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1
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Manav N, Sharma P, Mochan S, Malhotra L. Unraveling the unique amyloid-like aggregation behavior of the tumor suppressor p53 mutants in human cancers. Int J Biol Macromol 2025; 311:143883. [PMID: 40319958 DOI: 10.1016/j.ijbiomac.2025.143883] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/13/2025] [Revised: 04/19/2025] [Accepted: 04/30/2025] [Indexed: 05/07/2025]
Abstract
Missense mutations in the tumor suppressor p53 significantly disrupt its native structure and functions, playing a pivotal role in human cancer pathogenesis. Oncogenic mutant p53 (mutp53) not only loses its tumor-suppressive capabilities but also acquires oncogenic functions, driving cancer progression, metastasis, and chemoresistance. Despite extensive research on mutp53, the role of missense mutations in triggering amyloid-like aggregation of p53 remains an underexplored and fascinating area of study. To date, over 36 proteins are known to form amyloid-like aggregates due to abnormal folding, resulting in insoluble protein fibrils that contribute to various protein misfolding diseases, including cancer. However, the precise mechanisms by which aggregated proteins induce cancer remain inadequately understood. Notably, certain p53 mutations promote its aggregation, which has emerged as a critical factor in protein aggregation-induced oncogenesis. This review delves into the mechanisms underpinning mutp53 aggregation, emphasizing unique properties such as coaggregation, bio-isolation, prion-like cell-to-cell transmission, and chemoresistance promotion. Leveraging diverse in-silico, biophysical, and biochemical approaches, we comprehensively analyzed the aggregating potential of 26 mutp53 variants among 1297 missense mutations identified in human cancers. These findings shed light on the multifaceted roles of mutp53 aggregates in oncogenesis and tumor progression. Lastly, we present an integrative exploration of emerging therapeutic strategies designed to disaggregate mutp53 aggregates, offering promising directions for targeted cancer therapy. By addressing this enigmatic aspect of mutp53 biology, our review advances the understanding of protein aggregation in cancer and identifies avenues for innovative therapeutic interventions.
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Affiliation(s)
- Nisha Manav
- Department of Biochemistry, All India Institute of Medical Sciences, New Delhi 110029, India
| | - Pratibha Sharma
- Department of Neurology, Institute of Human Behaviour and Allied Sciences, Delhi 110095, India
| | - Sankat Mochan
- Department of Anatomy, University College of Medical Sciences, University of Delhi, Delhi 110095, India
| | - Lakshay Malhotra
- Department of Biochemistry, Sri Venkateswara College, University of Delhi, New Delhi 110021, India.
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2
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Naeimzadeh Y, Tajbakhsh A, Fallahi J. Understanding the prion-like behavior of mutant p53 proteins in triple-negative breast cancer pathogenesis: The current therapeutic strategies and future directions. Heliyon 2024; 10:e26260. [PMID: 38390040 PMCID: PMC10881377 DOI: 10.1016/j.heliyon.2024.e26260] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2023] [Revised: 01/20/2024] [Accepted: 02/09/2024] [Indexed: 02/24/2024] Open
Abstract
Breast cancer (BC) is viewed as a significant public health issue and is the primary cause of cancer-related deaths among women worldwide. Triple-negative breast cancer (TNBC) is a particularly aggressive subtype that predominantly affects young premenopausal women. The tumor suppressor p53 playsa vital role in the cellular response to DNA damage, and its loss or mutations are commonly present in many cancers, including BC. Recent evidence suggests that mutant p53 proteins can aggregate and form prion-like structures, which may contribute to the pathogenesis of different types of malignancies, such as BC. This review provides an overview of BC molecular subtypes, the epidemiology of TNBC, and the role of p53 in BC development. We also discuss the potential implications of prion-like aggregation in BC and highlight future research directions. Moreover, a comprehensive analysis of the current therapeutic approaches targeting p53 aggregates in BC treatment is presented. Strategies including small molecules, chaperone inhibitors, immunotherapy, CRISPR-Cas9, and siRNA are discussed, along with their potential benefits and drawbacks. The use of these approaches to inhibit p53 aggregation and degradation represents a promising target for cancer therapy. Future investigations into the efficacy of these approaches against various p53 mutations or binding to non-p53 proteins should be conducted to develop more effective and personalized therapies for BC treatment.
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Affiliation(s)
- Yasaman Naeimzadeh
- Department of Molecular Medicine, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, 7133654361, Iran
| | - Amir Tajbakhsh
- Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Jafar Fallahi
- Department of Molecular Medicine, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, 7133654361, Iran
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3
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Martínez M, Úbeda A, Martínez‑Botas J, Trillo M. Field exposure to 50 Hz significantly affects wild‑type and unfolded p53 expression in NB69 neuroblastoma cells. Oncol Lett 2022; 24:295. [PMID: 35949615 PMCID: PMC9353226 DOI: 10.3892/ol.2022.13415] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2022] [Accepted: 06/17/2022] [Indexed: 11/26/2022] Open
Abstract
Previous studies have shown that intermittent exposure to a 50 Hz, 100 µT sinusoidal magnetic field (MF) promotes proliferation of human neuroblastoma cells, NB69. This effect is mediated by activation of the epidermal growth factor receptor through a free radical-dependent activation of the p38 pathway. The present study investigated the possibility that the oxidative stress-sensitive protein p53 is a potential target of the MF, and that field exposure can affect the protein expression. To that end, NB69 cells were exposed to short intervals of 30 to 120 min to the aforementioned MF parameters. Two specific anti-p53 antibodies that allow discrimination between the wild and unfolded forms of p53 were used to study the expression and cellular distribution of both isoforms of the protein. The expression of the antiapoptotic protein Bcl-2, whose regulation is mediated by p53, was also analyzed. The obtained results revealed that MF exposure induced increases in p53 gene expression and in protein expression of the wild-type form of p53. Field exposure also caused overexpression of the unfolded form of p53, together with changes in the nuclear/cytoplasmic distribution of both forms of the protein. The expression of protein Bcl-2 was also significantly increased in response to the MF. As a whole, these results indicated that the MF is capable of interacting with the function, distribution and conformation of protein p53. Such interactions could be involved in previously reported MF effects on NB69 proliferation promotion.
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Affiliation(s)
- María Martínez
- Bioelectromagnetics Service, Department of Research, Ramón y Cajal University Hospital, Ramón Y Cajal Institute of Health Research, 28034 Madrid
| | - Alejandro Úbeda
- Bioelectromagnetics Service, Department of Research, Ramón y Cajal University Hospital, Ramón Y Cajal Institute of Health Research, 28034 Madrid
| | - Javier Martínez‑Botas
- Biochemistry Service, Department of Research, Ramón y Cajal University Hospital, Ramón Y Cajal Institute of Health Research, 28034 Madrid, Spain
| | - María Trillo
- Bioelectromagnetics Service, Department of Research, Ramón y Cajal University Hospital, Ramón Y Cajal Institute of Health Research, 28034 Madrid
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4
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Billant O, Friocourt G, Roux P, Voisset C. p53, A Victim of the Prion Fashion. Cancers (Basel) 2021; 13:E269. [PMID: 33450819 PMCID: PMC7828285 DOI: 10.3390/cancers13020269] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2020] [Revised: 01/07/2021] [Accepted: 01/08/2021] [Indexed: 12/17/2022] Open
Abstract
Identified in the late 1970s as an oncogene, a driving force leading to tumor development, p53 turned out to be a key tumor suppressor gene. Now p53 is considered a master gene regulating the transcription of over 3000 target genes and controlling a remarkable number of cellular functions. The elevated prevalence of p53 mutations in human cancers has led to a recurring questioning about the roles of mutant p53 proteins and their functional consequences. Both mutants and isoforms of p53 have been attributed dominant-negative and gain of function properties among which is the ability to form amyloid aggregates and behave in a prion-like manner. This report challenges the ongoing "prion p53" hypothesis by reviewing evidence of p53 behavior in light of our current knowledge regarding amyloid proteins, prionoids and prions.
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Affiliation(s)
| | - Gaëlle Friocourt
- Inserm, Université de Bretagne Occidentale, EFS, UMR 1078, GGB, F-29200 Brest, France;
| | - Pierre Roux
- CRBM, CNRS, UMR5234, 34293 Montpellier, France;
| | - Cécile Voisset
- Inserm, Université de Bretagne Occidentale, EFS, UMR 1078, GGB, F-29200 Brest, France;
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5
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Benor G, Fuks G, Chin S, Rueda OM, Mukherjee S, Arandkar S, Aylon Y, Caldas C, Domany E, Oren M. Transcriptional profiling reveals a subset of human breast tumors that retain wt TP53 but display mutant p53-associated features. Mol Oncol 2020; 14:1640-1652. [PMID: 32484602 PMCID: PMC7400784 DOI: 10.1002/1878-0261.12736] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2020] [Revised: 05/12/2020] [Accepted: 05/28/2020] [Indexed: 11/25/2022] Open
Abstract
TP53 gene mutations are very common in human cancer. While such mutations abrogate the tumor suppressive activities of the wild-type (wt) p53 protein, some of them also endow the mutant (mut) protein with oncogenic gain of function (GOF), facilitating cancer progression. Yet, p53 may acquire altered functionality even without being mutated; in particular, experiments with cultured cells revealed that wtp53 can be rewired to adopt mut-like features in response to growth factors or cancer-mimicking genetic manipulations. To assess whether such rewiring also occurs in human tumors, we interrogated gene expression profiles and pathway deregulation patterns in the METABRIC breast cancer (BC) dataset as a function of TP53 gene mutation status. Harnessing the power of machine learning, we optimized a gene expression classifier for ER+Her2- patients that distinguishes tumors carrying TP53 mutations from those retaining wt TP53. Interestingly, a small subset of wt TP53 tumors displayed gene expression and pathway deregulation patterns markedly similar to those of TP53-mutated tumors. Moreover, similar to TP53-mutated tumors, these 'pseudomutant' cases displayed a signature for enhanced proliferation and had worse prognosis than typical wtp53 tumors. Notably, these tumors revealed upregulation of genes which, in BC cell lines, were reported to be positively regulated by p53 GOF mutants. Thus, such tumors may benefit from mut p53-associated activities without having to accrue TP53 mutations.
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Affiliation(s)
- Gal Benor
- Department of Physics of Complex SystemsThe Weizmann Institute of ScienceRehovotIsrael
| | - Garold Fuks
- Department of Physics of Complex SystemsThe Weizmann Institute of ScienceRehovotIsrael
| | - Suet‐Feung Chin
- Cancer Research UK Cambridge Institute and Department of OncologyLi Ka Shing CentreUniversity of CambridgeCambridgeUK
| | - Oscar M. Rueda
- Cancer Research UK Cambridge Institute and Department of OncologyLi Ka Shing CentreUniversity of CambridgeCambridgeUK
| | - Saptaparna Mukherjee
- Department of Molecular Cell BiologyThe Weizmann Institute of ScienceRehovotIsrael
| | - Sharathchandra Arandkar
- Department of Molecular Cell BiologyThe Weizmann Institute of ScienceRehovotIsrael
- Advanced Centre for Treatment, Research and Education in Cancer (ACTREC)Tata Memorial CentreKhargharIndia
| | - Yael Aylon
- Department of Molecular Cell BiologyThe Weizmann Institute of ScienceRehovotIsrael
| | - Carlos Caldas
- Cancer Research UK Cambridge Institute and Department of OncologyLi Ka Shing CentreUniversity of CambridgeCambridgeUK
| | - Eytan Domany
- Department of Physics of Complex SystemsThe Weizmann Institute of ScienceRehovotIsrael
| | - Moshe Oren
- Department of Molecular Cell BiologyThe Weizmann Institute of ScienceRehovotIsrael
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6
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Sabapathy K, Lane DP. Understanding p53 functions through p53 antibodies. J Mol Cell Biol 2020; 11:317-329. [PMID: 30907951 PMCID: PMC6487784 DOI: 10.1093/jmcb/mjz010] [Citation(s) in RCA: 82] [Impact Index Per Article: 16.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2018] [Revised: 01/20/2019] [Accepted: 02/11/2019] [Indexed: 01/19/2023] Open
Abstract
TP53 is the most frequently mutated gene across all cancer types. Our understanding of its functions has evolved since its discovery four decades ago. Initially thought to be an oncogene, it was later realized to be a critical tumour suppressor. A significant amount of our knowledge about p53 functions have come from the use of antibodies against its various forms. The early anti-p53 antibodies contributed to the recognition of p53 accumulation as a common feature of cancer cells and to our understanding of p53 DNA-binding and transcription activities. They led to the concept that conformational changes can facilitate p53’s activity as a growth inhibitory protein. The ensuing p53 conformational-specific antibodies further underlined p53’s conformational flexibility, collectively forming the basis for current efforts to generate therapeutic molecules capable of altering the conformation of mutant p53. A subsequent barrage of antibodies against post-translational modifications on p53 has clarified p53’s roles further, especially with respect to the mechanistic details and context-dependence of its activity. More recently, the generation of p53 mutation-specific antibodies have highlighted the possibility to go beyond the general framework of our comprehension of mutant p53—and promises to provide insights into the specific properties of individual p53 mutants. This review summarizes our current knowledge of p53 functions derived through the major classes of anti-p53 antibodies, which could be a paradigm for understanding other molecular events in health and disease.
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Affiliation(s)
- Kanaga Sabapathy
- Laboratory of Molecular Carcinogenesis, Division of Cellular & Molecular Research, Humphrey Oei Institute of Cancer Research, National Cancer Centre Singapore, 11 Hospital Drive, Singapore, Singapore.,Cancer and Stem Cell Biology Program, Duke-NUS Medical School, 8 College Road, Singapore, Singapore.,Department of Biochemistry, National University of Singapore (NUS), 8 Medical Drive, Singapore, Singapore.,Institute of Molecular and Cellular Biology, 61 Biopolis Drive, Singapore, Singapore
| | - David P Lane
- p53 Laboratory (p53Lab), Agency for Science, Technology, and Research (A*STAR), Singapore, Singapore
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7
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Furth N, Bossel Ben-Moshe N, Pozniak Y, Porat Z, Geiger T, Domany E, Aylon Y, Oren M. Down-regulation of LATS kinases alters p53 to promote cell migration. Genes Dev 2016; 29:2325-30. [PMID: 26588988 PMCID: PMC4691886 DOI: 10.1101/gad.268185.115] [Citation(s) in RCA: 56] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
p53 is a pivotal tumor suppressor and a major barrier against cancer. We now report that silencing of the Hippo pathway tumor suppressors LATS1 and LATS2 in nontransformed mammary epithelial cells reduces p53 phosphorylation and increases its association with the p52 NF-κB subunit. Moreover, it partly shifts p53's conformation and transcriptional output toward a state resembling cancer-associated p53 mutants and endows p53 with the ability to promote cell migration. Notably, LATS1 and LATS2 are frequently down-regulated in breast cancer; we propose that such down-regulation might benefit cancer by converting p53 from a tumor suppressor into a tumor facilitator.
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Affiliation(s)
- Noa Furth
- Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel
| | - Noa Bossel Ben-Moshe
- Department of Physics of Complex Systems, The Weizmann Institute of Science, Rehovot 76100, Israel
| | - Yair Pozniak
- Department of Human Molecular Genetics and Biochemistry, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel
| | - Ziv Porat
- Flow Cytometry Unit, Department of Biological Services, The Weizmann Institute of Science, Rehovot 76100, Israel
| | - Tamar Geiger
- Department of Human Molecular Genetics and Biochemistry, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel
| | - Eytan Domany
- Department of Physics of Complex Systems, The Weizmann Institute of Science, Rehovot 76100, Israel
| | - Yael Aylon
- Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel
| | - Moshe Oren
- Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel
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8
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Buizza L, Cenini G, Lanni C, Ferrari-Toninelli G, Prandelli C, Govoni S, Buoso E, Racchi M, Barcikowska M, Styczynska M, Szybinska A, Butterfield DA, Memo M, Uberti D. Conformational altered p53 as an early marker of oxidative stress in Alzheimer's disease. PLoS One 2012; 7:e29789. [PMID: 22242180 PMCID: PMC3252333 DOI: 10.1371/journal.pone.0029789] [Citation(s) in RCA: 50] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2011] [Accepted: 12/05/2011] [Indexed: 11/18/2022] Open
Abstract
In order to study oxidative stress in peripheral cells of Alzheimer's disease (AD) patients, immortalized lymphocytes derived from two peculiar cohorts of patients, referring to early onset AD (EOSAD) and subjects harboured AD related mutation (ADmut), were used. Oxidative stress was evaluated measuring i) the typical oxidative markers, such as HNE Michel adducts, 3 Nitro-Tyrosine residues and protein carbonyl on protein extracts, ii) and the antioxidant capacity, following the enzymatic kinetic of superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione reductase (GRD). We found that the signs of oxidative stress, measured as oxidative marker levels, were evident only in ADmut but not in EOSAD patients. However, oxidative imbalance in EOSAD as well as ADmut lymphocytes was underlined by a reduced SOD activity and GRD activity in both pathological groups in comparison with cells derived from healthy subjects. Furthermore, a redox modulated p53 protein was found conformational altered in both EOSAD and ADmut B lymphocytes in comparison with control cells. This conformational altered p53 isoform, named “unfolded p53”, was recognized by the use of two specific conformational anti-p53 antibodies. Immunoprecipitation experiments, performed with the monoclonal antibodies PAb1620 (that recognizes p53wt) and PAb240 (that is direct towards unfolded p53), and followed by the immunoblotting with anti-4-hydroxynonenal (HNE) and anti- 3-nitrotyrosine (3NT) antibodies, showed a preferential increase of nitrated tyrosine residues in unfolded p53 isoform comparing to p53 wt protein, in both ADmut and EOSAD. In addition, a correlation between unfolded p53 and SOD activity was further found. Thus this study suggests that ROS/RNS contributed to change of p53 tertiary structure and that unfolded p53 can be considered as an early marker of oxidative imbalance in these patients.
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Affiliation(s)
- Laura Buizza
- Department of Biomedical Sciences and Biotechnologies, University of Brescia, Brescia, Italy
| | - Giovanna Cenini
- Department of Biomedical Sciences and Biotechnologies, University of Brescia, Brescia, Italy
- Sanders-Brown Centre on Aging, University of Kentucky, Lexington, Kentucky, United States of America
| | - Cristina Lanni
- Department of Experimental and Applied Pharmacology, University of Pavia, Pavia, Italy
| | | | - Chiara Prandelli
- Department of Biomedical Sciences and Biotechnologies, University of Brescia, Brescia, Italy
| | - Stefano Govoni
- Department of Experimental and Applied Pharmacology, University of Pavia, Pavia, Italy
| | - Erica Buoso
- Department of Experimental and Applied Pharmacology, University of Pavia, Pavia, Italy
| | - Marco Racchi
- Department of Experimental and Applied Pharmacology, University of Pavia, Pavia, Italy
| | | | - Maria Styczynska
- Medical Research Centre Polish Academy of Science, Warszawa, Poland
| | - Aleksandra Szybinska
- Laboratory of Neurodegeneration, International Institute of Molecular and Cell Biology, Warszawa, Poland
| | - David Allan Butterfield
- Sanders-Brown Centre on Aging, University of Kentucky, Lexington, Kentucky, United States of America
| | - Maurizio Memo
- Department of Biomedical Sciences and Biotechnologies, University of Brescia, Brescia, Italy
| | - Daniela Uberti
- Department of Biomedical Sciences and Biotechnologies, University of Brescia, Brescia, Italy
- * E-mail:
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9
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Abstract
TP53 mutations are the most frequent genetic alterations found in human cancer. For more than 20 years, TP53 mutation databases have collected over 30,000 somatic mutations from various types of cancer. Analyses of these mutations have led to many types of studies and have improved our knowledge about the TP53 protein and its function. The recent advances in sequencing methodologies and the various cancer genome sequencing projects will lead to a profound shift in database curation and data management. In this paper, we will review the current status of the TP53 mutation database, its application to various fields of research, and how data quality and curation can be improved. We will also discuss how the genetic data will be stored and handled in the future and the consequences for database management.
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10
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Abstract
Nerve Growth Factor is an essential protein that supports neuronal survival during development and influences neuronal function throughout adulthood, both in the central and peripheral nervous system. The unprocessed precursor of NGF, proNGF, seems to be endowed with biological functions distinct from those of the mature protein, such as chaperone-like activities and apoptotic and/or neurotrophic properties. We have previously suggested, based on Small Angle X-ray Scattering data, that recombinant murine proNGF has features typical of an intrinsically unfolded protein. Using complementary biophysical techniques, we show here new evidence that clarifies and widens this hypothesis through a detailed comparison of the structural properties of NGF and proNGF. Our data provide direct information about the dynamic properties of the pro-peptide and indicate that proNGF assumes in solution a compact globular conformation. The N-terminal pro-peptide extension influences the chemical environment of the mature protein and protects the protein from proteolytic digestion. Accordingly, we observe that unfolding of proNGF involves a two-steps mechanism. The distinct structural properties of proNGF as compared to NGF agree with and rationalise a different functional role of the precursor.
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Abstract
Currently, around 11 million people are living with a tumour that contains an inactivating mutation of TP53 (the human gene that encodes p53) and another 11 million have tumours in which the p53 pathway is partially abrogated through the inactivation of other signalling or effector components. The p53 pathway is therefore a prime target for new cancer drug development, and several original approaches to drug discovery that could have wide applications to drug development are being used. In one approach, molecules that activate p53 by blocking protein-protein interactions with MDM2 are in early clinical development. Remarkable progress has also been made in the development of p53-binding molecules that can rescue the function of certain p53 mutants. Finally, cell-based assays are being used to discover compounds that exploit the p53 pathway by either seeking targets and compounds that show synthetic lethality with TP53 mutations or by looking for non-genotoxic activators of the p53 response.
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12
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Bom APDA, Freitas MS, Moreira FS, Ferraz D, Sanches D, Gomes AMO, Valente AP, Cordeiro Y, Silva JL. The p53 core domain is a molten globule at low pH: functional implications of a partially unfolded structure. J Biol Chem 2009; 285:2857-66. [PMID: 19933157 PMCID: PMC2807339 DOI: 10.1074/jbc.m109.075861] [Citation(s) in RCA: 53] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
p53 is a transcription factor that maintains genome integrity, and its function is lost in 50% of human cancers. The majority of p53 mutations are clustered within the core domain. Here, we investigate the effects of low pH on the structure of the wild-type (wt) p53 core domain (p53C) and the R248Q mutant. At low pH, the tryptophan residue is partially exposed to the solvent, suggesting a fluctuating tertiary structure. On the other hand, the secondary structure increases, as determined by circular dichroism. Binding of the probe bis-ANS (bis-8-anilinonaphthalene-1-sulfonate) indicates that there is an increase in the exposure of hydrophobic pockets for both wt and mutant p53C at low pH. This behavior is accompanied by a lack of cooperativity under urea denaturation and decreased stability under pressure when p53C is in acidic pH. Together, these results indicate that p53C acquires a partially unfolded conformation (molten-globule state) at low pH (5.0). The hydrodynamic properties of this conformation are intermediate between the native and denatured conformation. 1H-15N HSQC NMR spectroscopy confirms that the protein has a typical molten-globule structure at acidic pH when compared with pH 7.2. Human breast cells in culture (MCF-7) transfected with p53-GFP revealed localization of p53 in acidic vesicles, suggesting that the low pH conformation is present in the cell. Low pH stress also tends to favor high levels of p53 in the cells. Taken together, all of these data suggest that p53 may play physiological or pathological roles in acidic microenvironments.
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Affiliation(s)
- Ana Paula D Ano Bom
- Centro Nacional de Ressonância Magnética Nuclear de Macromoléculas, Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-590, Brazil
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13
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Gordo S, Giralt E. Knitting and untying the protein network: modulation of protein ensembles as a therapeutic strategy. Protein Sci 2009; 18:481-93. [PMID: 19241367 DOI: 10.1002/pro.43] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Proteins constitute the working machinery and structural support of all organisms. In performing a given function, they must adopt highly specific structures that can change with their level of activity, often through the direct or indirect action of other proteins. Indeed, proteins typically function within an ensemble, rather than individually. Hence, they must be sufficiently flexible to interact with each other and execute diverse tasks. The discovery that errors within these groups can ultimately cause disease has led to a paradigm shift in drug discovery, from an emphasis on single protein targets to a holistic approach whereby entire ensembles are targeted.
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Affiliation(s)
- Susana Gordo
- Institute for Research in Biomedicine, Parc Científic de Barcelona, Barcelona, Spain
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14
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Gomes de Souza L, Miranda de Lima J, Dale Cotrim Guerreiro da Silva I, Manoukian Forones N. P53 Arg72Pro polymorphism in gastric cancer patients. J Gastrointest Cancer 2009; 40:41-5. [PMID: 19543839 DOI: 10.1007/s12029-009-9078-7] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2009] [Accepted: 05/31/2009] [Indexed: 12/27/2022]
Abstract
INTRODUCTION Polymorphism in codon 72, exon 4 of p53 may alter apoptosis and cancer progression. PATIENTS AND METHODS P53 Arg72Pro genotype was assessed by PCR from 84 gastric cancer patients and 185 controls. The control group was comparable in sex, race, age, smoking, and alcohol intake to the cancer group. RESULTS AND DISCUSSION There was no difference among the frequency of the alleles or genotypes between the groups. P53 Pro/Pro was associated to a lower risk of metastatic disease (p = 0.02) but not to lymph nodes metastasis or worst prognosis. Arg/Arg or Arg/Pro genotype may be associated to metastatic disease.
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Affiliation(s)
- Lessileia Gomes de Souza
- Oncology Group, Medicine Department, Universidade Federal de Sao Paulo (UNIFESP/EPM), São Paulo, Brazil.
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15
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Puca R, Nardinocchi L, Bossi G, Sacchi A, Rechavi G, Givol D, D'Orazi G. Restoring wtp53 activity in HIPK2 depleted MCF7 cells by modulating metallothionein and zinc. Exp Cell Res 2008; 315:67-75. [PMID: 18996371 DOI: 10.1016/j.yexcr.2008.10.018] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/13/2008] [Revised: 09/25/2008] [Accepted: 10/11/2008] [Indexed: 11/19/2022]
Abstract
The maintenance of p53 transactivation activity is important for p53 apoptotic function. We have shown that stable knockdown of HIPK2 induces p53 misfolding with inhibition of p53 target gene transcription. In this study we established a lentiviral-based system for doxycyclin (Dox)-induced conditional interference of HIPK2 expression to evaluate the molecular mechanisms involved in p53 deregulation. We found that HIPK2 knockdown induced metallothionein 2A (MT2A) upregulation as assessed by RT-PCR analysis, increased promoter acetylation, and increased promoter luciferase activity. The MT2A upregulation correlated with resistance to Adriamycin (ADR)-driven apoptosis and with p53 inhibition. Thus, acute knockdown of HIPK2 (HIPK2i) induced misfolded p53 protein in MCF7 breast cancer cells and inhibited p53 DNA-binding and transcription activities in response to ADR treatment. Previous works show that MT may modulate p53 activity through zinc exchange. Here, we found that inhibition of MT2A expression by siRNA in the HIPK2i cells restored p53 transcription activity. Similarly zinc supplementation to HIPK2i cells restored p53 transcription activity and drug-induced apoptosis. These data support the notion that MT2A is involved in p53 deregulation and strengthen the possibility that combination of chemotherapy and zinc might be useful to treat tumors with inactive wtp53.
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Affiliation(s)
- Rosa Puca
- Department of Experimental Oncology, Molecular Oncogenesis Laboratory, Regina Elena Cancer Institute, 00158 Rome, Italy
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16
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Helmbrecht K, Zeise E, Rensing L. Chaperones in cell cycle regulation and mitogenic signal transduction: a review. Cell Prolif 2008; 33:341-65. [PMID: 11101008 PMCID: PMC6496586 DOI: 10.1046/j.1365-2184.2000.00189.x] [Citation(s) in RCA: 211] [Impact Index Per Article: 12.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
Chaperones/heat shock proteins (HSPs) of the HSP90 and HSP70 families show elevated levels in proliferating mammalian cells and a cell cycle-dependent expression. They transiently associate with key molecules of the cell cycle control system such as Cdk4, Wee-1, pRb, p53, p27/Kip1 and are involved in the nuclear localization of regulatory proteins. They also associate with viral oncoproteins such as SV40 super T, large T and small t antigen, polyoma large and middle S antigen and EpsteinBarr virus nuclear antigen. This association is based on a J-domain in the viral proteins and may assist their targeting to the pRb/E2F complex. Small HSPs and their state of phosphorylation and oligomerization also seem to be involved in proliferation and differentiation. Chaperones/HSPs thus play important roles within cell cycle processes. Their exact functioning, however, is still a matter of discussion. HSP90 in particular, but also HSP70 and other chaperones associate with proteins of the mitogen-activated signal cascade, particularly with the Src kinase, with tyrosine receptor kinases, with Raf and the MAP-kinase activating kinase (MEK). This apparently serves the folding and translocation of these proteins, but possibly also the formation of large immobilized complexes of signal transducing molecules (scaffolding function).
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Affiliation(s)
- K Helmbrecht
- Institute of Cell Biology, Biochemistry and Biotechnology, University of Bremen, Germany
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17
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Grochová D, Šmardová J. The antimutagenic and cytoprotective effects of amifostine: the role of p53. J Appl Biomed 2007. [DOI: 10.32725/jab.2007.023] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022] Open
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18
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Abstract
The strongest and undisputed fact about p53 is the high frequency of p53 alterations in human cancer and that mutant p53 proteins constitute a complex family of several hundred proteins with heterogeneous properties. Beyond these observations, the p53 pathway and its regulation in a normal cell is like a desert trail, always moving with the wind of novel findings. The field is full of black boxes that are often ignored for sake of simplicity or because they do not fit with the current dominant view. Mutant p53 protein accumulation in tumours is the best example of a preconceived idea, as there is no experimental evidence to explain this observation. In this review, we will discuss several questions concerning the activity or selection of p53 mutations. The central domain of the p53 protein targeted by 80% of p53 mutations is associated with the DNA-binding activity of the p53 protein, but it is also the binding site for several proteins that play a key role in p53 regulation such as ASPP proteins or BclxL. The role of impaired DNA binding and/or protein interactions in tumour development has not been fully elucidated. Similarly, novel animal models carrying either missense p53 mutations or inducible p53 have provided abundant observations, some of which could challenge our view on p53 function as a tumour suppressor gene. Finally, the possible clinical applications of p53 will be discussed.
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Affiliation(s)
- T Soussi
- Department of Life Sciences, Université Pierre et Marie Curie-Paris, Paris, France.
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19
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Brazda V, Muller P, Brozkova K, Vojtesek B. Restoring wild-type conformation and DNA-binding activity of mutant p53 is insufficient for restoration of transcriptional activity. Biochem Biophys Res Commun 2006; 351:499-506. [PMID: 17070499 DOI: 10.1016/j.bbrc.2006.10.065] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2006] [Accepted: 10/11/2006] [Indexed: 11/25/2022]
Abstract
Most human tumors contain inactivated p53 protein, either by mutations and/or functional deactivation. Restoration of wild-type p53 function could be one of the key tools in new anticancer therapy. Using an electromobility shift assay, we investigated the effect of temperature on DNA binding of wild-type and mutant p53 proteins. We showed that analysis of the DNA-binding capacity of mutant p53 proteins is complicated by the temperature at which the assay is performed. Furthermore, neither ability to bind to DNA nor conformational analysis accurately defines the transcriptional activity of human tumor-derived p53 mutant proteins. That some mutants can bind DNA and adopt a wild-type conformation in vitro, but are transcriptionally inactive in vivo, points to the involvement of cellular factors required for transactivation. Therefore, the common use of purified proteins and in vitro determinations of DNA binding and conformation are not the best indicators of the functional properties of mutant p53.
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Affiliation(s)
- Vaclav Brazda
- Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic
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20
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Yi SY, Lee WJ. A p53 genetic polymorphism of gastric cancer: Difference between early gastric cancer and advanced gastric cancer. World J Gastroenterol 2006; 12:6536-9. [PMID: 17072987 PMCID: PMC4100644 DOI: 10.3748/wjg.v12.i40.6536] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the role of the polymorphism of p53 codon 72 in early gastric cancer (EGC) and advanced gastric cancer (AGC) in Korean patients.
METHODS: DNA was extracted from blood samples of gastric cancer patients (n = 291) and controls (n = 216). In the p53 codon 72 genotypes were determined by PCR-RFLP.
RESULTS: Patients with gastric cancer had a significantly higher frequency of the homozygous proline (Pro) allele than the control (P = 0.032). Patients with AGC had a significantly higher frequency of the Arg/Arg (arginine) allele (P = 0.038) than EGC and a similar Pro/Pro allele. The signet ring cell type had a higher frequency of the Pro/Pro allele than other types (P = 0.031). The Pro/Pro genotype carries a 3.9-fold increased risk of developing gastric cancer (95% CI, 1.3-15.4, P = 0.039) when compared to Arg/Arg and Arg/Pro genotypes and to develop EGC is a 5.25 fold increased risk (95% CI, 1.8-19.6, P = 0.021).
CONCLUSION: The Pro/Pro genotype of the p53 codon 72 polymorphism carries a higher risk for gastric cancer in general and is also associated with a much higher risk for EGC than AGC.
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Affiliation(s)
- Sun Young Yi
- Department of Internal Medicine and Medical Research Center, College of Medicine, Ewha Womans University, Seoul, Korea.
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21
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Römer L, Klein C, Dehner A, Kessler H, Buchner J. p53 – ein natürlicher Krebskiller: Einsichten in die Struktur und Therapiekonzepte. Angew Chem Int Ed Engl 2006. [DOI: 10.1002/ange.200600611] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023]
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22
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Römer L, Klein C, Dehner A, Kessler H, Buchner J. p53—A Natural Cancer Killer: Structural Insights and Therapeutic Concepts. Angew Chem Int Ed Engl 2006; 45:6440-60. [PMID: 16983711 DOI: 10.1002/anie.200600611] [Citation(s) in RCA: 81] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023]
Abstract
Every single day, the DNA of each cell in the human body is mutated thousands of times, even in absence of oncogenes or extreme radiation. Many of these mutations could lead to cancer and, finally, death. To fight this, multicellular organisms have evolved an efficient control system with the tumor-suppressor protein p53 as the central element. An intact p53 network ensures that DNA damage is detected early on. The importance of p53 for preventing cancer is highlighted by the fact that p53 is inactivated in more than 50 % of all human tumors. Thus, for good reason, p53 is one of the most intensively studied proteins. Despite the great effort that has been made to characterize this protein, the complex function and the structural properties of p53 are still only partially known. This review highlights basic concepts and recent progress in understanding the structure and regulation of p53, focusing on emerging new mechanistic and therapeutic concepts.
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Affiliation(s)
- Lin Römer
- Department Chemie, Technische Universität München, Lichtenbergstr. 4, 85747 Garching, Germany
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23
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Soussi T, Asselain B, Hamroun D, Kato S, Ishioka C, Claustres M, Béroud C. Meta-analysis of the p53 mutation database for mutant p53 biological activity reveals a methodologic bias in mutation detection. Clin Cancer Res 2006; 12:62-9. [PMID: 16397025 DOI: 10.1158/1078-0432.ccr-05-0413] [Citation(s) in RCA: 62] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
PURPOSE Analyses of the pattern of p53 mutations have been essential for epidemiologic studies linking carcinogen exposure and cancer. We were concerned by the inclusion of dubious reports in the p53 databases that could lead to controversial analysis prejudicial to the scientific community. EXPERIMENTAL DESIGN We used the universal mutation database p53 database (21,717 mutations) combined with a new p53 mutant activity database (2,300 mutants) to perform functional analysis of 1,992 publications reporting p53 alterations. This analysis was done using a statistical approach similar to that of clinical meta-analyses. RESULTS This analysis reveals that some reports of infrequent mutations are associated with almost normal activities of p53 proteins. These particular mutations are frequently found in studies reporting multiple mutations in one tumor, silent mutations, or lacking mutation hotspots. These reports are often associated with particular methodologies, such as nested PCR, for which key controls are not satisfactory. CONCLUSIONS We show the importance of accurate functional analysis before inferring any genetic variation. The quality of the p53 databases is essential in order to prevent erroneous analysis and/or conclusions. The availability of functional data from our new p53 web site (http://p53.free.fr and http://www.umd.be:2072/) will allow functional prescreening to identify potential artifactual data.
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Affiliation(s)
- Thierry Soussi
- Laboratoire de Génotoxicologie des tumeurs, UPMC, Dpt Pneumologie, Hôpital Tenon, Paris, France.
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24
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Lu-Hesselmann J, van Beuningen D, Meineke V, Franke E. Influences of TP53 expression on cellular radiation response and its relevance to diagnostic biodosimetry for mission environmental monitoring. RADIATION PROTECTION DOSIMETRY 2006; 122:237-43. [PMID: 17164278 DOI: 10.1093/rpd/ncl459] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/13/2023]
Abstract
TP53 is a transcriptional activator and regulates genomic instability and cellular responses to DNA damage in response to ionising radiation. The molecular mechanism behind p53-mediated responses, such as, apoptosis and genomic instability remains unclear. An in vitro model of biological effects to irradiation was established. In order to elucidate the functional role of TP53 under different stress-reaction pathways and identify possible biological indicators, p53 was stably transfected into HL-60 cells, which provides a p53 minus background. Significantly enhanced radiosensitivity and growth suppression were observed. G(2) accumulation was obtained. Radiation-induced apoptosis of HL-60 cells was significantly inhibited by TP53, indicating that, in the event of DNA damage, TP53 is able to prevent cell death of HL-60 leukaemia cells by sustaining an arrest of the cell cycle at G(2) phase. Further evidence will be presented to identify specific radiation-targeted genes or signals as possible biomarkers for early diagnosis of radiation damage as well as mission environmental monitoring.
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Affiliation(s)
- J Lu-Hesselmann
- Bundeswehr Institute of Medical Occupational and Environmental Safety, Scharnhorststrasse 13, 10115 Berlin, Germany.
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25
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Hamroun D, Kato S, Ishioka C, Claustres M, Béroud C, Soussi T. The UMD TP53 database and website: update and revisions. Hum Mutat 2006; 27:14-20. [PMID: 16278824 DOI: 10.1002/humu.20269] [Citation(s) in RCA: 109] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
Mutation of the p53 gene is the most frequent genetic alteration found in human cancer, but it is also the most frequently reported with more than 22,000 mutations published in 2,000 papers. In 1991, we developed a database and software to handle and analyze all this information. The database has been widely used for clinical analysis and molecular epidemiology. We have expanded the scope of the database by integrating structural, phylogenetic and biological information on wild-type (wt) and mutant TP53. Integration of the TP53 mutant activity database provides unique information that will be useful to both clinicians and scientists. All of this information is available from a new website (www.umd.be:2072/) that will generate a detailed informative page for every TP53 mutant in the database. New tools to check TP53 mutations and minimize errors found in the literature are also available.
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Affiliation(s)
- Dalil Hamroun
- Laboratoire de Génétique Moléculaire et Chromosomique, Institut Universitaire de Recherche Clinique et CHU, CNRS UPR 1142, Montpellier, France
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26
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Uberti D, Lanni C, Carsana T, Francisconi S, Missale C, Racchi M, Govoni S, Memo M. Identification of a mutant-like conformation of p53 in fibroblasts from sporadic Alzheimer's disease patients. Neurobiol Aging 2005; 27:1193-201. [PMID: 16165254 DOI: 10.1016/j.neurobiolaging.2005.06.013] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2004] [Revised: 04/29/2005] [Accepted: 06/21/2005] [Indexed: 11/28/2022]
Abstract
Here we show that fibroblasts from sporadic Alzheimer's disease (AD) patients specifically express an anomalous and detectable conformational state of p53 that makes these cells distinct from fibroblasts of age-matched non-AD subjects. In particular, we found that, in contrast to non-AD fibroblasts, p53 in AD fibroblasts is expressed at higher levels in resting condition, and presents a significant impairment of its DNA binding and transcriptional activity. All together, these findings figured out the presence of a mutant-like p53 phenotype. However, gene sequencing of the entire p53 gene from either AD or non-AD did not unravel point mutations. Based on immunoprecipitation studies with conformation-specific p53 antibodies (PAb1620 and PAb240), which discriminated folded versus unfolded p53 tertiary structure, we found that a significant amount of p53 assumed an unfolded tertiary structure in fibroblasts from AD patients. This conformational mutant-like p53 form was virtually undetectable in fibroblasts from non-AD patients. These data, independently from their relevance in understanding the etiopathogenesis of AD, might be useful for supporting AD diagnosis.
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Affiliation(s)
- Daniela Uberti
- Department of Biomedical Sciences and Biotechnologies, University of Brescia, Viale Europa 11, 25124 Brescia, Italy.
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27
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Abstract
The p53 gene is inactivated in about 50% of human cancers and the p53 protein is an essential component of the cell response induced by genotoxic stresses such as those generated by radiotherapy or chemotherapy. It is therefore highly likely that these alterations are an important component in tumor resistance to therapy. The particular characteristics of these alterations, 80% of which are missense mutations leading to functionally heterogeneous proteins, make p53 a unique gene in the class of tumor suppressor genes. A considerable number of mutant p53 proteins probably have an oncogenic activity per se and therefore actively participate in cell transformation. The fact that the apoptotic and antiproliferative functions of p53 can be dissociated in certain mutants also suggests another level of complexity in the relationships between p53 inactivation and neoplasia.
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Affiliation(s)
- T Soussi
- Laboratoire de Génotoxicologie des tumeurs, EA3493 IC-UPMC, Hôpital Tenon, Dpt Pneumologie, 26 rue d'Ulm, 75005 Paris, France.
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28
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Soussi T, Kato S, Levy PP, Ishioka C. Reassessment of theTP53 mutation database in human disease by data mining with a library ofTP53 missense mutations. Hum Mutat 2004; 25:6-17. [PMID: 15580553 DOI: 10.1002/humu.20114] [Citation(s) in RCA: 107] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
TP53 alteration is the most frequent genetic alteration found in human cancers. To date, more than 15,000 tumors with TP53 mutations have been published, leading to the description of more than 1,500 different TP53 mutants (http://p53.curie.fr). The frequency of these mutants is highly heterogeneous, with 11 hotspot mutants found more than 100 times, whereas 306 mutants have been reported only once. So far, little is known concerning the biological significance of these rare mutants, as the majority of biological studies have focused on classic hotspot mutants. In order to gain a deeper knowledge about the significance of all of these mutants, we have cross-checked each mutant of the TP53 mutation database for its activity, derived from a library of 2,314 TP53 mutants representing all possible amino acid substitutions caused by a point mutation. The transactivation activity of all of these mutant was analyzed with respect to eight transcription promoters [Kato S, et al., Proc Natl Acad Sci USA (2003)100:8424-8429]. Although the most frequent TP53 mutants sustain a clear loss of transactivation activity, more than 50% of the rare TP53 mutants display significant activity. Analysis in specific types of cancer or in normal skin patches demonstrates a similar distribution of TP53 loss of activity, with the exception of melanoma, in which the majority of TP53 mutants display significant activity. Our data indicate that TP53 mutants represent a highly heterogeneous population with a large diversity in terms of loss of transactivation activity that could account for the heterogeneous tumor phenotypes and the difficulty of clinical studies.
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Affiliation(s)
- Thierry Soussi
- Laboratoire de génotoxicologie des tumeurs, Département de Pneumologie, Hôpital Tenon, Paris, France.
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29
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Kanayasu T, Tsukinoki K, Jinbu Y, Kusama M, Watanabe Y. Association Between p53 Protein Expression and Chemosensitivity in Pretreatment Biopsy Specimens of Oral Squamous Cell Carcinoma. ACTA ACUST UNITED AC 2004. [DOI: 10.3353/omp.9.149] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
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Bradford CR, Zhu S, Ogawa H, Ogawa T, Ubell M, Narayan A, Johnson G, Wolf GT, Fisher SG, Carey TE. P53 mutation correlates with cisplatin sensitivity in head and neck squamous cell carcinoma lines. Head Neck 2003; 25:654-61. [PMID: 12884349 DOI: 10.1002/hed.10274] [Citation(s) in RCA: 116] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022] Open
Abstract
BACKGROUND A critical factor for successful organ preservation treatment in head and neck cancer may be selecting tumors that respond to chemotherapy and radiation. Previous results in patients indicated that tumors that overexpressed p53 were more sensitive to chemotherapy than those that did not overexpress p53. METHODS To determine the relationship of p53 mutations to sensitivity to cisplatin in vitro, 23 head and neck squamous cell carcinoma (HNSCC) cell lines were analyzed for cisplatin sensitivity, p53 expression, and p53 mutation status. RESULTS Mutations of the p53 gene were identified in 13 of 23 of the cell lines tested. Mutation of the p53 gene was significantly associated with high levels of expression of the p53 protein. The average ID(50) (drug dose required to inhibit 50% of cell growth) for cell lines with mutant p53 was 6.8 microM, whereas the average ID(50) for cell lines with wild-type p53 was 13.7 microM. CONCLUSIONS These in vitro data support a role for mutation of the p53 tumor suppressor gene as a marker for response to cisplatin in HNSCC.
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Affiliation(s)
- Carol R Bradford
- Department of Otolaryngology-Head and Neck Surgery, 1904 Taubman Center, University of Michigan, Ann Arbor, Michigan 48109-0312, USA.
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31
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Kim E, Deppert W. The complex interactions of p53 with target DNA: we learn as we go. Biochem Cell Biol 2003; 81:141-50. [PMID: 12897847 DOI: 10.1139/o03-046] [Citation(s) in RCA: 51] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023] Open
Abstract
The most import biological function of the tumor suppressor p53 is that of a sequence-specific transactivator. In response to a variety of cellular stress stimuli, p53 induces the transcription of an ever-increasing number of target genes, leading to growth arrest and repair, or to apoptosis. Long considered as a "latent" DNA binder that requires prior activation by C-terminal modification, recent data provide strong evidence that the DNA binding activity of p53 is strongly dependent on structural features within the target DNA and is latent only if the target DNA lacks a certain structural signal code. In this review we discuss evidence for complex interactions of p53 with DNA, which are strongly dependent on the dynamics of DNA structure, especially in the context of chromatin. We provide a model of how this complexity may serve to achieve selectivity of target gene regulation by p53 and how DNA structure in the context of chromatin may serve to modulate p53 functions.
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Affiliation(s)
- Ella Kim
- Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie, Universität Hamburg, Germany
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32
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Bensaad K, Le Bras M, Unsal K, Strano S, Blandino G, Tominaga O, Rouillard D, Soussi T. Change of conformation of the DNA-binding domain of p53 is the only key element for binding of and interference with p73. J Biol Chem 2003; 278:10546-55. [PMID: 12519788 DOI: 10.1074/jbc.m208233200] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Xenopus p53 has biological and biochemical properties similar to those of human p53, except for optimal temperature. The frog protein is fully active at 30 degrees C and inactive at 37 degrees C, leading to a temperature-sensitive behavior similar to that of the human mutant p53Ala(143) and the murine mutant p53Val(135). Using hybrid proteins between human and Xenopus expressed from artificial p53 minigenes, we have been able to demonstrate that change of conformation of the DNA-binding domain is the major determinant of this heat sensitivity. It has been reported that some human tumor-derived p53 mutants can engage in a physical association with p73, thus inhibiting its transactivating properties. The mechanism of this association remains to be elucidated. The nature of the mutant p53 that can engage in this association also remains controversial. Using the unique opportunity of the temperature sensitivity of Xenopus p53, we demonstrate that binding of and interference with p73 require a change of conformation in the p53 protein. This interaction occurs through the DNA-binding domain of p53 only when it is in a denatured state. These results reinforce the notion that mutant p53 with a conformational change can act as a down-regulator of the p73 pathway in human cancer and could confer a selective advantage to the tumor.
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Affiliation(s)
- Karim Bensaad
- Laboratoire de génotoxicologie des tumeurs, Institut Curie, 26 rue d'Ulm, 75005 Paris, France
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33
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Bell S, Klein C, Müller L, Hansen S, Buchner J. p53 contains large unstructured regions in its native state. J Mol Biol 2002; 322:917-27. [PMID: 12367518 DOI: 10.1016/s0022-2836(02)00848-3] [Citation(s) in RCA: 199] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
Abstract
The human tumor suppressor protein p53 is understood only to some extent on a structural level. We performed a comprehensive biochemical and biophysical structure-function analysis of p53 full-length protein and p53 fragments. The analysis showed that p53 and the fragments investigated form stable functional units. Full-length p53 and the tetrameric fragment N93p53 (residues 93-393) are, however, destabilized significantly compared to the monomeric core domain (residues 94-312) and the monomeric fragment p53C312 (residues 1-312). At the physiological temperature of 37 degrees C and in the absence of modifications or stabilizing partners, wild-type p53 is more than 50% unfolded correlating with a 75% loss in DNA-binding activity. Furthermore the analysis of CD spectra revealed that full-length p53 contains large unstructured regions in its N and C-terminal parts. Our results indicate that full-length p53 is a modular protein consisting of defined structured and unstructured regions. We propose that p53 belongs to the growing family of loosely folded or partially unstructured native proteins. The lack of a rigid structure combined with the low overall stability may allow the physiological interaction of p53 with a multitude of partner proteins and the regulation of its turnover.
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Affiliation(s)
- Stefan Bell
- Institut für Organische Chemie und Biochemie, Technische Universität München, Lichtenbergstr 4, 85747 Garching, Germany
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34
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North S, Pluquet O, Maurici D, El-Ghissassi F, Hainaut P. Restoration of wild-type conformation and activity of a temperature-sensitive mutant of p53 (p53(V272M)) by the cytoprotective aminothiol WR1065 in the esophageal cancer cell line TE-1. Mol Carcinog 2002; 33:181-8. [PMID: 11870884 DOI: 10.1002/mc.10038] [Citation(s) in RCA: 63] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
Abstract
The aminothiol WR1065, the active metabolite of the cytoprotector amifostine, exerts its antimutagenic effects through free-radical scavenging and other unknown mechanisms. In an earlier report, we showed that WR1065 activates wild-type p53 in MCF-7 cells, leading to p53-dependent arrest in the G(1) phase of the cell cycle. To determine whether WR1065 activates p53 by modulating protein conformation, we analyzed its effects on p53 conformation and activity in the esophageal cancer cell line TE-1. This cell line contains a mutation in codon 272 of p53 (p53(V272M), with methionine instead of a valine), conferring temperature-sensitive properties to the p53 protein. At the nonpermissive temperature (37 degrees C), p53(V272M) adopts the mutant p53 conformation (nonreactive with the antibody PAb1620), does not bind specifically to DNA, and is not activated in response to DNA-damaging treatment. However, treatment with 0.5-4 mM WR1065 partially restored wild-type conformation at 37 degrees C, stimulated DNA binding activity, and increased the expression of p53 target genes WAF-1, GADD45, and MDM2, leading to cell-cycle arrest in G(1). These results suggest that WR1065 activates p53 through a mechanism distinct from DNA-damage signaling, which involves modulation of p53 protein conformation.
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Affiliation(s)
- Sophie North
- Group of Molecular Carcinogenesis, International Agency for Research on Cancer, Lyon, France
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35
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Fleckenstein DS, Uphoff CC, Drexler HG, Quentmeier H. Detection of p53 gene mutations by single strand conformational polymorphism (SSCP) in human acute myeloid leukemia-derived cell lines. Leuk Res 2002; 26:207-14. [PMID: 11755471 DOI: 10.1016/s0145-2126(01)00107-2] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
We have identified new mutations in the p53 gene in 3/11 growth factor-independent and in 2/8 growth factor-dependent human acute myeloid leukemia (AML)-derived cell lines by single-strand conformational polymorphism (SSCP) and sequencing analysis. MEG-01 had a triplet deletion at codon 304; F-36P, NB-4 and MV4-11 showed point mutations at codon 344. F-36P had a second point mutation at codon 270 and NB-4 additionally at codon 319. M-MOK had a nucleotide substitution at codon 191. The frequency of p53 mutations in the cytokine-independent cell lines was comparable to that in the cytokine-dependent lines. These results suggest that loss of Wild type (wt) p53 is not the decisive event causing tumor cells to proliferate in vitro without externally added growth factors.
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MESH Headings
- Acute Disease
- Blotting, Western
- Cell Division/drug effects
- Codon/genetics
- DNA Mutational Analysis
- DNA, Neoplasm/genetics
- Genes, p53
- Granulocyte Colony-Stimulating Factor/pharmacology
- Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology
- Humans
- Interleukin-3/pharmacology
- Leukemia, Myeloid/genetics
- Leukemia, Myeloid/pathology
- Point Mutation
- Polymorphism, Single-Stranded Conformational
- RNA, Messenger/biosynthesis
- RNA, Messenger/genetics
- RNA, Neoplasm/biosynthesis
- RNA, Neoplasm/genetics
- Reverse Transcriptase Polymerase Chain Reaction
- Sequence Deletion
- Tumor Cells, Cultured/chemistry
- Tumor Cells, Cultured/drug effects
- Tumor Suppressor Protein p53/physiology
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Affiliation(s)
- Diana S Fleckenstein
- Department of Human and Animal Cell Cultures, DSMZ-German Collection of Microorganisms and Cell Cultures, Mascheroder Weg 1 B, D-38124 Braunschweig, Germany
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36
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Abstract
One protein--p53--plays nemesis to most cancers by condemning damaged cells to death or quarantining them for repair. But the activity of p53 relies on its intact native conformation, which can be lost following mutation of a single nucleotide. With thousands of such mutations identified in patients, how can a future cancer drug buttress this fragile protein structure and restore the cell's natural defence?
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Affiliation(s)
- A N Bullock
- Department of Biochemistry, University of Washington, Seattle, USA
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37
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Abstract
The p53 protein is a tumor suppressor often inactivated in cancer, which controls cell proliferation and survival through several coordinated pathways. The p53 protein is induced in response to many forms of cellular stress, genotoxic or not. p53 is a zinc-binding protein containing several reactive cysteines, and its key biochemical property, sequence-specific DNA binding, is dependent upon metal and redox regulation in vitro. In this review, we describe the main features of p53 as a metalloprotein and we discuss how metal binding and oxidation-reduction may affect p53 activity in vivo. In particular, we stress the possible involvement of thioredoxin, Ref-1 (redox factor 1), and metallothionein in the control of p53 protein conformation and activity. Furthermore, we also review the available evidence on the role of p53 as a transactivator or transrepressor of genes involved in the production and control of reactive oxygen intermediates. Overall, these data indicate that p53 lies at the center of a network of complex redox interactions. In this network, p53 can control the timely production of reactive oxygen intermediates (e.g., to initiate apoptosis), but this activity is itself under the control of changes in metal levels and in cellular redox status. This redox sensitivity may be one of the biochemical mechanisms by which p53 acts as a "sensor" of multiple forms of stress.
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Affiliation(s)
- P Hainaut
- Group of Molecular Carcinogenesis, International Agency for Research on Cancer, Lyon, France.
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38
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Eleuteri AM, Lupidi G, Angeletti M, Amici M, Marchini C, Pucciarelli S, Fioretti E. Structure--function relationships in bovine thymus 20S proteasome: a fluorimetric study. Int J Biol Macromol 2001; 28:321-30. [PMID: 11311722 DOI: 10.1016/s0141-8130(01)00132-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/18/2022]
Abstract
The structure--function relationships occurring on the bovine thymus 20S proteasome, which exhibits the features of an immunoproteasome, have been studied. The investigation has been performed, essentially, using a fluorimetric approach, taking advantage either of the sensitivity of the complex to sodium dodecil sulfate and chaotropic agents (urea and guanidine hydrochloride) or of the presence, on the molecule, of a high number of tryptophan residues. The results obtained indicate that the perturbation or the oxidation of these residues affect the catalytic events taking place on the thymus proteasome and that the functional effects determined by SDS and chaotropic agents most likely occur through a series of progressive structural modifications leading to an inactive molecule. The presence of structural intermediates in the proteasome inactivation process suggests that thymus proteasome is a molecule characterized, at the same time, by structural flexibility (modulation of active sites) and structural stability (maintaining of the quaternary structure) in agreement with its crucial role in the cell life cycle.
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Affiliation(s)
- A M Eleuteri
- Department of Molecular, Cellular and Animal Biology, Post-graduate School of Clinical Biochemistry, University of Camerino, 62032 Camerino MC, Italy.
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39
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Méplan C, Richard MJ, Hainaut P. Metalloregulation of the tumor suppressor protein p53: zinc mediates the renaturation of p53 after exposure to metal chelators in vitro and in intact cells. Oncogene 2000; 19:5227-36. [PMID: 11077439 DOI: 10.1038/sj.onc.1203907] [Citation(s) in RCA: 163] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023]
Abstract
The tumor suppressor p53 is a transcription factor which binds DNA through a structurally complex domain stabilized by a zinc atom. Zinc chelation disrupts the architecture of this domain, inducing the protein to adopt an immunological phenotype identical to that of many mutant forms of p53. In this report, we used 65Zn to show that incorporation of zinc within the protein was required for folding in the 'wild-type' conformation capable of specific DNA-binding. Using a cellular assay, we show that addition of extracellular zinc at concentrations within the physiological range (5 microM) was required for renaturation and reactivation of wild-type p53. Among other divalent metals tested (Cd2+, Cu2+, Co2+, Fe2+ and Ni2+), only Co2+ at 125 microM had a similar effect. Recombinant metallothionein (MT), a metal chelator protein, was found to modulate p53 conformation in vitro. In cultured cells, overexpression of MT by transfection could modulate p53 transcriptional activity. Taken together, these results suggest that zinc binding plays a regulatory role in the control of p53 folding and DNA-binding activity.
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Affiliation(s)
- C Méplan
- Group of Molecular Carcinogenesis, International Agency for Research on Cancer, 150 cours Albert Thomas, F-69372, Lyon Cedex 08, France
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40
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Bullock AN, Henckel J, Fersht AR. Quantitative analysis of residual folding and DNA binding in mutant p53 core domain: definition of mutant states for rescue in cancer therapy. Oncogene 2000; 19:1245-56. [PMID: 10713666 DOI: 10.1038/sj.onc.1203434] [Citation(s) in RCA: 324] [Impact Index Per Article: 13.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
The tumour suppressor p53 is mutated in half of all human cancers, most frequently with missense substitutions in its core domain. We present a new assessment of the mutation database based on quantitative folding and DNA-binding studies of the isolated core domain. Our data identify five distinct mutant classes that correlate with four well-defined regions of the core domain structure. On extrapolation to 37 degrees C the wild-type protein has a stability of 3.0 kcal/mol. This also emerges as an oncogenic threshold: all beta-sandwich mutants destabilized by this amount (50% denatured) are expected to promote cancer. Other weakly destabilizing mutations are restricted to loop 3 in the DNA-binding region. Drugs that stabilize mutant p53 folding have the potential to reactivate apoptotic signalling pathways in tumour cells either by transactivation-dependent or independent pathways. Using an affinity ligand as a proof of principle we have recovered the thermodynamic stability of the hotspot G245S. With reference states for the five mutant classes as a guide, future therapeutic strategies may similarly stabilize partially structured or binding states of mutant p53 that restore limited p53 pathways to tumour suppression.
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Affiliation(s)
- A N Bullock
- Cambridge University Chemical Laboratory and Cambridge Centre for Protein Engineering, Medical Research Council Centre, Hills Road, Cambridge CB2 2QH, UK
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41
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Méplan C, Richard MJ, Hainaut P. Redox signalling and transition metals in the control of the p53 pathway. Biochem Pharmacol 2000; 59:25-33. [PMID: 10605931 DOI: 10.1016/s0006-2952(99)00297-x] [Citation(s) in RCA: 127] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/17/2022]
Abstract
The p53 tumour suppressor protein exerts multiple, antiproliferative effects in response to genotoxic exposures. Reactive oxygen intermediates (ROI) play several distinct roles in the p53 pathway. First, they are important activators of p53 through their capacity to induce DNA strand breaks. Second, they regulate the DNA-binding activity of p53 by modulating the redox status of a critical set of cysteines in the DNA-binding domain, which are also involved in the coordination of zinc. Third, they play a role in the signalling pathways regulated by p53, as several genes encoding redox effectors are transcriptionally controlled by p53. In this review, we summarize the evidence for the involvement of ROI at these three levels. Emphasis is placed on the role of metals and ROI as potential regulators of p53 protein conformation and functions, and on the putative toxicological consequences of such a regulation.
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Affiliation(s)
- C Méplan
- Unit of Mechanisms of Carcinogenesis, International Agency for Research on Cancer, Lyon, France
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42
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Apenten RK, Galani D. Is the rate of sulfur-disulfide exchange between the native beta-Lactoglobulin and PDS related to protein conformational stability? Int J Food Sci Technol 1999. [DOI: 10.1046/j.1365-2621.1999.00390.x] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
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43
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Phillips HA. The role of the p53 tumour suppressor gene in human breast cancer. Clin Oncol (R Coll Radiol) 1999; 11:148-55. [PMID: 10465467 DOI: 10.1053/clon.1999.9032] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Affiliation(s)
- H A Phillips
- Department of Clinical Oncology, Western General Hospital, Edinburgh, UK
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44
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Wong KB, DeDecker BS, Freund SM, Proctor MR, Bycroft M, Fersht AR. Hot-spot mutants of p53 core domain evince characteristic local structural changes. Proc Natl Acad Sci U S A 1999; 96:8438-42. [PMID: 10411893 PMCID: PMC17534 DOI: 10.1073/pnas.96.15.8438] [Citation(s) in RCA: 173] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 05/19/1999] [Indexed: 11/18/2022] Open
Abstract
Most of the oncogenic mutations in the tumor suppressor p53 map to its DNA-binding (core) domain. It is thus a potential target in cancer therapy for rescue by drugs. To begin to understand how mutation inactivates p53 and hence to provide a structural basis for drug design, we have compared structures of wild-type and mutant p53 core domains in solution by NMR spectroscopy. Structural changes introduced by five hot-spot mutations (V143A, G245S, R248Q, R249S, and R273H) were monitored by chemical-shift changes. Only localized changes are observed for G245S, R248Q, R249S, and R273H, suggesting that the overall tertiary folds of these mutant proteins are similar to that of wild type. Structural changes in R273H are found mainly in the loop-sheet-helix motif and the loop L3 of the core domain. Mutations in L3 (G245S, R248Q, and R249S) introduce structural changes in the loop L2 and L3 as well as terminal residues of strands 4, 9, and 10. It is noteworthy that R248Q, which is often regarded as a contact mutant that affects only interactions with DNA, introduces structural changes as extensive as the other loop L3 mutations (G245S and R249S). These changes suggest that R248Q is also a structural mutant that perturbs the structure of loop L2-L3 regions of the p53 core domain. In contrast to other mutants, replacement of the core residue valine 143 to alanine causes chemical-shift changes in almost all residues in the beta-sandwich and the DNA-binding surface. Long-range effects of V143A mutation may affect the specificity of DNA binding.
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Affiliation(s)
- K B Wong
- Cambridge University Chemical Laboratory and Cambridge Centre for Protein Engineering, Medical Research Council Centre, Lensfield Road, Cambridge, CB2 1EW, United Kingdom
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45
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Abstract
Cancers frequently express mutant forms of the p53 transcription factor and tumor suppressor. Early observations indicated that mutant p53 can enhance the malignancy of tumor cells and immortalize primary cells. Immortalization is also frequently observed in primary cell cultures upon loss of wild-type (wt) p53, and since p53 acts as a tetramer and mutant p53 can hetero-oligomerize with the wild type, a significant number of effects are assigned to mutant p53 acting as a dominant-negative protein. Dominance depends on the ratio of the proteins as well as on the position of the mutated amino acid residue. Mutations that alter the tertiary structure can give rise to proteins capable of forcing upon wt p53 a non-wild-type conformation, and hetero-tetrameric complexes with altered conformation are impaired for DNA binding. Mutations that affect DNA contact sites compromise DNA binding in dependence on the affinity of the hetero-tetrameric complex for a p53 recognition motif. In addition to dominance, mutant p53 can exert oncogenic functions independently of the inactivation of wt p53. Such gain-of-function manifests itself in the enhancement of tumorigenicity, of metastatic potential, and of survival and therapy resistance of wt p53-null tumor cells. The significance of dominant-negative function and gain-of-function for the various cancer phenotypes, for prognosis and for the success of therapy are currently unclear and subject of study.
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Affiliation(s)
- K Roemer
- Department of Virology, University of Saarland Medical School, Homburg/Saar, Germany
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46
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Delphin C, Ronjat M, Deloulme JC, Garin G, Debussche L, Higashimoto Y, Sakaguchi K, Baudier J. Calcium-dependent interaction of S100B with the C-terminal domain of the tumor suppressor p53. J Biol Chem 1999; 274:10539-44. [PMID: 10187847 DOI: 10.1074/jbc.274.15.10539] [Citation(s) in RCA: 66] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023] Open
Abstract
In vitro, the S100B protein interacts with baculovirus recombinant p53 protein and protects p53 from thermal denaturation. This effect is isoform-specific and is not observed with S100A1, S100A6, or calmodulin. Using truncated p53 proteins in the N-terminal (p53(1-320)) and C-terminal (p53(73-393)) domains, we localized the S100B-binding region to the C-terminal region of p53. We have confirmed a calcium-dependent interaction of the S100B with a synthetic peptide corresponding to the C-terminal region of p53 (residues 319-393 in human p53) using plasmon resonance experiments on a BIAcore system. In the presence of calcium, the equilibrium affinity of the S100B for the C-terminal region of p53 immobilized on the sensor chip was 24 +/- 10 nM. To narrow down the region within p53 involved in S100B binding, two synthetic peptides, O1(357-381) (residues 357-381 in mouse p53) and YF-O2(320-346) (residues 320-346 in mouse p53), covering the C-terminal region of p53 were compared for their interaction with purified S100B. Only YF-O2 peptide interacts with S100B with high affinity. The YF-O2 motif is a critical determinant for the thermostability of p53 and also corresponds to a domain responsible for cytoplasmic sequestration of p53. Our results may explain the rescue of nuclear wild type p53 activities by S100B in fibroblast cell lines expressing the temperature-sensitive p53val135 mutant at the nonpermissive temperature.
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Affiliation(s)
- C Delphin
- Département de Biologie Moléculaire et Structurale du Commissariat à Energie Atomique, INSERM Unité 244, 38054 Grenoble, France
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47
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Abstract
The tumor suppressor p53 is conformationally unstable at physiological temperature. Even the activated p53delta30 variant, which lacks the self-inhibiting carboxy terminal domain, has a half-life of only 8 min at 37 degrees C in vitro. We have developed a genetic approach to identify p53 variants that stabilize the active conformation. The human p53delta30 gene was randomly mutated, and the resulting library was expressed in Escherichia coli under conditions that apparently denatured the parental protein. Stable p53 variants were identified based on their ability to specifically bind a p53 consensus site. The initial thermostable variants were randomly recombined by DNA shuffling, and substitutions that were functionally additive or synergistic were identified in a second more stringent round of screening. The DNA binding activity of N239Y/N268D/E336V p53delta30 variant has a half-life of 100 min at 37 degrees C, 12 times longer than that of the parental protein. The thermostable variants should be more amenable to crystallographic studies and more effective in gene therapies than the wild-type protein.
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48
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Salvat C, Aquaviva C, Jariel-Encontre I, Ferrara P, Pariat M, Steff AM, Carillo S, Piechaczyk M. Are there multiple proteolytic pathways contributing to c-Fos, c-Jun and p53 protein degradation in vivo? Mol Biol Rep 1999; 26:45-51. [PMID: 10363646 DOI: 10.1023/a:1006960021281] [Citation(s) in RCA: 41] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
The c-Fos and c-Jun oncoproteins and the p53 tumor suppressor protein are short-lived transcription factors. Several catabolic pathways contribute to their degradation in vivo. c-Fos and c-Jun are thus mostly degraded by the proteasome, but there is indirect evidence that, under certain experimental/physiological conditions, calpains participate in their destruction, at least to a limited extent. Lysosomes have also been reported to participate in the destruction of c-Fos. Along the same lines, p53 is mostly degraded following the ubiquitin/proteasome pathway and calpains also seem to participate in its degradation. Moreover, c-Fos, c-Jun and p53 turnovers are regulated upon activation of intracellular signalling cascades. All taken together, these observations underline the complexity of the mechanisms responsible for the selective destruction of proteins within cells.
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Affiliation(s)
- C Salvat
- Institut de Génétique Moléculaire, UMR5535 - CNRS, Montpellier, France
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49
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Yu MW, Yang SY, Chiu YH, Chiang YC, Liaw YF, Chen CJ. A p53 genetic polymorphism as a modulator of hepatocellular carcinoma risk in relation to chronic liver disease, familial tendency, and cigarette smoking in hepatitis B carriers. Hepatology 1999; 29:697-702. [PMID: 10051470 DOI: 10.1002/hep.510290330] [Citation(s) in RCA: 101] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/10/2023]
Abstract
This study evaluated whether the codon 72 p53 polymorphism was related to hepatocellular carcinoma (HCC). Genotypes of p53 were determined in 80 incident cases of HCC and 328 controls nested in a cohort study of 4,841 male chronic hepatitis B carriers. No overall increase in HCC risk with the Pro variant allele of the p53 polymorphism was apparent. However, there were synergistic effects on HCC development for the Pro allele with chronic liver disease and family history of HCC in first-degree relatives. Compared with subjects without the Pro allele and chronic liver disease, the increase in HCC risk associated with chronic liver disease among those without the Pro allele was only threefold. Subjects with both chronic liver disease and the Pro allele were at an increased risk of 7.60 (95% CI = 2.28-25.31). When subjects without family history of HCC and the Pro allele were considered as the reference group, there was no apparent increased risk of HCC for those without the Pro allele who had family history of HCC. Among those with both factors, there was a significantly increased risk of 3.29 (95% CI = 1.10-9.85). Both cigarette smoking and glutathione S-transferase M1 genotype modified the risk of HCC associated with the p53 polymorphism. Significantly increased risk associated with the p53 genotype was observed only among smokers who were glutathione S-transferase-null (Pro/Pro vs. Arg/Arg: odds ratio = 6.46; 95% CI = 1.55-26.94). The p53 polymorphism also interacted with the cytochrome P450 1A1 and carotenoid levels in smoking-related hepatocarcinogenesis.
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Affiliation(s)
- M W Yu
- School of Public Health, College of Public Health, National Taiwan University, Taipei, Taiwan
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50
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Covini N, Tamburin M, Consalez G, Salvati P, Benatti L. ZFM1/SF1 mRNA in rat and gerbil brain after global ischaemia. Eur J Neurosci 1999; 11:781-7. [PMID: 10103072 DOI: 10.1046/j.1460-9568.1999.00485.x] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
Cerebral ischaemia results in significant brain damage, but the molecular mechanisms associated with ischaemia-induced brain injury are not well defined. We have adopted an improved differential-display method to search for new ischaemia-related genes. Among the different cDNAs isolated following transient forebrain ischaemia in rat, PH3.3 was selected for further studies. The search for homologies revealed that it is the rat homologue to human zinc finger motif 1 (ZFM1), also called mammalian splicing factor 1 (SF1). With Northern blot, PH3.3 hybridized with three mRNA species of 2.3, 2.9 and 3.6 kb, significantly increased at 6 h and 5 days after the ischaemic insult. These findings were extended also to another animal model. In situ hybridization in ischaemic gerbils showed that PH3.3 mRNA was induced in the dentate gyrus as early as 4 h post-ischaemia. Expression peaked at 2 days in the whole hippocampus and cortex, and then progressively decreased towards sham levels. By day 4, expression had disappeared almost entirely from the cells in the CA1 region of the hippocampus, concomitant with the degeneration of pyramidal neurons. Interestingly, ZFM1/SF1 has been recently identified as activated following p53-induced apoptosis. Several lines of evidence suggest that p53 may play two roles in the post-ischaemic brain. The primary role of p53 is to activate DNA repair processes, but if repair fails, apoptosis will be initiated. Thus, ZFM1/SF1 may represent a relevant link between p53 and the neuroprotective/neurodegenerative processes which follow cerebral ischaemia.
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Affiliation(s)
- N Covini
- Pharmacia & Upjohn, CNS Research, 20014 Nerviano, Italy
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