RESUMO
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.
Assuntos
Neoplasias/metabolismo , Doenças Priônicas/metabolismo , Proteína Supressora de Tumor p53/genética , Amiloide/química , Amiloide/genética , Amiloide/metabolismo , Animais , Humanos , Mutação , Neoplasias/genética , Ligação Proteica , Dobramento de Proteína , Multimerização Proteica , Estrutura Quaternária de Proteína , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismoRESUMO
Over 50% of all human cancers lose p53 function. To evaluate the role of aggregation in cancer, we asked whether wild-type (WT) p53 and the hot-spot mutant R248Q could aggregate as amyloids under physiological conditions and whether the mutant could seed aggregation of the wild-type form. The central domains (p53C) of both constructs aggregated into a mixture of oligomers and fibrils. R248Q had a greater tendency to aggregate than WT p53. Full-length p53 aggregated into amyloid-like species that bound thioflavin T. The amyloid nature of the aggregates was demonstrated using x-ray diffraction, electron microscopy, FTIR, dynamic light scattering, cell viabilility assay, and anti-amyloid immunoassay. The x-ray diffraction pattern of the fibrillar aggregates was consistent with the typical conformation of cross ß-sheet amyloid fibers with reflexions of 4.7 Å and 10 Å. A seed of R248Q p53C amyloid oligomers and fibrils accelerated the aggregation of WT p53C, a behavior typical of a prion. The R248Q mutant co-localized with amyloid-like species in a breast cancer sample, which further supported its prion-like effect. A tumor cell line containing mutant p53 also revealed massive aggregation of p53 in the nucleus. We conclude that aggregation of p53 into a mixture of oligomers and fibrils sequestrates the native protein into an inactive conformation that is typical of a prionoid. This prion-like behavior of oncogenic p53 mutants provides an explanation for the negative dominance effect and may serve as a potential target for cancer therapy.
Assuntos
Amiloide/química , Mutação de Sentido Incorreto , Neoplasias/química , Príons , Multimerização Proteica , Proteína Supressora de Tumor p53/química , Substituição de Aminoácidos , Amiloide/genética , Amiloide/metabolismo , Benzotiazóis , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/terapia , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Tiazóis/química , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Difração de Raios XRESUMO
P53 is one of the most important tumor suppressor proteins in human cancers. Mutations in the TP53 gene are common features of malignant tumors and normally correlate to a more aggressive disease. In breast cancer, these gene alterations are present in approximately 20% of cases and are characteristically of missense type. In the present work we describe TP53 mutations in breast cancer biopsies and investigate whether wild and mutant p53 participate in protein aggregates formation in these breast cancer cases. We analyzed 88 biopsies from patients residing in the metropolitan area of Rio de Janeiro, and performed TP53 mutation screening using direct sequencing of exons 5-10. Seventeen mutations were detected, 12 of them were of missense type, 2 nonsenses, 2 deletions and 1 insertion. The presence of TP53 mutation was highly statistically associated to tumor aggressiveness of IDC cases, indicated here by Elston Grade III (p<0.0001). Paraffin embedded breast cancer tissues were analyzed for the presence of p53 aggregates through immunofluorescence co-localization assay, using anti-aggregate primary antibody A11, and anti-p53. Our results show that mutant p53 co-localizes with amyloid-like protein aggregates, depending on mutation type, suggesting that mutant p53 may form aggregates in breast cancer cells, in vivo.
Assuntos
Amiloide , Neoplasias da Mama , Genes p53 , Proteína Supressora de Tumor p53 , Adulto , Amiloide/química , Amiloide/metabolismo , Amiloide/ultraestrutura , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/ultraestrutura , DNA de Neoplasias/metabolismo , Feminino , Imunofluorescência , Regulação Neoplásica da Expressão Gênica , Estudos de Associação Genética , Humanos , Dados de Sequência Molecular , Mutação , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/ultraestruturaRESUMO
The tumor suppressor protein p53 is a nuclear protein that serves as an important transcription factor. The region responsible for sequence-specific DNA interaction is located in its core domain (p53C). Although full-length p53 binds to DNA as a tetramer, p53C binds as a monomer since it lacks the oligomerization domain. It has been previously demonstrated that two core domains have a dimerization interface and undergo conformational change when bound to DNA. Here we demonstrate that the interaction with a consensus DNA sequence provides the core domain of p53 with enhanced conformational stability at physiological salt concentrations (0.15 M). This stability could be either increased or abolished at low (0.01 M) or high (0.3 M) salt concentrations, respectively. In addition, interaction with the cognate sequence prevents aggregation of p53C into an amyloid-like structure, whereas binding to a nonconsensus DNA sequence has no effect on p53C stability, even at low ionic strength. Strikingly, sequence-specific DNA binding also resulted in a large stabilization of full-length p53, whereas nonspecific sequence binding led to no stabilization. The effects of cognate DNA could be mimicked by high concentrations of osmolytes such as glycerol, which implies that the stabilization is caused by the exclusion of water. Taken together, our results show an enhancement in protein stability driven by specific DNA recognition. When cognate DNA was added to misfolded protein obtained after a pressurization cycle, the original conformation was mostly recovered. Our results may aid the development of therapeutic approaches to prevent misfolded species of p53.
Assuntos
DNA/farmacologia , Dobramento de Proteína/efeitos dos fármacos , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismo , Naftalenossulfonato de Anilina/química , Sequência Consenso , DNA/metabolismo , Corantes Fluorescentes/química , Glicerol/química , Humanos , Pressão Hidrostática , Luz , Concentração Osmolar , Polidesoxirribonucleotídeos/química , Polidesoxirribonucleotídeos/metabolismo , Ligação Proteica/genética , Conformação Proteica/efeitos dos fármacos , Desnaturação Proteica , Estabilidade Proteica/efeitos dos fármacos , Proteínas Recombinantes/química , Espalhamento de Radiação , Espectrometria de Fluorescência , Proteína Supressora de Tumor p53/genética , Água/químicaRESUMO
Somatic mutations in the TP53 gene are the most frequently observed genetic alterations in human malignancies, including breast cancer, which is one of the leading causes of death among women in Brazil. In our study, we determined the frequency and the pattern of TP53 mutations in malignant breast tumors from 120 patients living in Rio de Janeiro, Brazil. TP53 mutations were found in 20% of the tumors, which contained a diversity of mutation types: missense (62.5%), nonsense (8.3%), silent (4.2%), intronic (12.5%), insertion (4.2%) and deletion (8.3%). Of a total of 15 missense mutations, 4 were observed at Arg248 and 2 at Cys242, which are directly involved in DNA binding and in zinc binding, respectively. A subgroup of 51 patients was analyzed with respect to the relation between the presence of TP53 mutations and classical risk factors and with tumor and patient characteristics. For this analysis, we used logistic regression and, in order to obtain more precise confidence intervals, they were recalculated using a bootstrap resampling technique. Our results demonstrate that these mutations are not statistically associated with the risk factors or the patients' characteristics. However, the presence of TP53 mutations is strongly associated with the aggressiveness of the tumors, measured by Elston classification (OR = 11.97 and 95% CI of 2.24-307.05). This finding is in agreement with previous studies, which report the presence of TP53 mutations in tumors with poor prognosis. This correlation between tumor aggressiveness and TP53 mutations could be a crucial variable for the treatment and prognosis of breast cancer.