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BRE, brain and reproductive organ-expressed protein, was found previously to bind the intracellular juxtamembrane domain of a ubiquitous death receptor, tumor necrosis factor receptor 1 (TNF-R1), and to down-regulate TNF-alpha-induced activation of NF-kappaB. Here we show that BRE also binds to another death receptor, Fas, and upon overexpression conferred resistance to apoptosis induced by TNF-alpha, anti-Fas agonist antibody, cycloheximide, and a variety of stress-related stimuli. However, down-regulation of the endogenous BRE by small interfering RNA increased apoptosis to TNF-alpha, but nottoetoposide, indicating that the physiological antiapoptotic role of this protein is specific to death receptor-mediated apoptosis. We further demonstrate that BRE mediates antiapoptosis by inhibiting the mitochondrial apoptotic machinery but without translocation to the mitochondria or nucleus or down-regulation of the cellular level of truncated Bid. Dissociation of BRE rapidly from TNF-R1, but not from Fas, upon receptor ligation suggests that this protein interacts with the death inducing signaling complex during apoptotic induction. Increased association of BREwith phosphorylated, sumoylated, and ubiquitinated proteins after death receptor stimulation was also detected. We conclude that in contrast to the truncated Bid that integrates mitochondrial apoptosis to death receptor-triggered apoptotic cascade, BRE inhibits the integration. We propose that BRE inhibits, by ubiquitination-like activity, components in or proximal to the death-inducing signaling complexes that are necessary for activation of the mitochondria.

Little is known about human anti-idiotypic antibodies. Phage display methodology was used to reconstruct these antibodies from lupus patients, which recognize a subset (T14(+)) of anti-DNA antibodies. Antigen-specific B cells were isolated from the blood using a peptide based on a complementarity determining region (V(H)CDR3) of the prototypic T14(+) antibody. cDNA fragments of the V(H) and V(L) genes prepared from the cells were expressed as phage displayed single chain Fv (scFv) fragments using the pCANTAB-5E phagemid vector. From a reactive clone obtained, the Ig genes used were identified to be V(H)3, D5-D3, J(H)4b, V(kappa)I and J(kappa)2. The heavy chain was highly mutated, especially in CDR3, which bears mutations mostly of the replacement type; this region is also unusual in being extremely long due to a D-D fusion. In contrast, a mouse hybridoma antibody, made to the same T14(+) peptide and transformed as a scFv fragment, uses a short V(H)CDR3 comprising five amino acids, three of which are tyrosines. Tyrosines may be important for antigen binding because two of these also exist in the human V(H)CDR3. The light chains of both antibodies may also contribute to the specificity of the protein, because their V(L) segments, including the CDRs, are highly homologous to each other.

A phosphorylcholine (PC)-binding IgG (Mab2) antibody produced by a hybridoma derived from a BALB/c mouse which had been immunized against Trichinella spiralis was found to bind to the immunizing antigen (TSC) but not to other PC-associated antigens such as pneumococcal antigen (PNC) and PC-conjugated ovalbumin (PC-OVA). Sequence analysis of the protein revealed the presence of a heavy chain (VH) which was very similar (differing in only four amino acids) to that of the M511 myeloma protein, and a light chain (VL) which was completely identical to that of the M167 myeloma protein. Several M511/M167+ proteins, including the prototypic M511 protein and PC-binding proteins of other families (TEPC 15 and W3207), were examined in their binding to the various PC-associated antigens. These were found to be largely indiscriminate although subtle differences were observed for some antigens with some of the antibodies. A comparison of the VH sequences of Mab2 and these proteins revealed that of the differences seen, the single most important substitution in Mab2 which could contribute to the unique specificity of the molecule is the glycine residue at 49H. None of the other proteins, including other PCV-binding proteins published to-date, which utilize the same VH segment (99 in total), has this substitution.