weeks of pregnancy, strong placensin signals were detected in both syncytiotrophoblasts and cytotrophoblasts (arrows). Staining using E-cadherin antibodies confirmed the identity of cytotrophoblasts (Inset showing higher magnification E-cadherin (E-Cad) staining). Lower panels depict sections stained with nonimmune IgG as negative controls. Scale bars, 100 μm.E Immunoblotting of placensin in human placental villi (term pregnancy). Samples from three individual patients were processed with or without N-glycosidase pre-treatment to reveal untreated and deglycosylated placensin.F For prokaryotic cell expression, a cDNA fragment corresponding to residues 2779 to 2912 of the human FBN2 coding region was appended with GST and 6-histidine tags and sub-cloned into the PGEX-6P-1 vector for expression in E. coli. Left panel: Coomassie blue staining of proteins in the SDS-PAGE gel before and after digestion with the PreScission protease. 1: Flow through; 2: Washes; 3: Before PreScission treatment; 4: After PreScission treatment. Right panel: Immunoblotting using placensin antibodies.G Immunoblotting of recombinant placensin generated in E. coli. and CHO cells. For CHO cell expression, placensin cDNA sequence was subcloned into an adenoviral vector pAV[Exp]-CMV downstream of the IgK signal peptide under the control of the CMV promoter for infection of cells before immunoblotting of media 2 days later. Recombinant placensin proteins secreted from CHO cells (b) showed higher molecular weight than those from bacteria (a). Following treatment with N-glycosidase to remove N-linked carbohydrate side chains, prokaryotic- and eukaryotic cell-derived placensin proteins showed similar sizes.
Figure 3A Prokaryotic cell-derived placensin stimulated cAMP production (at 10 min.), and PKA activity (at 30 min.) by primary mouse hepatocytes and hepatocyte-derived AML12 cells (biological replicates, n=5).B Placensin stimulation of glucose secretion (at 5h) and key gluconeogenesis gene transcripts (at 1h) in both cell types (biological replicates, n=4). Transcript levels for PEPCK and G6Pase were determined using quantitative RT-PCR. Treatment with glucagon (10 nM) served as positive controls.C Liver from adult female mice was perfused in situ with placensin (1.0 ug/pulse) or PBS before measurement of hepatic glucose output from inferior vena cava (biological replicates, n=3).D Adult female mice were injected intraperitoneally with placensin (30 ug/injection) with blood sampling at different time points for glucose levels (biological replicates, n=5).
Figure 4A Left panel: Immunoblotting of placensin content in HTR-8/SVneo, but not BeWo, JAR, and JEG3, cells. Right panel: Time-dependent secretion of placensin by HTR-8/SVneo, but not JAR, cells monitored using an ELISA (biological replicates, n=4-5).B Knockdown of FBN2 in HTR-8/SVneo cells led to decreases in FBN2 transcripts and placensin secretion. Cells were treated with FBN2 siRNA (SiFBN2; 20 nM) or control siRNA (SiC; 20 nM) for 6h. Following media change, cells and conditioned media were collected at 48h after incubation. Placensin transcripts and proteins were determined by RT-PCR and ELISA (biological replicates, n=8), respectively. Lower panel: immunoblotting of secreted placensin by HTR-8/Svneo.C Placensin stimulation of cAMP production and PKA activities by HTR-8/SVneo cells. Cells were treated with placensin for 10 min. before cAMP measurement (biological replicates, n=5). For PKA activities, cells were treated with placensin (50 ng/ml) for 30 min. Treatment with forskolin (10 uM) served as positive controls.D Placensin stimulation of transcript levels for MMP9 but not MMP2(biological replicates, n=12). Cells were treated with placensin for 24h before RT-PCR analyses of transcript levels.E Placensin stimulation of MMP9, but not MMP2, activities without affecting cell proliferation. After 24h of treatment with placensin, media were collected for zymographic and densitometric analyses as well as determination of total cell numbers (biological replicates, n=4).F Placensin stimulation of HTR-8/SVneo cell invasion. After placensin treatment for 24h, cell invasiveness was determined using the Transwell assay (left panel: micrographs of cells; right panel: fold changes in number of migrated cells, biological replicates, n=4). Cells were incubated with mitomycin C to rule out effects on cell proliferation. Scale bars: 100μm. C: control.
Figure 5A Immunoblotting of serum placensin from patients at second trimester (1-3), term pregnancy (4-6) using specific placensin antibodies.B Serum levels of placensin and glucagon at different weeks of pregnancy measured using specific ELISA in normal and GDM patients. Numbers in parentheses represent number of patients.