Mechanism of rescue of common p53 cancer mutations by second-site suppressor mutations

The core domain of p53 is extremely susceptible to mutations that lead to loss of function. We analysed the stability and DNA-binding activity of such mutants to understand the mechanism of second-site suppressor mutations. Double-mutant cycles show that N239Y and N268D act as 'global stability' suppressors by increasing the stability of the cancer mutants G245S and V143A-the free energy changes are additive. Conversely, the suppressor H168R is specific for the R249S mutation: despite destabilizing wild type, H168R has virtually no effect on the stability of R249S, but restores its binding affinity for the gadd45 promoter. NMR structural comparisons of R249S/H168R and R249S/T123A/H168R with wild type and R249S show that H168R reverts some of the structural changes induced by R249S. These results have implications for possible drug therapy to restore the function of tumorigenic mutants of p53: the function of mutants such as V143A and G245S is theoretically possible to restore by small molecules that simply bind to and hence stabilize the native structure, whereas R249S requires alteration of its mutant native structure.     

Structures of p53 cancer mutants and mechanism of rescue by second-site suppressor mutations

We have solved the crystal structures of three oncogenic mutants of the core domain of the human tumor suppressor p53. The mutations were introduced into a stabilized variant. The cancer hot spot mutation R273H simply removes an arginine involved in DNA binding without causing structural distortions in neighboring residues. In contrast, the "structural" oncogenic mutations H168R and R249S induce substantial structural perturbation around the mutation site in the L2 and L3 loops, respectively. H168R is a specific intragenic suppressor mutation for R249S. When both cancer mutations are combined in the same molecule, Arg(168) mimics the role of Arg(249) in wild type, and the wild type conformation is largely restored in both loops. Our structural and biophysical data provide compelling evidence for the mechanism of rescue of mutant p53 by intragenic suppressor mutations and reveal features by which proteins can adapt to deleterious mutations.     

Activating mutations in p53 produce a common conformational effect. A monoclonal antibody specific for the mutant form.

Point mutations in the p53 gene are the most frequently identified genetic change in human cancer. They convert murine p53 from a tumour suppressor gene into a dominant transforming oncogene able to immortalize primary cells and bring about full transformation in combination with an activated ras gene. In both the human and murine systems the mutations lie in regions of p53 conserved from man to Xenopus. We have developed a monoclonal antibody to p53 designated PAb240 which does not immunoprecipitate wild type p53. A series of different p53 mutants all react more strongly with PAb240 than with PAb246. The PAb240 reactive form of p53 cannot bind to SV40 large T antigen but does bind to HSP70. In contrast, the PAb246 form binds to T antigen but not to HSP70. PAb240 recognizes all forms of p53 when they are denatured. It reacts with all mammalian p53 and chicken p53 in immunoblots. We propose that immunoprecipitation of p53 by PAb240 is diagnostic of mutation in both murine and human systems and suggest that the different point mutations which convert p53 from a recessive to a dominant oncogene exert a common conformational effect on the protein. This conformational change abolishes T antigen binding and promotes self-oligomerization. These results are consistent with a dominant negative model where mutant p53 protein binds to and neutralizes the activity of p53 in the wild type conformation.     

All-codon scanning identifies p53 cancer rescue mutations

In vitro scanning mutagenesis strategies are valuable tools to identify critical residues in proteins and to generate proteins with modified properties. We describe the fast and simple All-Codon Scanning (ACS) strategy that creates a defined gene library wherein each individual codon within a specific target region is changed into all possible codons with only a single codon change per mutagenesis product. ACS is based on a multiplexed overlapping mutagenesis primer design that saturates only the targeted gene region with single codon changes. We have used ACS to produce single amino-acid changes in small and large regions of the human tumor suppressor protein p53 to identify single amino-acid substitutions that can restore activity to inactive p53 found in human cancers. Single-tube reactions were used to saturate defined 30-nt regions with all possible codon changes. The same technique was used in 20 parallel reactions to scan the 600-bp fragment encoding the entire p53 core domain. Identification of several novel p53 cancer rescue mutations demonstrated the utility of the ACS approach. ACS is a fast, simple and versatile method, which is useful for protein structure–function analyses and protein design or evolution problems.     

The transforming and suppressor functions of p53 alleles: effects of mutations that disrupt phosphorylation, oligomerization and nuclear translocation.

Mutant p53 alleles that have a recessive phenotype in human tumors can, in cooperation with an activated H-ras gene, transform rat embryo fibroblasts (REFs). Mutant p53 proteins differ from wild type, and from each other in conformation, localization and transforming potential. Missense mutations in codons 143, 175 and 275 confer strong transforming potential. A serine 135 p53 mutant has an intermediate transforming potential, while the histidine codon 273 allele transforms weakly, if at all. In contrast to the wild type p53 gene, mutant p53 alleles with strong transforming ability cannot suppress the transformation of REFs by other oncogenes. The His273 allele retains partial suppressor function in this assay. The relevance of p53 oligomerization, phosphorylation and nuclear translocation to the transforming potential of mutant p53 and to wild type p53 suppressor function were examined. The inability of mutant p53 polypeptides to form homodimers correlates with loss of transforming function. Monomeric variants of wild type p53 protein, however, retain the ability to suppress focus formation. Phosphorylation of serine residues 315 and 392 is not required for the transforming function of mutant p53, nor is serine 315 required for suppressor function when these alleles are constitutively expressed in REF assays. Nuclear translocation-defective mutant and wild type p53 proteins retain transforming and suppressor function in REF assays.     

APEX disease gene resequencing: mutations in exon 7 of the p53 tumor suppressor gene.

Detection of mutations in disease genes will be a significant application of genomic research. Methods for detecting mutations at the single nucleotide level are required in highly mutated genes such as the tumor suppressor p53. Resequencing of an individual patient's DNA by conventional Sanger methods is impractical, calling for novel methods for sequence analysis. Toward this end, an arrayed primer extension (APEX) method for identifying sequence alterations in primary DNA structure was developed. A two-dimensional array of immobilized primers (DNA chip) was fabricated to scan p53 exon 7 by single bases. Primers were immobilized with 200 microm spacing on a glass support. Oligonucleotide templates of length 72 were used to study individual APEX resequencing reactions. A template-dependent DNA polymerase extension was performed on the chip using fluorescein-labeled dideoxynucleotides (ddNTPs). Labeled primers were evanescently excited and the induced fluorescence was imaged by CCD. The average signal-to-noise ratio (S/N) observed was 30:1. Software was developed to analyze high-density DNA chips for sequence alterations. Deletion, insertion, and substitution mutations were detected. APEX can be used to scan for any mutation (up to two-base insertions) in a known region of DNA by fabricating a DNA chip comprising complementary primers addressing each nucleotide in the wild-type sequence. Since APEX is a parallel method for determining DNA sequence, the time required to assay a region is independent of its length. APEX has a high level of accuracy, is sequence-based, and can be miniaturized to analyze a large DNA region with minimal reagents.     

Skeletons in the p53 tumor suppressor closet: genetic evidence that p53 blocks bone differentiation and development

A series of in vitro tissue culture studies indicated that the p53 tumor suppressor promotes cellular differentiation, which could explain its role in preventing cancer. Quite surprisingly, however, two new in vivo studies provide genetic evidence that p53 blocks osteoblast differentiation and bone development. These interesting results and their biological and clinical implications are the focus of this comment.     

Tumor suppressor p53: analysis of wild-type and mutant p53 complexes.

It has been suggested that the dominant effect of mutant p53 on tumor progression may reflect the mutant protein binding to wild-type p53, with inactivation of suppressor function. To date, evidence for wild-type/mutant p53 complexes involves p53 from different species. To investigate wild-type/mutant p53 complexes in relation to natural tumor progression, we sought to identify intraspecific complexes, using murine p53. The mutant phenotype p53-246(0) was used because this phenotype is immunologically distinct from wild-type p53-246+ and thus permits immunological analysis for wild-type/mutant p53 complexes. The p53 proteins were derived from genetically defined p53 cDNAs expressed in vitro and also from phenotypic variants of p53 expressed in vivo. We found that the mutant p53 phenotype was able to form a complex with the wild type when the two p53 variants were cotranslated. When mixed in their native states (after translation), the wild-type and mutant p53 proteins did not exhibit any binding affinity for each other in vitro. Under identical conditions, complexes of wild-type human and murine p53 proteins were formed. For murine p53, both the wild-type and mutant p53 proteins formed high-molecular-weight complexes when translated in vitro. This oligomerization appeared to involve the carboxyl terminus, since truncated p53 (amino acids 1 to 343) did not form complexes. We suggest that the ability of the mutant p53 phenotype to complex with wild type during cotranslation may contribute to the transforming function of activated mutants of p53 in vivo.     

Structure of the rat p53 tumor suppressor gene.

Aberration within the p53 tumor suppressor gene is the most frequently identified genetic damage in human cancer. Regulatory functions proposed for the p53 protein include modulation of the cell cycle, cellular differentiation, signal transduction, and gene expression. Additionally, the p53 gene product may guard the genome against incorporation of damaged DNA. To facilitate study of its role in carcinogenesis using a common animal model, we determined the structure of the rat p53 gene. We identified 18 splice sites and defined 25 bases of the intervening sequences adjacent to these sites. We also discovered an allelic polymorphism that occurs within intron 5 of the gene. The rat gene approximates the mouse ortholog. It is 12 kb in length with the non-coding exon 1 separated from exon 2 by 6.2 kb of intervening sequence. The location and size of all rat gene introns approximate those of the mouse. Whereas the mouse and human genes each contain 11 exons, the rat p53 gene is composed of only 10. No intervening sequence occurs between the region of the rat gene corresponding to exons 6 and 7 of the mouse and human p53 genes. This implies intron 6 may be functionally insignificant for species in which it is retained. To extrapolate to p53 involvement in human tumorigenesis, we suggest that mutational events within intron 6 may not be of pathological significance unless splicing is hindered.     

Genetic selection for mutations that impair the co-operative binding of lambda repressor

Bacteriophage λ repressor binds co-operatively to adjacent pairs of DNA target sites. A novel combination of positive genetic selections, involving two different operon fusions derived from P22 challenge phages, was used to isolate mutant λ repressors that have lost the ability to bind co-operatively to tandem sites yet retain the ability to bind a strong, single site. These cb (co-operative binding) mutations result in 10 different amino acid changes, which define eight residues in the carboxyl-terminus of repressor. Because challenge phage derivatives may be applied to study essentially any specific protein-DNA interaction, analogous combinations of genetic selections may be used to explore the ways that a variety of proteins interact to assemble regulatory complexes.     

Expanding the prion concept to cancer biology: dominant-negative effect of aggregates of mutant p53 tumour suppressor

p53 is a key protein that participates in cell-cycle control, and its malfunction can lead to cancer. This tumour suppressor protein has three main domains; the N-terminal transactivation domain, the CTD (C-terminal domain) and the core domain (p53C) that constitutes the sequence-specific DBD (DNA-binding region). Most p53 mutations related to cancer development are found in the DBD. Aggregation of p53 into amyloid oligomers and fibrils has been shown. Moreover, amyloid aggregates of both the mutant and WT (wild-type) forms of p53 were detected in tumour tissues. We propose that if p53 aggregation occurred, it would be a crucial aspect of cancer development, as p53 would lose its WT functions in an aggregated state. Mutant p53 can also exert a dominant-negative regulatory effect on WT p53. Herein, we discuss the dominant-negative effect in light of p53 aggregation and the fact that amyloid-like mutant p53 can convert WT p53 into more aggregated species, leading into gain of function in addition to the loss of tumour suppressor function. In summary, the results obtained in the last decade indicate that cancer may have characteristics in common with amyloidogenic and prion diseases.     

新的抑癌基因ASPP对p53作用的研究进展

p53是一个肿瘤抑制蛋白,它是通过调节相关基因表达,诱导细胞凋亡。p53诱导细胞凋亡的机制多年来一直不太清楚,而最近发现的ASPP(apoptosis stimulating protein of p53)蛋白家族对p53诱导细胞凋亡的机制研究有了新的进展。本文就此作一综述。     

Activation of the p53 tumor suppressor protein

The p53 tumor suppressor gene plays an important role in preventing cancer development, by arresting or killing potential tumor cells. Mutations within the p53 gene, leading to the loss of p53 activity, are found in about half of all human cancers, while many of the tumors that retain wild type p53 carry mutations in the pathways that allow full activation of p53. In either case, the result is a defect in the ability to induce a p53 response in cells undergoing oncogenic stress. Significant advances have been made recently in our understanding of the molecular pathways through which p53 activity is regulated, bringing with them fresh possibilities for the design of cancer therapies based on reactivation of the p53 response.     

The intragenic suppressor mutation Leu59Phe compensates for the effect of detrimental mutations in the jasmonate receptor COI1

The phytohormones jasmonates (JAs) control plant development, growth, and defense against insects and pathogens. The Arabidopsis JA receptor Coronatine Insensitive 1 (COI1) interacts with ARABIDOPSIS SKP-LIKE1 (ASK1)/ASK2 to form the SCF^COI1 E3 ligase and mediate JA responses. Here, we performed a genetic suppressor screen using the leaky coi1-2 (COI1^Leu245Phe) mutant for restored sensitivity to JA, and identified the intragenic suppressor mutation Leu59Phe, which was in the region connecting the F-box and leucine-rich repeats domains of COI1. The L59F substitution not only restores the COI1^L245F function, but also the COI1^Gly434Glu (coi1-22^rsp) function in JA responses, through recovering their interactions with ASK1 or ASK2 and their protein levels. The L59F change itself could not enhance the interactions between COI1 and ASK1/2, nor affect JA responses. The present study reveals that the Leu59Phe substitution compensates for the effect of some deleterious mutations in the JA receptor COI1.