Structure – function studies of the p53 protein
The main objective of our work is to understand the role of mutant p53 in human tumorigenesis. Mutations in the gene of the tumor suppressor p53 represent the most frequent genetic alterations in human cancer, affecting about 50% of all individual tumors. The major effect of these mutations is the elimination of the various tumor suppressing functions demonstrated for wild-type p53. In addition, there is accumulating evidence for an active role of mutant p53 in tumorigenesis, resulting from a "gain of function" for at least some mutant p53 proteins. Mutant p53 gain of function becomes evident because of an increased tumorigenic potential of tumor cells expressing mutant p53 genes, as well as alterations in growth properties of such cells in culture. Furthermore, data available indicate that the presence of certain p53 mutations in several types of human cancer correlates with less favourable patient prognosis. Based on these observations, we have undertaken the analysis of the heterogeneityof mutant p53.
Some mutant p53 display a change of conformation
The structural difference between the various mutant TP53 was initially identified using monoclonal antibodies able to discriminate mutations that change TP53 folding and mutations in the residues involved in DNA recognition (Gannon et al., 1990; Legros et al., 1994). Two classes of mutations have been distinguished on the basis of various in vitro assays and the three-dimensional structure of the protein (Cho et al., 1994):
The determination of the 3-dimensional crystal structure of p53 has also shed light on these two classes of mutants. As shown in Figure 6, crystallography of the central region of p53 with its DNA recognition motif has allowed a very precise definition of this interaction. Two types of regions have been identified :
Conformational studies with a new set of p53 monoclonal antibodies
Using a set of overlapping peptides of the human p53 protein, we analysed the epitopes recognized by 18 monoclonal antibodies specific for human p53 , that were produced in our laboratory. We showed that most of these epitopes corresponded to linear antigenic determinants which lie predominantly in the amino- or carboxy-terminus of the p53 protein.
Using a truncated p53 (residues 66 to 361), we selected eight new monoclonal antibodies directed to thecentral part of the protein. We then identified the epitopes recognized by seven out of these eight antibodies with a set of overlapping peptides. One of these antibodies had an epitope similar to PAb240, whereas the others recognized novel and diverse antigenic determinants. Using a series of 19 p53 mutants, we showed that the behavior of several of the new monoclonal antibodies was similar to that of PAb240 despite their various epitope localizations.
Use of hybrids proteins to solve the function of the DNA binding domain in the gain of function of mutant p53 and how it is involved in the conformational change of p53