Journal: Oncotarget

Article Title: Regulation of tumor growth by circulating full-length chromogranin A

doi: 10.18632/oncotarget.12237

Figure Lengend Snippet: A. Induction of protease nexin-1 mRNA in endothelial cells . HUVECs were treated with various doses of recombinant CgA for 20 h. The presence of PN-1 transcripts in cells was analyzed by real time PCR. PN-1 mRNA expression is reported as fold change compared to untreated cells. The cumulative results of 3 experiments are shown: in each experiment each sample was analyzed 5-6 times by real-time PCR (each in triplicate). Box-plots with median, interquartiles and 5-95 percentile values are shown (dotted line, mean value of controls). ***, P < 0.001, by t test (2-tailed). B. Effect of PN1 gene silencing on the activity of CgA in the endothelial spheroid sprouting assay . HUVEC cells spheroids were transfected with PN-1 siRNA mixture (siPN1) or with control siRNA (siCtrl) as described in Methods. After transfection, spheroids were treated with or without and 0.2 nM recombinant CgA 1-439 for 16 hours. The number of sprouts was from each spheroid was then counted. Cumulative results of two independent experiments (8-16 spheroids/experiment). The number of spheroids used is indicated in each panel ( n ). *, P < 0.05; **, P < 0.01 by t test. C. - D. Effect of anti-PN-1 antibodies on the activity of CgA in the endothelial spheroid sprouting assay . Cumulative results of two independent experiments (8 spheroids/experiment, total n = 16). Spheroids were incubated with or without recombinant CgA (0.2 nM) in the absence or the presence of anti-PN-1 polyclonal immunoglobulins (goat IgGs anti-human Serpin E2/PN1), or control immunoglobulins (normal goat IgGs) as indicated. Bars: mean ± SE. ****, P < 0.0001, ***, P < 0.001, by t test (2-tailed). Representative microphotographs of spheroids are shown in ( C ) (arrows: endothelial cell sprouts). E. Effect of anti-PN-1 antibodies on the anti-tumor activity of CgA . WEHI-164- or TS/A-tumor-bearing mice were treated with recombinant CgA (i.p.) in combination with anti-PN-1, or with control immunoglobulins (normal goat IgGs) at the indicated doses. Antibodies were given 2.5 h before CgA. Arrows indicate the day of treatment. Tumor volume (mean ± SE). The area under the curve for each mouse was calculated using the GraphPad Prism Software. Statistical analysis was performed by Mann-Whitney test on the calculated areas (6 animals per group; **, P < 0.01; *, P < 0.05).

Article Snippet: Goat IgGs anti-mouse serpin E2/PN1, goat IgGs anti-human serpin E2/PN1 and normal goat immunoglobulins were from R&D System.

Techniques: Recombinant, Real-time Polymerase Chain Reaction, Expressing, Activity Assay, Transfection, Incubation, Software, MANN-WHITNEY

Journal: Nature chemical biology

Article Title: Targeting a Helix-in-Groove Interaction Between E1 and E2 Blocks Ubiquitin Transfer

doi: 10.1038/s41589-020-0625-7

Figure Lengend Snippet: ( a ) Structure of the binding interface between the UFD of S. pombe Uba1 (interacting residues colored pink) and the alpha-1 helix (aa 1–18, colored by amino acid property) of the E2 Ubc15 (cyan), as revealed by the x-ray structure of the S. pombe Uba1/Ubc15 complex (PDB: 5KNL) . ( b ) Amino acid sequences of the SAH-Ubc15 E7R staple-scanning library, which was generated by inserting all-hydrocarbon i, i+4 or i, i+7 staples sequentially along the length of the Ubc15 E7R peptide. X, S5-pentenyl alanine; 8, R5-octenyl alanine; B, norleucine (replacement for methionine, whose sulfur residue decreases the efficiency of ruthenium-catalyzed olefin metathesis). ( c ) Helical wheel depiction of SAH-Ubc15 E7R peptides and their staple positions (helical residues span from aa 5–18). ( d ) Inhibition of UBE1-mediated thioester transfer of ubiquitin to the E2 enzyme UBE2D2 by the indicated SAH-UBC15 E7R stapled peptides. Thioester transfer activity was monitored by the shift in E2 molecular weight from 17 kDa (free UBE2D2) to 25 kDa (UBE2D2~ubiquitin conjugate), as detected by gel electrophoresis and silver stain. Vehicle lane contained 1.5% DMSO and control lane lacked UBE1 protein. The bar graph represents mean inhibition relative to vehicle control ± s.e.m., as quantified and averaged across three independent biological replicates. Uncropped gel for panel d is available online. Source data for the thioester transfer assay plot is available online.

Article Snippet: For UBA3 thioester transfer assays, 15 nM NAE1/UBA3 was substituted for UBE1 (Boston Biochem E-313), 135 nM UBE2M for UBE2D2 (Boston Biochem E2–656), and 1 μM NEDD8 (Boston Biochem UL-812) for ubiquitin.

Techniques: Binding Assay, Generated, Residue, Inhibition, Ubiquitin Proteomics, Activity Assay, Molecular Weight, Nucleic Acid Electrophoresis, Silver Staining, Control

Journal: Nature chemical biology

Article Title: Targeting a Helix-in-Groove Interaction Between E1 and E2 Blocks Ubiquitin Transfer

doi: 10.1038/s41589-020-0625-7

Figure Lengend Snippet: ( a ) Amino acid sequences of stapled peptides corresponding to the N terminus and alpha-1 helix of UBE2D2 (aa 1–16), UBE2G2 (aa 1–14), and UBE2A (aa 1–17), which incorporate the optimal staple position identified for SAH-Ubc15–11. X, S5-pentenyl alanine; 8, R5-octenyl alanine; B, norleucine. ( b ) Comparative dose-responsive inhibition of UBE1 by the indicated SAH-E2 h1 peptides, as monitored by thioester transfer assay, gel electrophoresis, and silver stain. ( c ) Quantitation of the data in b by ImageJ analysis. Data are mean percent inhibition ± s.e.m. for three independent biological replicates. ( d ) Circular dichroism spectra of UBE2A (BSTPARRRLBRDFKRLQ, where B is norleucine) and SAH-UBE2A, demonstrating induction of α-helicity by insertion of the i, i+7 staple. ( e ) Inhibition of UBE1-mediated thioester transfer of ubiquitin to the E2 enzyme UBE2D2 by SAH-UBE2A but not the corresponding unmodified template peptide, UBE2A. Data are mean inhibition ± s.e.m. for three independent biological replicates. ( f ) Comparative protease resistance of SAH-UBE2A and UBE2A upon treatment with proteinase K, as measured by HPLC analysis. SAH-UBE2A displays a 23-fold longer half-life than UBE2A. Data are mean ± s.d. for experiments performed in triplicate. ( g ) SAH-UBE2A but not UBE2A was significantly protected from deuterium exchange upon incubation with UBE1. The relative difference in deuterium uptake after 10 sec of labeling is shown. The difference in uptake was calculated from the mean deuterium level for UBE2A or SAH-UBE2A in the presence of UBE1 minus that of the peptide alone. Mean deuterium levels were obtained from at least two independent biological replicates of each condition. ( h ) C-terminally biotinylated SAH-UBE2A directly bound to recombinant human UBE1 (MW 118 kDa), as demonstrated by streptavidin capture, gel electrophoresis, and silver stain. ( i ) C-terminally FITCylated SAH-UBE2A bound to UBE1 with a K d of 384 ± 43 nM, as assessed by fluorescence polarization assay. Error bars are s.d. for assays conducted in technical triplicate and performed three times with independent preparations of peptide and protein (all 9 points for each dosing level are plotted). Uncropped gels for panels b , e , and h are available online. Source data for thioester transfer assays, CD spectra, protease resistance testing, HXMS analyses, and FP plot are available online.

Article Snippet: For UBA3 thioester transfer assays, 15 nM NAE1/UBA3 was substituted for UBE1 (Boston Biochem E-313), 135 nM UBE2M for UBE2D2 (Boston Biochem E2–656), and 1 μM NEDD8 (Boston Biochem UL-812) for ubiquitin.

Techniques: Inhibition, Nucleic Acid Electrophoresis, Silver Staining, Quantitation Assay, Circular Dichroism, Ubiquitin Proteomics, Incubation, Labeling, Recombinant, Fluorescence

Journal: Nature chemical biology

Article Title: Targeting a Helix-in-Groove Interaction Between E1 and E2 Blocks Ubiquitin Transfer

doi: 10.1038/s41589-020-0625-7

Figure Lengend Snippet: (a) Sequence compositions of the unmodified UBE2A template peptide and stapled constructs SAH-UBE2A, SAH-UBE2A R7E , and SAH-UBE2A R7A/L9A/B10A . (b) Comparative effects of UBE2A, SAH-UBE2A, SAH-UBE2A R7E , and SAH-UBE2A R7A/L9A/B10A on UBE1-mediated thioester transfer of ubiquitin to the E2 enzyme UBE2D2. Whereas SAH-UBE2A (10 μM) completely inhibits UBE1 activity, triple mutagenesis abrogates the inhibitory effect and single R7E mutagenesis has an intermediate effect. (c) Progressive loss of UBE1 protection from deuterium exchange into SAH-UBE2A upon single R7E and triple R7A/L9A/B10A mutagenesis. The relative difference in deuterium uptake after 10 sec of labeling is shown. The difference in uptake was calculated from the mean deuterium level for each SAH-UBE2A peptide in the presence of UBE1 minus that of the peptide alone. Mean deuterium levels were obtained from at least two independent biological replicates of each condition. (d) Difference in deuterium uptake plots demonstrate the relative deuterium incorporation of SAH-UBE2A/UBE1, SAH-UBE2A R7E /UBE1, or SAH-UBE2A R7A/L9A/B10A /UBE1 minus the relative deuterium incorporation of UBE1 at 10 min of deuterium labeling. The shaded gray region indicates differences in deuteration that are below the meaningful differences threshold. Consistent with results of both the thioester transfer assay (b) and peptide deuterium exchange (c), and the predicted SAH-UBE2A/UBE1 binding mode, triple mutagenesis essentially abrogates the effect of SAH-UBE2A on UBE1 conformation, with single R7E mutagenesis having an intermediate influence. Data are representative of three independent replicates for SAH-UBE2A, and two independent replicates for SAH-UBE2A R7E and SAH-UBE2A R7A/L9A/B10A . Each peptide, from N- to C-terminus, was given a peptide number to simplify the presentation. The peptide list and residue identity of each peptide can be found in . Uncropped gel for panel b is available online. Source data for the HXMS analyses are available online.

Article Snippet: For UBA3 thioester transfer assays, 15 nM NAE1/UBA3 was substituted for UBE1 (Boston Biochem E-313), 135 nM UBE2M for UBE2D2 (Boston Biochem E2–656), and 1 μM NEDD8 (Boston Biochem UL-812) for ubiquitin.

Techniques: Sequencing, Construct, Ubiquitin Proteomics, Activity Assay, Mutagenesis, Labeling, Binding Assay, Residue

Journal: bioRxiv

Article Title: Downregulation of a cell polarity protein potentiates Chikungunya Virus infection in host cells

doi: 10.1101/2023.07.24.550336

Figure Lengend Snippet: The CHIKV RNA genome has two open reading frames (ORFs) separated by a junctional region (J). The 5’ ORF (ORF 1) codes for four non-structural proteins (nsP1-4). The 3’ ORF (ORF 2) codes for 5 structural proteins, namely capsid (C), E3, E2, 6K, and E1. The 6K has a ribosomal slippery site which leads to the production of a transframe (TF) protein (Firth et al., 2008) (upper panel, A). Schematic of 6K and TF sequences and the ribosome slip site (F at 48th and L at 49th position) (lower panel, A). Potential PDZ binding motifs (PBMs) present in TF as predicted by different softwares (B). Predicted interactions between TF and Scribble PDZ1, Scribble PDZ4, and HTRA1 (C). RMSF plot for TF interactions with Scribble and HTRA1 (D). Binding of TF C-terminal residues with ScPDZ1.

Article Snippet: Total protein was quantified using the bicinchoninic acid (BCA) assay, followed by SDS-PAGE and Western blotting with a primary antibody against Scribble (1:500, Santa Cruz sc55543), beta-actin (1:1000, Cell signaling Technology 4967), beta-tubulin (1:200, Santa Cruz sc55529), CHIKV E2 (1:3000, The Native antigen company MAB12130-200), and EGFP (1:3000, Bio Bharati BB-AB0065).

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Downregulation of a cell polarity protein potentiates Chikungunya Virus infection in host cells

doi: 10.1101/2023.07.24.550336

Figure Lengend Snippet: HEK293T cells were infected with CHIKV at 0.1 MOI. Scribble levels were analyzed at 24 and 48 hpi using western blot and quantitated using densitometry (A). Twenty-four hours post-infection, the cells were fixed, and immunostaining was performed for Scribble and CHIKV capsid and E2 proteins. Inset shows zoomed images of an E2 expressing cell. Arrowheads indicate Scribble puncta (B). Effect of TF on Scribble expression. HEK293T cells were transfected with pEGFPC1-TF and its mutants. Only vector and pEGFPC1-6K were also transfected, and Scribble levels were analyzed 48 h post-transfection using western blot and quantitated using densitometry (C). Electron microscopy (EM) imaging of CHIKV-infected HEK293T cells. HEK293T cells were infected with CHIKV at 1.0 MOI and Scribble was visualized using a gold-labeled secondary antibody (D-i). EM images of an infected cell at higher magnification (D-ii). Immunostaining for ubiquitin and Scribble in CHIKV infected HEK293T cells (E). Data represents mean ± SEM of two biological replicates.

Article Snippet: Total protein was quantified using the bicinchoninic acid (BCA) assay, followed by SDS-PAGE and Western blotting with a primary antibody against Scribble (1:500, Santa Cruz sc55543), beta-actin (1:1000, Cell signaling Technology 4967), beta-tubulin (1:200, Santa Cruz sc55529), CHIKV E2 (1:3000, The Native antigen company MAB12130-200), and EGFP (1:3000, Bio Bharati BB-AB0065).

Techniques: Infection, Western Blot, Immunostaining, Expressing, Transfection, Plasmid Preparation, Electron Microscopy, Imaging, Labeling