A split-ubiquitin-based assay detects the influence of mutations on the conformational stability of the p53 DNA binding domain in vivo

Many mutations in the human tumor suppressor p53 affect the function of the protein by destabilizing the structure of its DNA binding domain. To monitor the effects of those mutations in vivo the stability of the DNA binding domain of p53 and some of its mutants was investigated with the split-ubiquitin (split-Ub) method. The split-Ub-derived in vivo data on the relative stability of the mutants roughly correlate with the quantitative data from in vitro denaturation experiments as reported in the literature. A variation of this assay allows visualizing the difference in stability between the wild-type p53 core and the mutant p53(V143A) by a simple growth assay.     

Structure and Function of p53-DNA Complexes with Inactivation and Rescue Mutations: A Molecular Dynamics Simulation Study

The tumor suppressor protein p53 can lose its function upon DNA-contact mutations (R273C and R273H) in the core DNA-binding domain. The activity can be restored by second-site suppressor or rescue mutations (R273C_T284R, R273H_T284R, and R273H_S240R). In this paper, we elucidate the structural and functional consequence of p53 proteins upon DNA-contact mutations and rescue mutations and the underlying mechanisms at the atomic level by means of molecular dynamics simulations. Furthermore, we also apply the docking approach to investigate the binding phenomena between the p53 protein and DNA upon DNA-contact mutations and rescue mutations. This study clearly illustrates that, due to DNA-contact mutants, the p53 structure loses its stability and becomes more rigid than the native protein. This structural loss might affect the p53-DNA interaction and leads to inhibition of the cancer suppression. Rescue mutants (R273C_T284R, R273H_T284R and R273H_S240R) can restore the functional activity of the p53 protein upon DNA-contact mutations and show a good interaction between the p53 protein and a DNA molecule, which may lead to reactivate the cancer suppression function. Understanding the effects of p53 cancer and rescue mutations at the molecular level will be helpful for designing drugs for p53 associated cancer diseases. These drugs should be designed so that they can help to inhibit the abnormal function of the p53 protein and to reactivate the p53 function (cell apoptosis) to treat human cancer.     

Transactivation by temperature-dependent p53 mutants in yeast and human cells

The p53 protein plays an important role in cancer prevention. In response to stress signals, p53 controls essential cell functions by regulating expression of its target genes. Full or partial loss of the p53 function in cancer cells usually results from mutations of the p53 gene. Some of them are temperature-dependent, allowing reactivation of the p53 function in certain temperature. These mutations can alter general transactivation ability of the p53 protein or they modify its transactivation only towards specific genes. We analyzed transactivation of several target genes by 23 temperature-dependent p53 mutants and stratified them into four functional groups. Seventeen p53 mutants exhibited temperature-dependency and discriminative character in human and yeast cells. Despite the differences of yeast and human cells, they allowed similar transactivation rates to the p53 mutants, thus providing evidence that functional analysis of separated alleles in yeast is valuable tool for assessment of the human p53 status.     

Transforming activity of mutant human p53 alleles

Mutant forms of the p53 gene have been shown to cooperate with an activated ras gene in transforming primary cells in culture. The aberrant proteins encoded by p53 mutants are thought to act in a dominant negative manner in these assays. In vivo data, however, reveal that where p53 has undergone genetic change in tumors, both alleles have been affected. We previously identified a case of human acute myelogenous leukemia (AML) in which both alleles of the p53 gene had undergone independent missense mutations (at codons 135 cys to ser and 246 met to val). In these blasts, p53 mutations appear to be acting recessively. We have assayed the transforming potential of these p53 mutations, as well as that of another mutation at codon 273, also identified in a human neoplasm. Both mutations from the AML blasts (codon 135 and codon 246) confer transforming ability on the mutant protein. While transformation assays may define functionally different subsets of p53 mutations, the overexpression phenotype of mutants in this assay may not accurately reflect the pathological effects of p53 mutations in vivo.     

Gain of Function of p53 Cancer Mutants in Disrupting Critical DNA Damage Response Pathways

Loss of the tumor suppression activity of p53 is required for the progression of most human cancers. In this context, p53 gene is somatically mutated in about half of all human cancers; in the rest human cancers, p53 is mostly inactivated due to the disruption of pathways important for its activation. Most p53 cancer mutations are missense mutations within the core domain, leading to the expression of full-length mutant p53 protein. The expression of p53 mutants is usually correlated with the poor prognosis of the cancer patients. Accumulating evidence has indicated that p53 cancer mutants not only lose the tumor suppression activity of WT p53, but also gain novel oncogenic activities to promote tumorigenesis and drug resistance. Therefore, to improve current cancer therapy, it is critical to elucidate the gain-of-functions of p53 cancer mutants. By analyzing the humanized p53 mutant knock-in mouse models, we have identified a new gain of function of the common p53 cancer mutants in inducing genetic instability by disrupting ATM-mediated cellular responses to DNA double-stranded break (DSB) damage. Considering that some current cancer therapies such as radiotherapy kills the cancer cells by inducing DSBs in their genome DNA, our findings will have important implications on the treatment of human cancers that express common p53 mutants.