Journal: Cell reports

Article Title: The HLA-II immunopeptidome of SARS-CoV-2

doi: 10.1016/j.celrep.2023.113596

Figure Lengend Snippet:

Article Snippet: pcDNA3 myc CIITA , Addgene , Cat#14650.

Techniques: Recombinant, Virus, Protease Inhibitor, Mass Spectrometry, Bicinchoninic Acid Protein Assay, Software

Journal: The Journal of Biological Chemistry

Article Title: Molecular basis of cullin-3 (Cul3) ubiquitin ligase subversion by vaccinia virus protein A55

doi: 10.1074/jbc.RA118.006561

Figure Lengend Snippet: A55 directly binds to cullin-3 via its N-terminal BB domain. A–C, representative immunoblots following immunoprecipitation ( IP ) of cleared lysates from HEK293T–REx cell lines ( A ) expressing empty vector ( EV ), B14–TAP ( B14 ), or TAP–A55 ( A55 ), the TAP tag comprising STREP and FLAG epitopes; ( B ) expressing B14–TAP or TAP–A55 and transfected with plasmids encoding myc-Cul3 or myc-Cul5; ( C ) expressing EV, B14–TAP, TAP–A55, TAP–A55BB, or TAP–A55 Kelch. Cells were lysed in Nonidet P-40 ( A and C ) or RIPA buffer ( B ). Immunoprecipitates were subjected to SDS-PAGE and immunoblotting. A and C, FLAG IP and immunoblotting for co-IP of endogenous Cul3. B, Myc IP and immunoblotting for co-IP of TAP-tagged B14 or A55. Input, cleared lysate. Data shown are representative of at least three independent experiments. Signals arising from the light chain (*) or heavy chain (**) of the antibody used for IP are marked. D, SEC-MALS analyses showing the SEC elution profiles ( thin lines ) and molecular mass distribution ( thick lines ) across the elution peaks for A55BB ( peak 1, green , theoretical molecular mass 30 kDa and observed molecular mass 60 kDa), Cul3NΔ22 ( peak 2, blue, theoretical molecular mass 46 kDa and observed molecular mass 45 kDa), and A55BB and Cul3NΔ22 together ( peak 3, red, theoretical molecular mass 76 kDa and observed molecular mass 141 kDa) when eluting from a Superdex 200 10/300 GL column. Peak 4 is assumed to be excess Cul3NΔ22. E, Coomassie-stained SDS-PAGE analysis of peaks 1–3 from D .

Article Snippet: The mammalian expression vectors pcDNA-myc- CUL3 (19893) and pcDNA-myc- CUL5 (19895) were purchased from Addgene.

Techniques: Western Blot, Immunoprecipitation, Expressing, Plasmid Preparation, Transfection, SDS Page, Co-Immunoprecipitation Assay, Staining

Journal: The Journal of Biological Chemistry

Article Title: Molecular basis of cullin-3 (Cul3) ubiquitin ligase subversion by vaccinia virus protein A55

doi: 10.1074/jbc.RA118.006561

Figure Lengend Snippet: ITC studies show that A55 binds to Cul3 with nanomolar to sub-nanomolar affinity. A–D, representative ITC titration curves showing interactions between A55BB and Cul3NΔ22 ( A ) or Cul3N ( B ) and between KLHL3, a human BTB related to A55, and Cul3NΔ22 ( C ) or Cul3N ( D ). The top figure in each panel shows the baseline-corrected differential power ( DP ) versus time. The bottom figure of each panel is the normalized binding curve showing integrated changes in enthalpy (Δ H ) against molar ratio. The corresponding dissociation constant ( K D ), number of binding sites ( N ), enthalpy change (Δ H ), and change in Gibbs free energy (Δ G ) for each representative experiment are shown. All experiments were performed at least twice independently.

Article Snippet: The mammalian expression vectors pcDNA-myc- CUL3 (19893) and pcDNA-myc- CUL5 (19895) were purchased from Addgene.

Techniques: Titration, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Molecular basis of cullin-3 (Cul3) ubiquitin ligase subversion by vaccinia virus protein A55

doi: 10.1074/jbc.RA118.006561

Figure Lengend Snippet: Comparison of the dissociation constants ( K D ), enthalpic change (Δ H ), and entropic change ( T Δ S ) for BB–Cul3 interaction Experiments for this study were performed at least twice, and mean ± S.E. is shown.

Article Snippet: The mammalian expression vectors pcDNA-myc- CUL3 (19893) and pcDNA-myc- CUL5 (19895) were purchased from Addgene.

Techniques: Comparison

Journal: The Journal of Biological Chemistry

Article Title: Molecular basis of cullin-3 (Cul3) ubiquitin ligase subversion by vaccinia virus protein A55

doi: 10.1074/jbc.RA118.006561

Figure Lengend Snippet: A55 and cellular BB domains share conserved modes of dimerization and Cul3 binding. A, structure of the A55/Cul3NΔ22 heterodimer in the asymmetric unit as ribbon diagram . Cul3 is in cyan and the three domains of A55 (BTB, three-box and BACK) are in green, orange, and gray , respectively. Helices α1–α12 from A55 are labeled in black with the exception of α10, which is hidden behind α9 in the picture. Helices α1–α16 from Cul3 are labeled in red. B, A55/Cul3 dimer formed by applying crystallographic 2-fold symmetry. C, overlay of three BB/Cul3 complex structures (KLHL3/Cul3, PDB code 4HXI ; KLHL11/Cul3, PDB code 4APF ; and A55/Cul3). The structures are aligned to the Cul3 part of the A55/Cul3 complex only. A55, KLHL3, KLHL11, and Cul3 are in green, purple, magenta, and cyan , respectively, and the three sub-domains are marked. Additional helices at the C terminus of the KLHL11 BACK domain are shown as semi-transparent helices. D, comparison of the dimers formed by A55, KLHL3, and KLHL11 BB domains, colored as in C .

Article Snippet: The mammalian expression vectors pcDNA-myc- CUL3 (19893) and pcDNA-myc- CUL5 (19895) were purchased from Addgene.

Techniques: Binding Assay, Labeling, Comparison

Journal: The Journal of Biological Chemistry

Article Title: Molecular basis of cullin-3 (Cul3) ubiquitin ligase subversion by vaccinia virus protein A55

doi: 10.1074/jbc.RA118.006561

Figure Lengend Snippet: Structure-based sequence alignment of the A55, KLHL3, and KLHL11 BB domains. Columns are shaded based on amino acid similarity. Secondary structural elements for A55 are shown above the aligned sequences and colored as in A . Residues at the A55/Cul3 interface are underlined in blue . Residues selected for subsequent mutagenesis studies of A55 are marked by stars at bottom: the two conserved sites (Phe-54 and Asp-56) are marked by purple stars , and the nonconserved site (Ile-48) is marked by a red star . Residues from KLHL11 that are involved in Cul3–NTE binding are marked by magenta triangles .

Article Snippet: The mammalian expression vectors pcDNA-myc- CUL3 (19893) and pcDNA-myc- CUL5 (19895) were purchased from Addgene.

Techniques: Sequencing, Mutagenesis, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Molecular basis of cullin-3 (Cul3) ubiquitin ligase subversion by vaccinia virus protein A55

doi: 10.1074/jbc.RA118.006561

Figure Lengend Snippet: Conserved and nonconserved interactions at the interface between A55 and Cul3. A, A55BB/Cul3NΔ22 complex structure with two key Cul3-binding sites in the BTB domain boxed in black (enlarged in B–F ) and red (enlarged in G ). B, structural overlay of the φ X (D/E) motifs from A55, KLHL3, KLHL11, and SPOP. C–F, surface of Cul3 colored by residue hydrophobicity from yellow (hydrophobic) to white (polar) . Hydrophobic binding pockets are shown for Phe-54 of A55, Met-83 of KLHL3, Phe-130 of KLHL11, and Met-233 of SPOP, which are equivalent to the φ residue of the φ X (D/E) motif, and for Ile-48 of A55 and its equivalent residues Ala-77, Pro-124, and Ala-227 in KLHL3, KLHL11, and SPOP, respectively. G, overlay of the hydrogen bond formed between Tyr-125 of Cul3 and Asp-99 of A55 with equivalent residues Ser-128, Asp-181, and Asp-278 in KLHL3, KLHL11, and SPOP, respectively.

Article Snippet: The mammalian expression vectors pcDNA-myc- CUL3 (19893) and pcDNA-myc- CUL5 (19895) were purchased from Addgene.

Techniques: Binding Assay, Residue

Journal: The Journal of Biological Chemistry

Article Title: Molecular basis of cullin-3 (Cul3) ubiquitin ligase subversion by vaccinia virus protein A55

doi: 10.1074/jbc.RA118.006561

Figure Lengend Snippet: I48E mutation significantly impairs A55 binding to Cul3. A, representative thermal melt curves of WT A55BB and mutants F54E, D99A, I48A, and I48E from DSF studies. Curves are offset along the vertical axis for clarity. All experiments were performed in triplicate. B, comparison of the melting temperatures for WT A55BB ( green ), F54E ( red ), D99A ( blue ), I48A ( orange ), and I48E ( black ) mutants. Upper and lower panels display T m values for the first and second melting events, respectively. Error bars show the standard errors of the mean from experiments performed in triplicate. C–J, representative ITC titration curves showing binding of A55BB mutants F54E ( C and D ), D99A ( E and F ), I48A ( G and H ), and I48E ( I and J ) to Cul3NΔ22 and Cul3N, respectively. Integrated changes in enthalpy (Δ H ) are plotted against molar ratio of titrant. The corresponding dissociation constant ( K D ), number of binding sites ( N ), enthalpy change (Δ H ), and change in Gibbs free energy (Δ G ) for each representative experiment are shown. All experiments were performed at least twice independently. Raw data for C–J are shown in Fig. S7 .

Article Snippet: The mammalian expression vectors pcDNA-myc- CUL3 (19893) and pcDNA-myc- CUL5 (19895) were purchased from Addgene.

Techniques: Mutagenesis, Binding Assay, Comparison, Titration

Journal: The Journal of Biological Chemistry

Article Title: Molecular basis of cullin-3 (Cul3) ubiquitin ligase subversion by vaccinia virus protein A55

doi: 10.1074/jbc.RA118.006561

Figure Lengend Snippet: Comparison of the dissociation constants ( K D ) for WT and mutant A55BB for Cul3 Experiments were performed n times and the mean ± S.E. is shown.

Article Snippet: The mammalian expression vectors pcDNA-myc- CUL3 (19893) and pcDNA-myc- CUL5 (19895) were purchased from Addgene.

Techniques: Comparison, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Molecular basis of cullin-3 (Cul3) ubiquitin ligase subversion by vaccinia virus protein A55

doi: 10.1074/jbc.RA118.006561

Figure Lengend Snippet: Cul3-binding residues of A55 are conserved across orthopoxvirus orthologues but not in VACV paralogues C2 and F3. Multiple sequence alignment of the A55 BB domains against its orthologues from selected poxviruses and two other VACV BTB–Kelch proteins, C2 and F3. Columns are shaded based on amino acid similarity. Secondary structural elements for A55 are shown above the aligned sequences and colored as in A . Residues at the A55/Cul3 interface are underlined in blue . Residues aligned with A55-Ile-48 and the φ X (D/E) motif are boxed in red .

Article Snippet: The mammalian expression vectors pcDNA-myc- CUL3 (19893) and pcDNA-myc- CUL5 (19895) were purchased from Addgene.

Techniques: Binding Assay, Sequencing

Journal: Scientific Reports

Article Title: Tau Internalization is Regulated by 6-O Sulfation on Heparan Sulfate Proteoglycans (HSPGs)

doi: 10.1038/s41598-018-24904-z

Figure Lengend Snippet: Tau uptake is regulated by quaternary structure. ( a ) 2N4R monomer, oligomer, and fibrillized proteins show distinct banding patterns on a non-reducing SDS-PAGE gel. 2N4R Fibrils before (top) and after (bottom) sonication as visualized by negative-stain EM. Bar represents 500 nm ( b ) DLS of 2N4R monomer, oligomer, fibril, and sonicated fibril species. Experiments were performed in triplicate and the error shown is SD. ( c ) Uptake of 2N4R quaternary structures in H4 cells after 1 h at 37 °C. ( d ) Uptake of 2N4R quaternary structures in iPS-derived neurons after 1 h at 37 °C. For all uptake experiments, three independent experiments were performed in duplicate, identical control experiments were performed at 4 °C and subtracted from 37 °C data to generate final curves shown, and the error shown is SEM.

Article Snippet: Transfection of H4 cells with Sulf1 (Addgene #13003) or Sulf2 (Addgene #13004) were performed with Lipofectamine 3000 (Invitrogen) according to the manufacturers instructions and cells were assayed 48 h later.

Techniques: SDS Page, Sonication, Staining, Derivative Assay, Control

Journal: Scientific Reports

Article Title: Tau Internalization is Regulated by 6-O Sulfation on Heparan Sulfate Proteoglycans (HSPGs)

doi: 10.1038/s41598-018-24904-z

Figure Lengend Snippet: 6-O sulfation regulates tau uptake in CNS culture. ( a ) Internalization of 2N4R-488 (50 nM, 1 h at 37 °C) into H4 cells is strongly inhibited by incubation with heparin, heparan sulfate, and 2- O -desulfated heparin as compared to 6- O -desulfated heparin or chondroitin sulfate (0.05 mg/ml for all derivatives). ( b ) Internalization of 2N4R-488 (200 nM, 1 h at 37 °C) into iPS-derived neurons is inhibited by incubation with heparin, heparan sulfate, and 2- O -desulfated heparin as compared to 6- O -desulfated heparin or chondroitin sulfate (0.5 mg/ml for all derivatives). All uptake experiments were performed in duplicate over three independent experiments with the data combined. Lines on the graphs represent WT mean +/− 3 standard deviations, error bars represent SEM, and a one-way ANOVA analysis with Dunnett’s method was used to determine significance compared to the negative control *p-value ≤ 0.05, **p-value ≤ 0.01, ***p-value ≤ 0.001, ns = not significant. ( c ) Internalization of 2N4R-488 (200 nM, 30 min at 37 °C) into mouse slice culture is strongly inhibited by incubation with 2- O -desulfated heparin, but not 6- O -desulfated heparin (0.5 mg/ml). Hoechst stain is used to label nuclei. ( d ) Quantification of the median 488 fluorescence intensity for tau uptake in mouse slice culture. Two independent experiments were performed with multiple images (>5) from each condition. Error bars represent SEM. ( e ) Uptake of 2N4R-488 (50 nM, 1 h at 37 °C) tau is reduced when Sulf1 or Sulf2 is overexpressed in H4 cells as compared to WT or mock transfected cells. Uptake experiments were performed in duplicate over three independent experiments with the data normalized to WT and combined. Error bars represent SEM. ( f ) ICC confirms that Sulf1 and Sulf2 are overexpressed (red) in H4 cells. Hoechst stain is used to label nuclei.

Article Snippet: Transfection of H4 cells with Sulf1 (Addgene #13003) or Sulf2 (Addgene #13004) were performed with Lipofectamine 3000 (Invitrogen) according to the manufacturers instructions and cells were assayed 48 h later.

Techniques: Incubation, Derivative Assay, Negative Control, Staining, Fluorescence, Transfection

Journal: The Journal of Clinical Investigation

Article Title: METTL1-mediated m 7 G modification of valine tRNAs drives metabolic adaptation in pancreatic ductal adenocarcinoma cells

doi: 10.1172/JCI204312

Figure Lengend Snippet: ( A – E ) Distribution of ATP5H ( A ), NDUFC1 ( B ), NDUFV2 ( C ), SLC25A4 ( D ), and UCP2 ( E ) mRNAs across ribosome fractions (free RNA/RNP, 40S/60S/80S, light polysome, heavy polysome) from sgNC and sgMETTL1 BxPC-3 and MiaPaCa-2 cells, quantified by qRT-PCR. Bar graphs show the proportion of untranslated and translated fractions. ** P < 0.01 and *** P < 0.001, by 2-tailed, unpaired t test. ( F ) Relative protein levels of NDUFV2 and UCP2 in PDAC control and METTL1-KO cells by Western blotting. ( G ) Relative protein levels of NDUFV2 and UCP2 in PDAC control and valine tRNA–KD cells by Western blotting. ( H and I ) Oxygen consumption rate in control and METTL1-KO groups from BxPC-3 ( H ) and MiaPaCa-2 ( I ) cells by Seahorse Mito Stress testing. *** P < 0.001, by 1-way ANOVA followed by Dunnett’s multiple-comparison test. ( J and K ) Oxygen consumption rate in control and valine tRNA–KD group from BxPC-3 ( J ) and MiaPaCa-2 ( K ) cells by Seahorse Mito Stress testing. *** P < 0.001, by1-way ANOVA followed by Dunnett’s multiple-comparison test. Data are representative of 3 independent experiments. All data are presented as the mean ± SD.

Article Snippet: The NDUFV2 expression vector (pcDNA3.1/NDUFV2/myc-His) was obtained from Addgene (127491).

Techniques: Quantitative RT-PCR, Control, Western Blot, Comparison