Critical residues of epitopes recognized by several anti-p53 monoclonal antibodies correspond to key residues of p53 involved in interactions with the mdm2 protein.

The aim of this work was to study the reactivity of antibodies directed against the N-terminus of p53 protein. First, we analysed the cross-reactivity of anti-p53 antibodies from human, mouse and rabbit sera with peptides derived from human, mouse and Xenopus p53. Next, we characterized more precisely a series of monoclonal antibodies directed against the N-terminal part of p53 and produced by immunizing mice with either full length human or Xenopus p53. For each of these mAbs we localized the epitope recognized on human p53 by the Spot method of multiple peptide synthesis, defined critical residues on p53 involved in the interaction by alanine scanning replacement experiments and determined kinetic parameters using real-time interaction analysis. These antibodies could be divided into two groups according to their epitopic and kinetic characteristics and their cross-reactivity with murine p53. Our results indicate that critical residues involved in the interaction of some of these mAbs with p53 correspond to key residues on p53 involved in its interaction with the mdm2 protein. These antibodies could, therefore, represent powerful tools for the study of p53 regulation.

Regulation of the specific DNA binding activity of Xenopus laevis p53: evidence for conserved regulation through the carboxy-terminus of the protein.

Recombinant human p53 isolated either from E. coli or from insect cells is poorly active for binding to DNA but it can be dramatically stimulated by phosphorylation, antibody binding to the carboxy-terminal negative regulatory domain, short peptides derived from this negative regulatory domain or short single strands of DNA. We report here that Xenopus p53 has a very similar behavior. Using a new set of monoclonal antibodies directed either to the amino- or the carboxy-terminus of Xenopus p53, we demonstrate that the frog protein can be activated by specific carboxy-terminus monoclonal antibodies in order to bind to human p53 DNA response element. In addition, we report that such activation of both humans and frogs protein can also be achieved by small peptides derived from the carboxy-terminus of both p53. Although, the sequence of this region is not conserved in the various p53 species, the presence of conserved basic residues indicates that such activation is charge-dependent. This is confirmed by the finding that small poly-lysine peptides can activate both human and Xenopus p53. In vivo expression of Xenopus p53 indicates that this protein is able to transactivate a wide variety of human p53 response elements as long as the experiments are performed at 32 degrees C since activity at 37 degrees C, a temperature well above the natural temperature of Xenopus, is lost. Finally, we demonstrate that human mdm2 is able to down regulate the transcriptional activity of Xenopus p53.

New monoclonal antibodies directed against the propart segment of human prorenin as a tool for the exploration of prorenin conformation.

Six monoclonal antibodies (MAbs) directed against human prorenin were produced by immunizing BALB/c mice with a peptide corresponding to the sequence (-17 to +9) of prorenin. The new MAbs were screened for their ability to first bind to the immobilized peptide and then to prorenin previously captured by an anti-total renin MAb. The specificity of the MAbs was confirmed by the total lack of binding to active renin. Using BIAcore technology, equilibrium affinity constants of the MAbs were determined and ranged from 3.2 x 10(8) to 5.7 x 10(9) l/mol. Immunoradiometric assays (IRMA) for prorenin were performed using the anti-total renin MAb and the anti-prorenin MAbs. The best results were obtained when an anti-prorenin MAb was immobilized and the anti-total renin MAb was used as tracer in a one-step procedure. Moreover, the signal was significantly increased by the presence of the renin inhibitor SR 43845 suggesting that the inhibitor-induced conformational change of prorenin could be detected by the MAbs.