Journal: Nature Communications

Article Title: Epstein-Barr virus-driven B cell lymphoma mediated by a direct LMP1-TRAF6 complex

doi: 10.1038/s41467-023-44455-w

Figure Lengend Snippet: a Schematic depiction of recombinant His-tagged TRAF and GST-LMP1 proteins used in this study. The CTAR1 and CTAR2 core sequences are highlighted in red. Relevant previously described direct interaction partners are shown. CC, coiled coil. b TRAF1, 2, 3, and 5 interact with the PxQxT motif of CTAR1, whereas TRAF6 directly binds to the CTAR2 domain. Recombinant His-TRAF proteins were detected via their His-tags on immunoblots (IB) of pulldowns with the indicated GST-LMP1 proteins. Uncropped blots with molecular weights in Supplementary Fig. . Representative results are shown. Number of independent experiments: TRAF1, n = 2; TRAF2, n = 3; TRAF3, n = 3; TRAF5, n = 3; TRAF6, n = 3. c Mutation of the CTAR2 sequence P 379 VQLSY abolishes the TRAF6 interaction with LMP1. LMP1-derived peptides and CD40-derived controls were immobilized on membranes in duplicate and incubated with recombinant His-TRAF6 310-522 (His-T6) or His-TRAF2 311-501 (His-T2). Peptide binding of TRAF6 or TRAF2 proteins was detected by TRAF-specific antibodies. The data are representative of two independent experiments. d Design of the AlphaScreen PPI assay for the detection and quantification of direct TRAF6 binding to LMP1. e LMP1 residues P 379 , V 380 , Q 381 and Y 384 are essential for TRAF6 binding to LMP1 in AlphaScreen PPI assays. Data are mean values ± standard deviation (SD) of three independent experiments. Statistics: two-way ANOVA. p -values: * p ≤ 0.05, *** p ≤ 0.001, **** p ≤ 0.0001. Source data and exact p -values in the Source Data file. f – i Quantitative analysis of TRAF6 interaction with LMP1, CD40, and RANK. His-TRAF6 310-522 was tested at different concentrations in AlphaScreen PPI assays with GST-LMP1 181-386 , GST-CD40 216-277 , GST-RANK 314-355 , and the indicated mutants. K D values are given for measurable interactions. Data are mean values ± SD of three (CD40), four (LMP1), or six (RANK) independent experiments. Curve fitting: Prism, one site-specific binding with hill slope. Source data in the Source Data file.

Article Snippet: AlphaScreen PPI signals were measured in a CLARIOstar reader (BMG Labtech GmbH).

Techniques: Recombinant, Western Blot, Mutagenesis, Sequencing, Derivative Assay, Incubation, Binding Assay, Amplified Luminescent Proximity Homogenous Assay, Standard Deviation

Journal: Nature Communications

Article Title: Epstein-Barr virus-driven B cell lymphoma mediated by a direct LMP1-TRAF6 complex

doi: 10.1038/s41467-023-44455-w

Figure Lengend Snippet: a Mutations within the receptor-interacting surface of TRAF6 affect binding to LMP1. The TRAF6 mutants R 392 A, K 469 A, F 471 A, and Y 473 A were tested LMP1 in AlphaScreen PPI experiments with GST-LMP1 181-386 . Data are mean values ± SD of three independent experiments. Statistics: one-way ANOVA. b TRAF6 R 392 A differentiates between CD40 and LMP1/RANK. AlphaScreen PPI experiments with GST-CD40 216-277 . Data are mean values ± SD of three independent experiments. Statistics: one-way ANOVA. c TRAF6 R 392 A and Y 471 A do not bind to RANK. AlphaScreen PPI experiments with GST-RANK 314-355 . Data are mean values ± SD of four independent experiments. Statistics: one-way ANOVA. d TRAF6 mutants that fail to interact with LMP1 in PPI assays are also unable to bind cellular LMP1. HEK293 cells were co-transfected with HA-LMP1 and the indicated Flag-TRAF6 mutants. Flag-TRAF6 was immunoprecipitated (IP) via its Flag-tag and coprecipitated HA-LMP1 was detected on immunoblots by an α-HA antibody. Uncropped blots in Supplementary Fig. . For statistical analysis LMP1 signals were digitalized and quantified (graph). Data are mean values ± SD of three independent experiments. Statistics: one-way ANOVA. e TRAF6 R 392 A, F 471 A, and Y 473 A fail to interact with LMP1 wild-type clusters in HeLa cells (large panel). TRAF6 recruitment is dependent on amino acids 371 − 386 of LMP1 (small panel). Confocal microscopy images show representative cells of three independent experiments. Quantitative data are mean values ± SD of ten randomly selected cells per transfection of one representative experiment (graph). Scale bars: 10 µm. Statistics: one-way ANOVA. p -values: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Source data and exact p-values in the Source Data file.

Article Snippet: AlphaScreen PPI signals were measured in a CLARIOstar reader (BMG Labtech GmbH).

Techniques: Binding Assay, Amplified Luminescent Proximity Homogenous Assay, Transfection, Immunoprecipitation, FLAG-tag, Western Blot, Confocal Microscopy

Journal: Nature Communications

Article Title: Epstein-Barr virus-driven B cell lymphoma mediated by a direct LMP1-TRAF6 complex

doi: 10.1038/s41467-023-44455-w

Figure Lengend Snippet: a Alignment of the RANK-derived TRAF6-interacting sequence of the TRAF6 inhibitor peptide DRQIKIWFQNRRMKWKK-RKIPTEDEY with the TRAF6-binding sequences of LMP1, CD40, and the TRAF2-binding sequence of LMP1. b TRAF6 binding to LMP1 is efficiently inhibited by the TRAF6 inhibitor peptide. Left, AlphaScreen PPI assay-based dose-response curve of TRAF6 inhibitor peptide (green) with recombinant GST-LMP1 181-386 and His-TRAF6 310-522 . The Antennapedia leader peptide DRQIKIWFQNRRMKWKK served as a negative control (gray). Right, TRAF6 binding is absent for the LMP1 null mutant A 379 xAxxA. Data are mean values ± SD of four independent experiments. Dose-response curve fitting: 4-parameter fit. Statistics for controls (right): paired T-test, two-tailed. c The TRAF6 inhibitor peptide does not affect TRAF2 binding to LMP1. GST-LMP1 and His-TRAF2 311-501 were incubated in the presence of 30 µM of TRAF6 inhibitor or control peptide in AlphaScreen PPI experiments. Data are mean values ± SD of three independent experiments. Statistics: one-way ANOVA. d Peptide-mediated inhibition of TRAF6 binding to CD40 is less effective than that to LMP1. Left, AlphaScreen PPI experiments with GST-CD40 and His-TRAF6 310-522 . Right panel, absent TRAF6 binding to the CD40 null mutant A 233 xAxxA. Data are mean values ± SD of two independent experiments. Dose-response curve fitting: 4-parameter fit. e Inhibition of LMP1-dependent cell proliferation of LCLs by the TRAF6 inhibitor peptide. Cells were incubated for four days in the presence of 100 µM of TRAF6 inhibitor peptide or control peptide as indicated. MTT viability assays. The averages of the control replicates were set to 100% viability for each cell line. Data are mean values ± SD of biological triplicates. Statistics: unpaired T-test, two-tailed. p -values: *p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, n.s. (not significant). Source data and exact p -values in the Source Data file.

Article Snippet: AlphaScreen PPI signals were measured in a CLARIOstar reader (BMG Labtech GmbH).

Techniques: Derivative Assay, Sequencing, Binding Assay, Amplified Luminescent Proximity Homogenous Assay, Recombinant, Negative Control, Mutagenesis, Two Tailed Test, Incubation, Control, Inhibition

Journal: ACS Chemical Biology

Article Title: Structure-Guided DOT1L Probe Optimization by Label-Free Ligand Displacement

doi: 10.1021/cb500796d

Figure Lengend Snippet: Development of nonradiometric biochemical and cellular assays for DOT1L. (a–c) AlphaScreen proximity bead-based assay demonstrating adaptability to high-throughput screening ( Z ′ calculated by 1 – ((3σ FED1 + σ DMSO )/absolute value(μ FED1 – μ DMSO )), and expected comparable potency differentiation of known inhibitors. (d–f) Fluorescence polarization assay demonstrating significant assay robustness (Z′ calculated with above formula) and separation of weak DOT1L inhibitors ( SAH ) from more potent compounds ( FED1 and EPZ004777 ). (g–i) High-content imaging assay evaluating H3K79me2 abundance by immunofluorescence in A431 cells after 4 days of indicated DOT1L inhibitors, with diminished H3K79me2 compared to DMSO. Assay is robust (Z′ calculated as above) and reports cellular EC 50 . Biochemical assays were performed in duplicate, and high-content assays were performed as four replicates.

Article Snippet: The addition of 10 uL of 2× this solution to the plates (AlphaScreen plates, PerkinElmer #6005359) was performed with a liquid handler.

Techniques: Amplified Luminescent Proximity Homogenous Assay, Bead-based Assay, High Throughput Screening Assay, Fluorescence, Imaging, Immunofluorescence

Journal: Cell reports

Article Title: Mechanism and evolutionary origins of alanine-tail C-degron recognition by E3 ligases Pirh2 and CRL2-KLHDC10.

doi: 10.1016/j.celrep.2023.113100

Figure Lengend Snippet: Figure 1. Pirh2 and KLHDC10 directly bind to Ala-tails (A) Domain arrangement of human Pirh2: NTM, N-terminal module; RING, really interesting new gene domain; CTD, C-terminal domain. (B) In vitro GST pull-down assay using the specified GST-Pirh2 truncation constructs and recombinantly purified GFP or GFP-Ala6. Anti-GST and anti-GFP immunoblots are indicated. (C) AlphaScreen assay to assess Pirh2-Ala-tail binding. Reactants were GST fusions with Pirh2 fragments containing both the NTM and RING domains (Pirh21–195) or the NTM alone (Pirh21–137) and a biotinylated Ala-tail peptide (MDELYKAAAAAA). Proximity-induced fluorescence signal (see STAR Methods) was monitored in the presence of increasing amounts of a competing, non-biotinylated Ala-tail peptide to determine IC50 values from the dose-response curve. Each data point is a technical triplicate, and error bars represent ± SD. The y axis presents the normalized AlphaScreen signal as arbitrary fluorescence units (AFUs). (D) Domain arrangement of human KLHDC10. (E) In vitro GST pull-down assay using the GST-KLHDC10 and GST-KLHDC2 constructs and recombinantly purified GFP or GFP-Ala6. Anti-GST and anti-GFP immunoblots are indicated. (F) AlphaScreen assay to assess KLHDC10-Ala-tail binding. As in (C) but using GST-KLHDC10 and GST-KLHDC10b1-88–442 instead. See also Figures S1, S2, S10, and S11.

Article Snippet: AlphaScreen signal was measured using PHERAstar microplate reader (BMG Labtech) at RT.

Techniques: In Vitro, Pull Down Assay, Construct, Western Blot, Amplified Luminescent Proximity Homogenous Assay, Binding Assay