Members of the transforming growth factor-␤ (TGF-␤)superfamily signal through unique cell membrane receptor serine-threonine kinases to activate downstream targets. TRAP1 is a previously described 96-kDa cytoplasmic protein shown to bind to TGF-␤ receptors and suggested to play a role in TGF-␤ signaling. We now fully characterize the binding properties of TRAP1, and show that it associates strongly with inactive heteromeric TGF-␤ and activin receptor complexes and is released upon activation of signaling. Moreover, we demonstrate that TRAP1 plays a role in the Smad-mediated signal transduction pathway, interacting with the common mediator, Smad4, in a ligand-dependent fashion. While TRAP1 has only a small stimulatory effect on TGF-␤ signaling in functional assays, deletion constructs of TRAP1 inhibit TGF-␤ signaling and diminish the interaction of Smad4 with Smad2. These are the first data to identify a specific molecular chaperone for Smad4, suggesting a model in which TRAP1 brings Smad4 into the vicinity of the receptor complex and facilitates its transfer to the receptor-activated Smad proteins.

No abstract

We have investigated the role of Smad family proteins, known to be important cytoplasmic mediators of signals from the transforming growth factor–β (TGF-β) receptor serine/threonine kinases, in TGF-β–dependent differentiation of hematopoietic cells, using as a model the human promyelocytic leukemia cell line, HL-60. TGF-β–dependent differentiation of these cells to monocytes, but not retinoic acid–dependent differentiation to granulocytes, was accompanied by rapid phosphorylation and nuclear translocation of Smad2 and Smad3. Vitamin D3 also induced phosphorylation of Smad2/3 and monocytic differentiation; however the effects were indirect, dependent on its ability to induce expression of TGF-β1. Simultaneous treatment of these cells with TGF-β1 and all-trans-retinoic acid (ATRA), which leads to almost equal numbers of granulocytes and monocytes, significantly reduced the level of phospho–Smad2/3 and its nuclear accumulation, compared with that in cells treated with TGF-β1 alone. TGF-β1 and ATRA activate P42/44 mitogen-activated protein (MAP) kinase with nearly identical kinetics, ruling out its involvement in these effects on Smad phosphorylation. Addition of the inhibitor-of-protein serine/threonine phosphatases, okadaic acid, blocks the ATRA-mediated reduction in TGF-β–induced phospho-Smad2 and shifts the differentiation toward monocytic end points. In HL-60R mutant cells, which harbor a defective retinoic acid receptor–α (RAR-α), ATRA is unable to reduce levels of TGF-β–induced phospho-Smad2/3, coincident with its inability to differentiate these cells along granulocytic pathways. Together, these data suggest a new level of cross-talk between ATRA and TGF-β, whereby a putative RAR-α–dependent phosphatase activity limits the levels of phospho-Smad2/3 induced by TGF-β, ultimately reducing the levels of nuclear Smad complexes mediating the TGF-β–dependent differentiation of the cells to monocytic end points.

Human cytomegalovirus (HCMV) is a major cause of disability in congenitally infected infants and in the immunosuppressed. There is currently no licensed prophylactic HCMV vaccine. The HCMV envelope glycoprotein B (gB) is considered a major vaccine target antigen based on its critical role in mediating viral-host cell fusion and thus viral entry. The natural conformation of HCMV gB within the viral envelope is a trimer, but there has been no reported success in producing a recombinant trimeric gB suitable for vaccine use. Phase II clinical trials of a monomeric recombinant gB protein demonstrated 50% efficacy in preventing HCMV infection in seronegative women of reproductive age, and in reducing viremia in solid organ transplantation recipients. We now report the production of a uniformly trimeric recombinant HCMV gB protein in Chinese ovary cells, as demonstrated by Western blot analysis under modified non-reducing conditions and size exclusion chromatography with multiangle scattering. Immunization of mice with trimeric HCMV gB induced up to 11-fold higher serum titers of total gB-specific IgG relative to monomeric HCMV gB using Alum + CpG as adjuvants. Further, trimeric HCMV gB elicited 50-fold higher complement-independent and 20-fold higher complement-dependent HCMV neutralizing titers compared to monomeric HCMV gB using the fibroblast cell line, MRC-5, and up to 6-fold higher complement-independent and -dependent HCMV neutralizing titers using the epithelial cell line, ARPE-19. The markedly enhanced HCMV neutralizing activity in response to trimeric HCMV gB was also observed using an additional four distinct clinical HCMV isolates. These data support a role for trimeric HCMV gB as an important component for clinical testing of a prophylactic HCMV vaccine.

Infectious mononucleosis and B-cell transformation in response to infection with Epstein-Barr virus (EBV) is dependent upon binding of the EBV envelope glycoprotein gp350 to CD21 on B-cells. Gp350-specific antibody comprises most of the EBV neutralizing activity in the serum of infected patients, making this protein a promising target antigen for a prophylactic EBV vaccine. We describe a novel, tetrameric gp350-based vaccine that exhibits markedly enhanced immunogenicity relative to its monomeric counterpart. Plasmid DNA was constructed for synthesis, within transfected CHO cells, of a tetrameric, truncated (a.a. 1-470) gp350 protein (gp3501-470). Tetrameric gp3501-470 induced ~20-fold higher serum titers of gp3501-470-specific IgG and >19-fold enhancements in neutralizing titers at the highest dose, and was >25-fold more immunogenic on a per-weight basis than monomeric gp3501-470. Further, epidermal immunization with plasmid DNA encoding gp3501-470 tetramer induced 8-fold higher serum titers of gp3501-470-specific IgG relative to monomer. Tetrameric gp3501-470 binding to human CD21 was >24-fold more efficient on a per-weight basis than monomer, but neither tetramer nor monomer mediated polyclonal human B-cell activation. Finally, the introduction of strong, universal tetanus toxoid (TT)-specific CD4+ T-cell epitopes into the tetrameric gp3501-470 had no effect on the gp3501-470-specific IgG response in naïve mice, and resulted in suppressed gp3501-470-specific IgG responses in TT-primed mice. Collectively, these data suggest that tetrameric gp3501-470 is a potentially promising candidate for testing as a prophylactic EBV vaccine, and that protein multimerization, using the approach described herein, is likely to be clinically relevant for enhancing the immunogenicity of other proteins of vaccine interest.