Primary structure and tissue expression of TRUSS. (A) Predicted amino acid sequence of TRUSS. Consensus TRAF2 binding sequences are boxed, and the predicted dileucine motifs are underlined. Sequences showing similarity to leucine zippers (one mismatch in each sequence) are in italics. (B) Expression of TRUSS protein in transfected COS-7 cells. Approximately 4 × 10⁷ cells were lysed, immunoprecipitated with either rabbit anti-TRUSS NH₂ terminus antibody or nonimmune rabbit IgG (NIgG), and analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting using rabbit anti-TRUSS-COOH antibody. WCL, whole-cell lysate; IP, imimmunoprecipitation. (C) Expression of endogenous TRUSS in primary cultured mouse macrophages and cell lines. (D) Mouse tissue and cell distribution of TRUSS mRNA as determined by Northern blotting with the full-length TRUSS cDNA. A cDNA probe for β-actin was used to determine RNA loading. B. M. Macrophages, bone marrow macrophages. (E) Schematic showing the proposed organization of TRUSS, the locations of the five predicted TRAF binding regions (white boxes), and the COOH-terminal dileucine repeats (black boxes).

TRUSS interacts with both the membrane-proximal region and the death domain of TNF-R1. (A) COS-7 cells were cotransfected with pFLAG.TNF-R1 and pHA.TRUSS, lysed, immunoprecipitated with hamster anti-TNF-R1 antagonist antibody, and analyzed by SDS-polyacrylamide gel electrophoresis. TRUSS was detected by Western blotting with anti-HA antibody. WCL, whole-cell lysate; IP, immunoprecipitation; NIgG, nonimmune rabbit IgG. (B) TRUSS interacts with TNF-R1 in U937 cells. Approximately 5 × 10⁸ U937 cells were stimulated with human TNF-α for up to 10 min and lysed. Coimmunoprecipitating TRUSS and TRADD were detected by Western blotting. (C) Schematic representation of the GST-TNF-R1 cytoplasmic domain fusion proteins that were tested for their ability to bind TRUSS. MPR, membrane-proximal region; DD, death domain. (D) Interaction of TRUSS with the cytoplasmic domain of TNF-R1. COS-7 cells were transfected with 3 μg of pHA.TRUSS, lysed, and incubated with equal amounts of glutathione-Sepharose beads coated with the indicated GST-TNF-R1 cytoplasmic domain deletion mutants or GST alone. Coprecipitating proteins were detected by Western blotting with anti-HA antibody. (E) COS-7 cells were transfected with plasmids encoding either (i) FLAG-tagged full-length TNF-R1 (FLAG.TNF-R1 1-425), (ii) the FLAG-tagged TNF-R1 cytoplasmic domain (FLAG.TNF-R1 207-425), or (iii) the FLAG-tagged TNF-R1death domain (FLAG.TNF-R1 294-425). Lysates were incubated with equal amounts of GST-TRUSS- or GST-coated Sepharose beads, and the coprecipitates were analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting with an anti-FLAG antibody to detect TNF-R1.

The N-terminal region of TRUSS interacts with the cytoplasmic domain of TNF-R1. (A) Schematic representation of the HA-tagged TRUSS deletion mutants that were tested for their ability to coimmunoprecipitate with FLAG-tagged TNF-R1. (B) HEK293 cells were cotransfected with 1.0 μg of the indicated HA-tagged TRUSS deletion mutants and 0.5 μg of FLAG-tagged full-length TNF-R1. Lysates were immunoprecipitated with goat anti-mouse TNF-R1 antibody and analyzed by SDS-polyacrylamide gel electrophoresis. Coimmunoprecipitating TRUSS was detected by Western blotting with anti-HA antibody. TNF-R1 was detected in each immunoprecipitate by Western blotting with an anti-TNF-R1 antibody. WCL, whole-cell lysate.

Transient overexpression of HA.TRUSS activates transcription factor NF-κB in NIH 3T3 cells. (A) NIH 3T3 cells were cotransfected with the indicated amounts of pHA.TRUSS or empty vector and with a fixed amount of the NF-κB luciferase reporter construct. After 18 h, the cells were incubated in the presence (black bars) or absence (white bars) of mouse TNF-α (10 ng/ml) for a further 6 h before lysis and analysis of luciferase activity. (B) NIH 3T3 cells were cotransfected as described above with 0.1 μg of pHA.TRUSS and the NF-κB luciferase reporter construct. The cells were then stimulated with mouse TNF-α (10 ng/ml; black bars) or human TNF-α (10 ng/ml; striped bars) for 6 h or were left unstimulated (white bars) prior to luciferase activity analysis. (C) NIH 3T3 cells were transfected with 1 μg of pHA.TRUSS and then stimulated with mouse TNF-α (rmTNF), human TNF-α (rhTNF) (both at 10 ng/ml), or medium alone for 15 min. NF-κB activity in nuclear extracts was determined by EMSA. p65-containing NF-κB complexes were verified by supershifting with 1 μg of anti-p65 antibody. The lower portion of each panel shows the level of expression of TRUSS as determined by Western blotting. r, recombinant.

TRUSS is localized in a proximal position to RIP and TRAF2 in a signaling cascade leading to NF-κB activation. (A) Effect of dominant inhibitory RIP (RIP_559-671). NIH 3T3 cells were cotransfected as indicated with 1 μg of pHA.TRUSS, RIP_559-671, or both constructs together with the NF-κB luciferase reporter construct. After 18 h the cells were either stimulated with either mouse TNF-α (10 ng/ml; black bars) or medium alone (white bars) for 6 h prior to luciferase activity analysis. (B) Effect of dominant inhibitory TRAF2 (TRAF2_87-501). NIH 3T3 cells were cotransfected with 1 μg of pHA.TRUSS, TRAF2_87-501, or both constructs together with the NF-κB luciferase reporter construct. After 18 h, the cells were stimulated with either mouse TNF-α (10 ng/ml; black bars) or medium alone (white bars) for 6 h prior to luciferase activity analysis. The lower portion of each panel shows the level of TRUSS expression as determined by Western blotting of whole-cell lysates.

TRUSS is necessary for TNF-α-stimulated NF-κB activation. (A) NIH 3T3 cells were cotransfected with the 1.0 μg of plasmids encoding the indicated TRUSS deletion mutants and the NF-κB luciferase reporter construct. At 18 h posttransfection, the cells were left unstimulated (white bars) or were stimulated with mouse TNF-α (10 ng/ml; black bars) for 6 h prior to luciferase activity analysis. (B) NIH 3T3 cells transfected with the indicated amounts of pHA.TRUSS1-723 or empty vector. After 18 h the cells were stimulated with mouse TNF-α (10 ng/ml; black bars) or medium alone (white bars) for 6 h prior to luciferase assay. The lower portion of each panel shows the level of expression of TRUSS as determined by Western blotting.

TRUSS interacts with TRADD, TRAF2, RIP, IKKα, IKKβ, and IKKγ. (A) Interaction of TRUSS with TRADD, TRAF2, and components of the IKK complex. Results of GST- and His-tagged pull-down assays are shown in the left column. COS-7 cells were transfected with plasmids encoding Myc-tagged TRADD, FLAG-tagged TRAF2, FLAG-tagged IKK-α, or Myc-tagged IKK-β, lysed, and incubated with GST-TRUSS or GST-coated Sepharose beads. Interactions between IKK-γ and TRUSS were detected by incubating recombinant His-tagged IKK-γ with lysates from HA-tagged-TRUSS-transfected COS-7 cells. The complexes were captured by binding to Ni-agarose beads. All coprecipitating proteins were detected by SDS-polyacrylamide gel electrophoresis and Western blotting with the antibodies indicated. Results of coimmunoprecipitation assays are shown in the right column. COS-7 cells were cotransfected with plasmids encoding Myc-tagged TRADD, FLAG-tagged TRAF2, IKK-α, IKK-β-,or IKK-γ together with HA-tagged TRUSS, lysed, and immunoprecipitated with anti-HA antibody (or anti-IKK-γ). Coimmunoprecipitating proteins were detected by immunoblotting with the relevant antibody as indicated. WCL, whole-cell lysate; IP, immunoprecipitation; NIgG, nonimmune rabbit IgG; NTA, nitrilotriacetic acid. (B) RIP is present in a complex with TRUSS. COS-7 cells were cotransfected with HA.TRUSS and FLAG-tagged RIP, lysed, and immunoprecipitated with anti-HA antibody or anti-FLAG antibody. Coimmunoprecipitating proteins were detected by Western blotting with anti-HA (TRUSS) or anti-FLAG (RIP) antibodies. (C) TRUSS coimmunoprecipitates with endogenous TRADD, TRAF-2, and IKK in HEK293 cells. HEK293 cells were transfected with HA-tagged TRUSS (top) or empty vector (bottom), lysed, and immunoprecipitated with anti-TNF-R1, anti-TRADD, anti-TRAF2, anti-IKK-α, anti-RIP, or with anti-DR3 antibody as a negative control. Coprecipitating TRUSS was detected by Western blotting.