Journal: Blood

Article Title: Activation of lymphoma-associated MyD88 mutations via allostery-induced TIR-domain oligomerization

doi: 10.1182/blood-2014-05-573188

Figure Lengend Snippet: Lymphoma-associated MyD88 mutants and their isolated TIR domains constitutively hyperactivate NF-κB signaling. (A) Structure of a human MyD88 TIR monomer (Protein Data Bank accession number 2JS7) with lymphoma-associated mutations highlighted in red. The α helices are shown in beige and β sheets in light pink. The BB loop is shown in cyan. (B-F) HEK293 cells were transfected with plasmids for full-length (B-C) or TIR-domain–only (D-F) constructs of lymphoma-associated MyD88 mutants, in parallel with NF-κB–inducible firefly luciferase and constitutive Renilla luciferase reporters, and TLR4 and MD-2 plasmids (F only). Cells were harvested 24 hours later (B-E) or stimulated with LPS (100 ng/mL) for another 24 hours (F). Luciferase activity was measured by DLA (B,D,F) or separately transfected cell lysates loaded on SDS polyacrylamide gel electrophoresis (PAGE) for immunoblot (C,E). For panels B, D, and F, data presented are means + standard deviation (SD) of triplicate samples, and for panels B to F, data shown are representative of at least 3 independent experiments. EV, empty vector; IB, immunoblot.

Article Snippet: Human embryonic kidney 293 (HEK293) cells (European Collection of Cell Cultures), MyD88-deficient cells (HEK293-I3A; a kind gift from George R. Stark, Department of Molecular Genetics, Lerner Research Institute, Cleveland, OH), WT MyD88 (OCI-LY19, BJAB, U2392), or heterozygously MYD88 -mutated (OCI-LY10, TMD8, HBL1 with L265P; HLY1 with S119C, SUDHL2 with S122R) or homozygously L265P-mutated (OCI-LY3) DLBCL cell lines (from Manfred Kögl [Boehringer Ingelheim RCV, Vienna] or Stephan Hailfinger [Department of Biochemistry, University of Tübingen]) were cultured as described in the supplemental Methods. siRNA knockdown of endogenous MyD88 Qiagen control (#1027280) or MYD88 (Hs_MYD88_10 SI02100902) small interfering RNA (siRNA) oligonucleotides were transfected (Lipofectamine, Life Technologies) at 40 nM together with MyD88 TIR-domain plasmids (amino acids 155-294, N-terminal streptavidin-hemagglutinin tag) and NF-κB firefly luciferase and constitutive Renilla luciferase in 24-well format.

Techniques: Isolation, Transfection, Construct, Luciferase, Activity Assay, Polyacrylamide Gel Electrophoresis, Western Blot, Standard Deviation, Plasmid Preparation

Journal: Blood

Article Title: Activation of lymphoma-associated MyD88 mutations via allostery-induced TIR-domain oligomerization

doi: 10.1182/blood-2014-05-573188

Figure Lengend Snippet: Augmented heterodimerization of lymphoma-associated mutants and WT MyD88 TIR domain. (A-D) MyD88-deficient HEK293-I3A cells were transfected with plasmids for full-length (A-B) or TIR-domain–only (C-D) constructs of lymphoma-associated MyD88 mutants, in parallel with NF-κB–inducible firefly luciferase and a constitutive Renilla luciferase reporters. Cells were harvested 48 hours later; 1 lysate aliquot was used for luciferase activity measurement by DLA (A,C) and another for immunoblot upon 15% SDS-PAGE separation (B,D). Data presented are means + SD of triplicate samples, and data shown are representative of at least 3 independent experiments. (E-F) The L265P mutation leads to increased TIR-TIR oligomerization. LUMIER luciferase analysis from HEK293T cells transfected with Protein A–tagged and Renilla luciferase–tagged WT or L265P mutant full-length (E) or TIR-domain–only (amino acid sequence 155-294) (F) MyD88 constructs. Forty-eight hours posttransfection, cells were lysed and raw luciferase measured in 10% of the lysate sample. The remainder was used for Protein A purification on immunoglobulin G magnetic beads and subsequent measurement of bound luciferase. Data represent ratios of bound vs raw luciferase for each transfection upon subtraction of background (Protein A–only control bait) combined from 7 identical experiments. Means ± SDs are shown and differences tested using a Mann-Whitney U t test. EV, empty vector; IB, immunoblot.

Article Snippet: Human embryonic kidney 293 (HEK293) cells (European Collection of Cell Cultures), MyD88-deficient cells (HEK293-I3A; a kind gift from George R. Stark, Department of Molecular Genetics, Lerner Research Institute, Cleveland, OH), WT MyD88 (OCI-LY19, BJAB, U2392), or heterozygously MYD88 -mutated (OCI-LY10, TMD8, HBL1 with L265P; HLY1 with S119C, SUDHL2 with S122R) or homozygously L265P-mutated (OCI-LY3) DLBCL cell lines (from Manfred Kögl [Boehringer Ingelheim RCV, Vienna] or Stephan Hailfinger [Department of Biochemistry, University of Tübingen]) were cultured as described in the supplemental Methods. siRNA knockdown of endogenous MyD88 Qiagen control (#1027280) or MYD88 (Hs_MYD88_10 SI02100902) small interfering RNA (siRNA) oligonucleotides were transfected (Lipofectamine, Life Technologies) at 40 nM together with MyD88 TIR-domain plasmids (amino acids 155-294, N-terminal streptavidin-hemagglutinin tag) and NF-κB firefly luciferase and constitutive Renilla luciferase in 24-well format.

Techniques: Transfection, Construct, Luciferase, Activity Assay, Western Blot, SDS Page, Mutagenesis, Sequencing, Purification, Magnetic Beads, Control, MANN-WHITNEY, Plasmid Preparation

Journal: Blood

Article Title: Activation of lymphoma-associated MyD88 mutations via allostery-induced TIR-domain oligomerization

doi: 10.1182/blood-2014-05-573188

Figure Lengend Snippet: Aggregation of constitutively active lymphoma-associated TIR mutants in lymphoma cell lines. (A-B) HEK293 cells were transfected with plasmids for mCitrine-TIR (yellow pseudocolor) or mutants and full-length MyD88-mCerulean (blue pseudocolor, B). Twenty-four hours later, cells were fixed and visualized under the confocal microscope at ×1000 magnification. Shown are representative confocal images of at least 3 independent experiments. (C-G) Augmented amount of MyD88 in aggregates in different DLBCL cell lines. Cell lysates were fractionated by centrifugation and lysate and pellet fraction analyzed by SDS-PAGE followed by anti-MyD88 (C-E) or IRAK1 (G) and anti-tubulin immunoblot (see blots in panels E and G). Relative MyD88 or IRAK1 and tubulin levels were quantified by charge-coupled device detection and plotted as the tubulin-normalized ratios of lysate (WCL) vs pellet MyD88 or IRAK1. (C-D) Data were combined from 9 experiments and are shown as min-to-max box-and-whisker plots. In panel C, WT MyD88 DLBCL cell lines (white, OCI-LY19, BJAB, U2392) were compared with MyD88-mutated cell lines (burgundy, OCI-LY10, TMD8, HBL1, OCI-LY3 with L265P; HLY1 with S119C, SUDHL2 with S122R). In panel D, cells were plotted according to L265P mutational status. White, WT MyD88 (OCI-LY19, BJAB, U2392); red, L265P heterozygous (OCI-LY10, TMD8, HBL1); burgundy, L265P homozygous (OCI-LY3). (E) Comparison of unmutated OCI-LY19 (white) vs L265P homozygous OCI-LY3 (red) lysate vs pellet ratio from 9 combined experiments and showing a representative immunoblot. (F) Differences in MyD88 lysate expression in L265P-mutated OCI-LY3 cells are not due to lower mRNA expression as assessed by quantitative PCR in OCI-LY19 (white) and OCI-LY3 (burgundy) cells. One representative of 3 independent experiments was done in triplicate (mean + upper limit). (G) MyD88-containing aggregates also contain IRAK1. Comparison of unmutated OCI-LY19 (white) vs L265P homozygous OCI-LY3 (red) lysate vs pellet ratio from 3 combined experiments and showing a representative immunoblot. IB, immunoblot; WCL, whole-cell lysate.

Article Snippet: Human embryonic kidney 293 (HEK293) cells (European Collection of Cell Cultures), MyD88-deficient cells (HEK293-I3A; a kind gift from George R. Stark, Department of Molecular Genetics, Lerner Research Institute, Cleveland, OH), WT MyD88 (OCI-LY19, BJAB, U2392), or heterozygously MYD88 -mutated (OCI-LY10, TMD8, HBL1 with L265P; HLY1 with S119C, SUDHL2 with S122R) or homozygously L265P-mutated (OCI-LY3) DLBCL cell lines (from Manfred Kögl [Boehringer Ingelheim RCV, Vienna] or Stephan Hailfinger [Department of Biochemistry, University of Tübingen]) were cultured as described in the supplemental Methods. siRNA knockdown of endogenous MyD88 Qiagen control (#1027280) or MYD88 (Hs_MYD88_10 SI02100902) small interfering RNA (siRNA) oligonucleotides were transfected (Lipofectamine, Life Technologies) at 40 nM together with MyD88 TIR-domain plasmids (amino acids 155-294, N-terminal streptavidin-hemagglutinin tag) and NF-κB firefly luciferase and constitutive Renilla luciferase in 24-well format.

Techniques: Transfection, Microscopy, Centrifugation, SDS Page, Western Blot, Whisker Assay, Comparison, Expressing, Real-time Polymerase Chain Reaction

Journal: Blood

Article Title: Activation of lymphoma-associated MyD88 mutations via allostery-induced TIR-domain oligomerization

doi: 10.1182/blood-2014-05-573188

Figure Lengend Snippet: Lymphoma-associated TIR-domain mutants are located at stable communication hubs in the TIR domain. (A-B) HEK293 cells were transfected with plasmids for designed MyD88 mutants and NF-κB–inducible firefly luciferase and constitutive Renilla luciferase reporter plasmids. Twenty-four hours later, cells were lysed and luciferase activity measured (A) or the lysates were run on SDS-PAGE and analyzed by anti-MyD88 immunoblot (B). (C-D) The MyD88 TIR domain contains highly stable RMSF minima. The 40-nanosecond (C) or 100-nanosecond (D) molecular dynamics simulations were done on WT (black dotted line) and mutant (colored as shown) MyD88 NMR structure 2js7 ensemble (composed of 20 structures each, 20 simulations each); shown are averaged data (C) or the single most representative conformer (D). RMSF over the TIR-domain residues were plotted. RMSF profiles are highly similar and show RMSF minima (black arrows pointing to residues 177, 204, 231/232, 265, and 303/304). (E-F) RMSF minima are shown as green or orange (in case they coincide with lymphoma-associated mutations) spheres and map to a plane describing MyD88 dimer formation (see schematic representation, panel F) as proposed by Bovijn et al.20 Communication hubs are indicated by arrows and coincide with RMSF minima or locate in the interaction plane within the MyD88 dimer. Some lymphoma-associated mutations directly map to hub positions (orange spheres) or locate within this plane (red spheres), with the exception of S143 and T294 (red). Hub positions for which so far no lymphoma-associated mutations have been reported are shown as green spheres. The region of higher flexibility in WT (black dotted line) vs mutated (colored as shown) TIR domains in molecular dynamics simulation (F) is shown in dark blue in panel E. EV, empty vector; IB, immunoblot.

Article Snippet: Human embryonic kidney 293 (HEK293) cells (European Collection of Cell Cultures), MyD88-deficient cells (HEK293-I3A; a kind gift from George R. Stark, Department of Molecular Genetics, Lerner Research Institute, Cleveland, OH), WT MyD88 (OCI-LY19, BJAB, U2392), or heterozygously MYD88 -mutated (OCI-LY10, TMD8, HBL1 with L265P; HLY1 with S119C, SUDHL2 with S122R) or homozygously L265P-mutated (OCI-LY3) DLBCL cell lines (from Manfred Kögl [Boehringer Ingelheim RCV, Vienna] or Stephan Hailfinger [Department of Biochemistry, University of Tübingen]) were cultured as described in the supplemental Methods. siRNA knockdown of endogenous MyD88 Qiagen control (#1027280) or MYD88 (Hs_MYD88_10 SI02100902) small interfering RNA (siRNA) oligonucleotides were transfected (Lipofectamine, Life Technologies) at 40 nM together with MyD88 TIR-domain plasmids (amino acids 155-294, N-terminal streptavidin-hemagglutinin tag) and NF-κB firefly luciferase and constitutive Renilla luciferase in 24-well format.

Techniques: Transfection, Luciferase, Activity Assay, SDS Page, Western Blot, Mutagenesis, Plasmid Preparation

Journal: British Journal of Pharmacology

Article Title: Hunting for the function of orphan GPCRs – beyond the search for the endogenous ligand

doi: 10.1111/bph.12942

Figure Lengend Snippet: Identified functions of class A orphan GPCRs

Article Snippet: Role in lymphoma cell growth via ERK activation , Gene expression profiling analysis, FACS, ISH, confocal microscopy, qPCR , HeLa (transient) OCI-Ly19 (transient) , Ansell et al ., 2012.

Techniques: Expressing, Mouse Assay, Western Blot, MTT Assay, Inhibition, In Vivo, Luciferase, Activation Assay, Gene Expression, Confocal Microscopy, Transmission Assay, Reporter Assay, Microscopy, GTPγS Binding Assay, Binding Assay, Ligand Binding Assay, Transgenic Assay, Immunohistochemistry, Microarray, Northern Blot, Control, Knockdown, Mutagenesis