No abstract

These results demonstrate that the p53 response is constitutively regulated in normal cells by Mdm2 and that disruption of the interaction alone is sufficient to stabilise the p53 protein and activate the p53 response. Our mini protein approach provides a powerful new method to activate p53 without causing DNA damage. More broadly, it establishes a powerful general method for determining the biological consequences of the specific disruption of protein-protein interactions in cells.

Mdm2 and MDMX are two structurally related p53-binding proteins which show the highest level of sequence similarity in the N-terminal p53-binding domains. Apart from its ability to inhibit p53 mediated transcription, a feature it shares with mdm2, very little is known about the physiological functions of MDMX. It is clearly distinct from mdm2 since its expression appears not to be regulated by p53 and it cannot compensate for lack of mdm2 in early development. We present data on the structural similarity between the p53 binding pockets of mdm2 and MDMX using p53-and phage-selected peptides. From the results we conclude that our recently devised innovative approach to reverse the mdm2-mediated inhibition of p53's transactivation function in vivo would probably target MDMX as well. Strategies for selectively targeting mdm2 and MDMX are suggested and a possible mechanism for regulating the p53-mdm2/MDMX interactions by protein phosphorylation is discussed.

From a panel of 4 murine monoclonal antibodies directed against keratin 19 various antibody combinations were evaluated in solid-phase enzyme-linked sandwich immunoassays for detection of soluble keratin 19 fragments in patient sera. One of these antibody combinations, comprised of the monoclonal antibodies Ks 19.1 and BM 19.21, was selected for further development to a routine test (Enzymun-Test CYFRA 21-1) because of its high diagnostic sensitivity and specificity for non-small cell lung carcinoma (NSCLC). Both antibodies are specific for keratin 19, no reactivity could be observed with cytokeratin 8 or 18. The epitopes of the two antibodies were determined to be within helix 2B of the rod romain. The epitope sequences lie within the sequence 311-335 for the catcher antibody Ks 19.1 and 346-367 for the detector antibody BM 19.21. These sequences are unique, as could be confirmed from sequence databases. The standard material for the assay was prepared from a cytoskeleton fraction of cultivated MCF-7 cells. Subsequent digestion of this fraction with chymotrypsin yielded a soluble and stable standard material. Both the standard material and the serum analyte appeared as oligomers when analysed on gel chromatography: the serum analyte appeared exclusively at a M(r) of 100 +/- 10 kD, whereas the standard material eluted in fractions corresponding to 100 +/- 10 kD and 450 kD. Due to the precise definition of the antigen and the localisation of the antibody binding sequences, Enzymun-Test CYFRA 21-1 is one of the best characterised tumor markers so far.

To generate tools for monitoring processing and folding in keratin intermediate filaments, a group of monoclonal antibodies reacting with the intermediate filament protein keratin 19 were studied using different approaches to define the structure and localization of their epitopes. The binding pattern to bacterially expressed human keratin 19 fragments allowed the definition of minimal amino acid sequences required for antibody binding. The screening of overlapping 15-residue peptides confirmed and further specified the epitope locations for a subset of the tested antibodies. In addition, the epitope of an antibody with apparent species-restricted specificity (LE64) was revealed by isolating and characterizing a full-length keratin 19 clone from a PtK2 cDNA library. Taken together with species cross-reactivity of individual antibodies and sequence information obtained by probing a phage display library, specific amino acid residues could be highlighted as likely to be involved in the antibody binding.