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Qi Y, Tan Z, Chen H, Xiao Z, Zhang L, Wu B, Liu C, Gao Y, Yang X, Wu L, Lu L, Wang H. Pyrimidine synthase CAD deamidates and inactivates p53. Cell Res 2025:10.1038/s41422-025-01112-9. [PMID: 40240485 DOI: 10.1038/s41422-025-01112-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2024] [Accepted: 03/30/2025] [Indexed: 04/18/2025] Open
Affiliation(s)
- Yue Qi
- Obstetrics and Gynecology Hospital, State Key Laboratory of Genetic Engineering, School of Life Sciences, Children's Hospital, Fudan University, Shanghai, China
| | - Zizheng Tan
- Shanghai Key Laboratory of Metabolic Remodeling and Health, Institute of Metabolism and Integrative Biology, Fudan University, Shanghai, China
| | - Hanyu Chen
- Obstetrics and Gynecology Hospital, State Key Laboratory of Genetic Engineering, School of Life Sciences, Children's Hospital, Fudan University, Shanghai, China
| | - Ziqi Xiao
- Shanghai Key Laboratory of Metabolic Remodeling and Health, Institute of Metabolism and Integrative Biology, Fudan University, Shanghai, China
| | - Liang Zhang
- Obstetrics and Gynecology Hospital, State Key Laboratory of Genetic Engineering, School of Life Sciences, Children's Hospital, Fudan University, Shanghai, China
| | - Boxuan Wu
- Shanghai Key Laboratory of Metabolic Remodeling and Health, Institute of Metabolism and Integrative Biology, Fudan University, Shanghai, China
| | - Chennan Liu
- Shanghai Key Laboratory of Metabolic Remodeling and Health, Institute of Metabolism and Integrative Biology, Fudan University, Shanghai, China
| | - Yunqian Gao
- Pediatric Translational Medicine Institute, Shanghai Children's Medical Center, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
| | - Xueyan Yang
- Obstetrics and Gynecology Hospital, State Key Laboratory of Genetic Engineering, School of Life Sciences, Children's Hospital, Fudan University, Shanghai, China
| | - Lingqian Wu
- The Center for Medical Genetics, Hunan Key Laboratory of Medical Genetics & Hunan Key Laboratory of Animal Models for Human Diseases, School of Life Sciences, Central South University, Changsha, Hunan, China
| | - Lei Lu
- Shanghai Key Laboratory of Metabolic Remodeling and Health, Institute of Metabolism and Integrative Biology, Fudan University, Shanghai, China.
| | - Hongyan Wang
- Obstetrics and Gynecology Hospital, State Key Laboratory of Genetic Engineering, School of Life Sciences, Children's Hospital, Fudan University, Shanghai, China.
- Shanghai Key Laboratory of Metabolic Remodeling and Health, Institute of Metabolism and Integrative Biology, Fudan University, Shanghai, China.
- Prenatal Diagnosis Center of Shenzhen Maternity & Child Healthcare Hospital, Shenzhen, Guangzhou, China.
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2
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Fallatah MMJ, Demir Ö, Law F, Lauinger L, Baronio R, Hall L, Bournique E, Srivastava A, Metzen LT, Norman Z, Buisson R, Amaro RE, Kaiser P. Pyrimidine Triones as Potential Activators of p53 Mutants. Biomolecules 2024; 14:967. [PMID: 39199355 PMCID: PMC11352488 DOI: 10.3390/biom14080967] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/29/2024] [Revised: 07/22/2024] [Accepted: 08/05/2024] [Indexed: 09/01/2024] Open
Abstract
p53 is a crucial tumor suppressor in vertebrates that is frequently mutated in human cancers. Most mutations are missense mutations that render p53 inactive in suppressing tumor initiation and progression. Developing small-molecule drugs to convert mutant p53 into an active, wild-type-like conformation is a significant focus for personalized cancer therapy. Prior research indicates that reactivating p53 suppresses cancer cell proliferation and tumor growth in animal models. Early clinical evidence with a compound selectively targeting p53 mutants with substitutions of tyrosine 220 suggests potential therapeutic benefits of reactivating p53 in patients. This study identifies and examines the UCI-1001 compound series as a potential corrector for several p53 mutations. The findings indicate that UCI-1001 treatment in p53 mutant cancer cell lines inhibits growth and reinstates wild-type p53 activities, including DNA binding, target gene activation, and induction of cell death. Cellular thermal shift assays, conformation-specific immunofluorescence staining, and differential scanning fluorometry suggest that UCI-1001 interacts with and alters the conformation of mutant p53 in cancer cells. These initial results identify pyrimidine trione derivatives of the UCI-1001 series as candidates for p53 corrector drug development.
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Affiliation(s)
| | - Özlem Demir
- Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA 92093, USA
| | - Fiona Law
- Department of Biological Chemistry, University of California Irvine, Irvine, CA 92697, USA
| | - Linda Lauinger
- Department of Biological Chemistry, University of California Irvine, Irvine, CA 92697, USA
| | - Roberta Baronio
- Department of Biological Chemistry, University of California Irvine, Irvine, CA 92697, USA
| | - Linda Hall
- Department of Biological Chemistry, University of California Irvine, Irvine, CA 92697, USA
| | - Elodie Bournique
- Department of Biological Chemistry, University of California Irvine, Irvine, CA 92697, USA
| | - Ambuj Srivastava
- Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA 92093, USA
| | - Landon Tyler Metzen
- Department of Biological Chemistry, University of California Irvine, Irvine, CA 92697, USA
| | - Zane Norman
- Department of Biological Chemistry, University of California Irvine, Irvine, CA 92697, USA
| | - Rémi Buisson
- Department of Biological Chemistry, University of California Irvine, Irvine, CA 92697, USA
| | - Rommie E. Amaro
- Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA 92093, USA
| | - Peter Kaiser
- Department of Biological Chemistry, University of California Irvine, Irvine, CA 92697, USA
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3
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Tjader NP, Beer AJ, Ramroop J, Tai MC, Ping J, Gandhi T, Dauch C, Neuhausen SL, Ziv E, Sotelo N, Ghanekar S, Meadows O, Paredes M, Gillespie J, Aeilts A, Hampel H, Zheng W, Jia G, Hu Q, Wei L, Liu S, Ambrosone CB, Palmer JR, Carpten JD, Yao S, Stevens P, Ho WK, Pan JW, Fadda P, Huo D, Teo SH, McElroy JP, Toland AE. Association of ESR1 germline variants with TP53 somatic variants in breast tumors in a genome-wide study. MEDRXIV : THE PREPRINT SERVER FOR HEALTH SCIENCES 2023:2023.12.06.23299442. [PMID: 38106140 PMCID: PMC10723566 DOI: 10.1101/2023.12.06.23299442] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/19/2023]
Abstract
Background In breast tumors, somatic mutation frequencies in TP53 and PIK3CA vary by tumor subtype and ancestry. HER2 positive and triple negative breast cancers (TNBC) have a higher frequency of TP53 somatic mutations than other subtypes. PIK3CA mutations are more frequently observed in hormone receptor positive tumors. Emerging data suggest tumor mutation status is associated with germline variants and genetic ancestry. We aimed to identify germline variants that are associated with somatic TP53 or PIK3CA mutation status in breast tumors. Methods A genome-wide association study was conducted using breast cancer mutation status of TP53 and PIK3CA and functional mutation categories including TP53 gain of function (GOF) and loss of function mutations and PIK3CA activating/hotspot mutations. The discovery analysis consisted of 2850 European ancestry women from three datasets. Germline variants showing evidence of association with somatic mutations were selected for validation analyses based on predicted function, allele frequency, and proximity to known cancer genes or risk loci. Candidate variants were assessed for association with mutation status in a multi-ancestry validation study, a Malaysian study, and a study of African American/Black women with TNBC. Results The discovery Germline x Mutation (GxM) association study found five variants associated with one or more TP53 phenotypes with P values <1×10-6, 33 variants associated with one or more TP53 phenotypes with P values <1×10-5, and 44 variants associated with one or more PIK3CA phenotypes with P values <1×10-5. In the multi-ancestry and Malaysian validation studies, germline ESR1 locus variant, rs9383938, was associated with the presence of TP53 mutations overall (P values 6.8×10-5 and 9.8×10-8, respectively) and TP53 GOF mutations (P value 8.4×10-6). Multiple variants showed suggestive evidence of association with PIK3CA mutation status in the validation studies, but none were significant after correction for multiple comparisons. Conclusions We found evidence that germline variants were associated with TP53 and PIK3CA mutation status in breast cancers. Variants near the estrogen receptor alpha gene, ESR1, were significantly associated with overall TP53 mutations and GOF mutations. Larger multi-ancestry studies are needed to confirm these findings and determine if these variants contribute to ancestry-specific differences in mutation frequency.
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Affiliation(s)
- Nijole P. Tjader
- Department of Cancer Biology and Genetics, The Ohio State University College of Medicine, Columbus, OH 43210, USA
| | - Abigail J. Beer
- Department of Cancer Biology and Genetics, The Ohio State University College of Medicine, Columbus, OH 43210, USA
| | - Johnny Ramroop
- The City College of New York, City University of New York, New York, NY, USA
| | - Mei-Chee Tai
- Cancer Research Malaysia, Subang Jaya, Selangor 47500, Malaysia
| | - Jie Ping
- Division of Epidemiology, Vanderbilt Epidemiology Center, Vanderbilt-Ingram Cancer Center, Nashville, TN 37203
| | - Tanish Gandhi
- Biomedical Sciences, The Ohio State University College of Medicine, Columbus, OH 43210, USA
- The Ohio State University Medical School, Columbus, OH, 43210, USA
| | - Cara Dauch
- Department of Cancer Biology and Genetics, The Ohio State University College of Medicine, Columbus, OH 43210, USA
- The Ohio State University Wexner Medical Center, Clinical Trials Office, Columbus, OH 43210, USA
| | - Susan L. Neuhausen
- Beckman Research Institute of City of Hope, Department of Population Sciences, Duarte, CA, USA
| | - Elad Ziv
- University of California, Helen Diller Family Comprehensive Cancer Center, San Francisco, San Francisco, CA, USA
- University of California, Department of Medicine, San Francisco, San Francisco, CA, USA
- University of California San Francisco, Institute for Human Genetics, San Francisco, CA, USA
| | - Nereida Sotelo
- Department of Cancer Biology and Genetics, The Ohio State University College of Medicine, Columbus, OH 43210, USA
| | - Shreya Ghanekar
- Department of Cancer Biology and Genetics, The Ohio State University College of Medicine, Columbus, OH 43210, USA
| | - Owen Meadows
- Biomedical Sciences, The Ohio State University College of Medicine, Columbus, OH 43210, USA
| | - Monica Paredes
- Biomedical Sciences, The Ohio State University College of Medicine, Columbus, OH 43210, USA
| | - Jessica Gillespie
- The Ohio State University Comprehensive Cancer Center, Columbus, OH, USA
| | - Amber Aeilts
- Department of Internal Medicine, Division of Human Genetics, The Ohio State University, Columbus, OH, 43210, USA
| | - Heather Hampel
- Department of Medical Oncology & Therapeutics Research, City of Hope National Medical Center, Duarte, CA, USA
| | - Wei Zheng
- Division of Epidemiology, Vanderbilt Epidemiology Center, Vanderbilt-Ingram Cancer Center, Nashville, TN 37203
| | - Guochong Jia
- Division of Epidemiology, Vanderbilt Epidemiology Center, Vanderbilt-Ingram Cancer Center, Nashville, TN 37203
| | - Qiang Hu
- Department of Biostatistics and Bioinformatics, Roswell Park Comprehensive Cancer Center, Buffalo, NY, USA
| | - Lei Wei
- Department of Biostatistics and Bioinformatics, Roswell Park Comprehensive Cancer Center, Buffalo, NY, USA
| | - Song Liu
- Department of Biostatistics and Bioinformatics, Roswell Park Comprehensive Cancer Center, Buffalo, NY, USA
| | - Christine B. Ambrosone
- Department of Cancer Control and Prevention, Roswell Park Comprehensive Cancer Center, Buffalo, NY, USA
| | - Julie R. Palmer
- Slone Epidemiology Center at Boston University, Boston, MA, USA
| | - John D. Carpten
- City of Hope Comprehensive Cancer Center, Duarte, CA, USA
- Department of Integrative Translational Sciences, City of Hope, Duarte, CA
| | - Song Yao
- Department of Cancer Control and Prevention, Roswell Park Comprehensive Cancer Center, Buffalo, NY, USA
| | - Patrick Stevens
- The Ohio State University Comprehensive Cancer Center, Bioinformatics Shared Resource, Columbus, OH, USA
| | - Weang-Kee Ho
- Cancer Research Malaysia, Subang Jaya, Selangor 47500, Malaysia
- School of Mathematical Sciences, Faculty of Science and Engineering, University of Nottingham Malaysia, Semenyih, Selangor 43500, Malaysia
| | - Jia Wern Pan
- Cancer Research Malaysia, Subang Jaya, Selangor 47500, Malaysia
| | - Paolo Fadda
- The Ohio State University Comprehensive Cancer Center, Genomics Shared Resource, Columbus, OH, USA
| | - Dezheng Huo
- Department of Public Health Sciences, University of Chicago, Chicago, IL, 60637, USA
| | - Soo-Hwang Teo
- Cancer Research Malaysia, Subang Jaya, Selangor 47500, Malaysia
- Faculty of Medicine, University Malaya Cancer Research Institute, University of Malaya, Kuala Lumpur 50603, Malaysia
| | - Joseph Paul McElroy
- The Ohio State University Center for Biostatistics, Department of Biomedical Informatics, Columbus, OH, USA
| | - Amanda Ewart Toland
- Department of Cancer Biology and Genetics, The Ohio State University College of Medicine, Columbus, OH 43210, USA
- The Ohio State University Comprehensive Cancer Center, Columbus, OH, USA
- Department of Internal Medicine, Division of Human Genetics, The Ohio State University, Columbus, OH, 43210, USA
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Fallatah MMJ, Law FV, Chow WA, Kaiser P. Small-molecule correctors and stabilizers to target p53. Trends Pharmacol Sci 2023; 44:274-289. [PMID: 36964053 PMCID: PMC10511064 DOI: 10.1016/j.tips.2023.02.007] [Citation(s) in RCA: 18] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2023] [Revised: 02/26/2023] [Accepted: 02/27/2023] [Indexed: 03/26/2023]
Abstract
The tumor suppressor p53 is the most frequently mutated protein in human cancer and tops the list of high-value precision oncology targets. p53 prevents initiation and progression of cancer by inducing cell-cycle arrest and various forms of cell death. Tumors have thus evolved ways to inactivate p53, mainly by TP53 mutations or by hyperactive p53 degradation. This review focuses on two types of p53 targeting compounds, MDM2 antagonists and mutant p53 correctors. MDM2 inhibitors prevent p53 protein degradation, while correctors restore tumor suppressor activity of p53 mutants by enhancing thermodynamic stability. Herein we explore both novel and repurposed p53 targeting compounds, discuss their mode of action, and examine the challenges in advancing them to the clinic.
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Affiliation(s)
- Maryam M J Fallatah
- Department of Biological Chemistry, University of California, Irvine, Irvine, CA 92697, USA; Chao Family Comprehensive Cancer Center, University of California, Irvine, Irvine, CA 92697, USA
| | - Fiona V Law
- Department of Biological Chemistry, University of California, Irvine, Irvine, CA 92697, USA; Chao Family Comprehensive Cancer Center, University of California, Irvine, Irvine, CA 92697, USA
| | - Warren A Chow
- Chao Family Comprehensive Cancer Center, University of California, Irvine, Irvine, CA 92697, USA; Division of Hematology/Oncology, Department of Medicine, University of California, Irvine, Irvine, CA 92697, USA
| | - Peter Kaiser
- Department of Biological Chemistry, University of California, Irvine, Irvine, CA 92697, USA; Chao Family Comprehensive Cancer Center, University of California, Irvine, Irvine, CA 92697, USA.
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5
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Chattopadhyay G, Bhowmick J, Manjunath K, Ahmed S, Goyal P, Varadarajan R. Mechanistic insights into global suppressors of protein folding defects. PLoS Genet 2022; 18:e1010334. [PMID: 36037221 PMCID: PMC9491731 DOI: 10.1371/journal.pgen.1010334] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2022] [Revised: 09/09/2022] [Accepted: 07/11/2022] [Indexed: 01/14/2023] Open
Abstract
Most amino acid substitutions in a protein either lead to partial loss-of-function or are near neutral. Several studies have shown the existence of second-site mutations that can rescue defects caused by diverse loss-of-function mutations. Such global suppressor mutations are key drivers of protein evolution. However, the mechanisms responsible for such suppression remain poorly understood. To address this, we characterized multiple suppressor mutations both in isolation and in combination with inactive mutants. We examined six global suppressors of the bacterial toxin CcdB, the known M182T global suppressor of TEM-1 β-lactamase, the N239Y global suppressor of p53-DBD and three suppressors of the SARS-CoV-2 spike Receptor Binding Domain. When coupled to inactive mutants, they promote increased in-vivo solubilities as well as regain-of-function phenotypes. In the case of CcdB, where novel suppressors were isolated, we determined the crystal structures of three such suppressors to obtain insight into the specific molecular interactions responsible for the observed effects. While most individual suppressors result in small stability enhancements relative to wildtype, which can be combined to yield significant stability increments, thermodynamic stabilisation is neither necessary nor sufficient for suppressor action. Instead, in diverse systems, we observe that individual global suppressors greatly enhance the foldability of buried site mutants, primarily through increase in refolding rate parameters measured in vitro. In the crowded intracellular environment, mutations that slow down folding likely facilitate off-pathway aggregation. We suggest that suppressor mutations that accelerate refolding can counteract this, enhancing the yield of properly folded, functional protein in vivo.
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Affiliation(s)
| | - Jayantika Bhowmick
- Molecular Biophysics Unit, Indian Institute of Science, Bangalore,
India
| | - Kavyashree Manjunath
- Centre for Chemical Biology and Therapeutics, Institute For Stem Cell
Science and Regenerative Medicine, Bangalore, India
| | - Shahbaz Ahmed
- Molecular Biophysics Unit, Indian Institute of Science, Bangalore,
India
| | - Parveen Goyal
- Institute for Stem Cell Science and Regenerative Medicine, Bangalore,
India
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6
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Structural Basis of Mutation-Dependent p53 Tetramerization Deficiency. Int J Mol Sci 2022; 23:ijms23147960. [PMID: 35887312 PMCID: PMC9316806 DOI: 10.3390/ijms23147960] [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: 06/28/2022] [Revised: 07/13/2022] [Accepted: 07/15/2022] [Indexed: 02/01/2023] Open
Abstract
The formation of a tetrameric assembly is essential for the ability of the tumor suppressor protein p53 to act as a transcription factor. Such a quaternary conformation is driven by a specific tetramerization domain, separated from the central DNA-binding domain by a flexible linker. Despite the distance, functional crosstalk between the two domains has been reported. This phenomenon can explain the pathogenicity of some inherited or somatically acquired mutations in the tetramerization domain, including the widespread R337H missense mutation present in the population in south Brazil. In this work, we combined computational predictions through extended all-atom molecular dynamics simulations with functional assays in a genetically defined yeast-based model system to reveal structural features of p53 tetramerization domains and their transactivation capacity and specificity. In addition to the germline and cancer-associated R337H and R337C, other rationally designed missense mutations targeting a significant salt-bridge interaction that stabilizes the p53 tetramerization domain were studied (i.e., R337D, D352R, and the double-mutation R337D plus D352R). The simulations revealed a destabilizing effect of the pathogenic mutations within the p53 tetramerization domain and highlighted the importance of electrostatic interactions between residues 337 and 352. The transactivation assay, performed in yeast by tuning the expression of wild-type and mutant p53 proteins, revealed that p53 tetramerization mutations could decrease the transactivation potential and alter transactivation specificity, in particular by better tolerating negative features in weak DNA-binding sites. These results establish the effect of naturally occurring variations at positions 337 and 352 on p53’s conformational stability and function.
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7
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Ding D, Green AG, Wang B, Lite TLV, Weinstein EN, Marks DS, Laub MT. Co-evolution of interacting proteins through non-contacting and non-specific mutations. Nat Ecol Evol 2022; 6:590-603. [PMID: 35361892 PMCID: PMC9090974 DOI: 10.1038/s41559-022-01688-0] [Citation(s) in RCA: 25] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2021] [Accepted: 01/31/2022] [Indexed: 01/08/2023]
Abstract
Proteins often accumulate neutral mutations that do not affect current functions but can profoundly influence future mutational possibilities and functions. Understanding such hidden potential has major implications for protein design and evolutionary forecasting but has been limited by a lack of systematic efforts to identify potentiating mutations. Here, through the comprehensive analysis of a bacterial toxin-antitoxin system, we identified all possible single substitutions in the toxin that enable it to tolerate otherwise interface-disrupting mutations in its antitoxin. Strikingly, the majority of enabling mutations in the toxin do not contact and promote tolerance non-specifically to many different antitoxin mutations, despite covariation in homologues occurring primarily between specific pairs of contacting residues across the interface. In addition, the enabling mutations we identified expand future mutational paths that both maintain old toxin-antitoxin interactions and form new ones. These non-specific mutations are missed by widely used covariation and machine learning methods. Identifying such enabling mutations will be critical for ensuring continued binding of therapeutically relevant proteins, such as antibodies, aimed at evolving targets.
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Affiliation(s)
- David Ding
- Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA
- Department of Systems Biology, Harvard Medical School, Boston, MA, USA
| | - Anna G Green
- Department of Systems Biology, Harvard Medical School, Boston, MA, USA
- Department of Biomedical Informatics, Harvard Medical School, Boston, MA, USA
| | - Boyuan Wang
- Department of Pharmacology, UT Southwestern Medical Center, Dallas, TX, USA
| | - Thuy-Lan Vo Lite
- Harvard-MIT Division of Health Sciences and Technology, Harvard Medical School, Boston, MA, USA
| | | | - Debora S Marks
- Department of Systems Biology, Harvard Medical School, Boston, MA, USA
| | - Michael T Laub
- Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA.
- Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA, USA.
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8
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Kerle IA, Jägerhuber L, Secci R, Pfarr N, Blüm P, Roesch R, Götze KS, Weichert W, Bassermann F, Ruland J, Winter C. Circulating Tumor DNA Profiling of a Diffuse Large B Cell Lymphoma Patient with Secondary Acute Myeloid Leukemia. Cancers (Basel) 2022; 14:cancers14061371. [PMID: 35326522 PMCID: PMC8946858 DOI: 10.3390/cancers14061371] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/21/2022] [Revised: 03/01/2022] [Accepted: 03/03/2022] [Indexed: 01/01/2023] Open
Abstract
Diffuse large B cell lymphomas (DLBCL) are the most common neoplasia of the lymphatic system. Circulating cell-free DNA released from tumor cells (ctDNA) has been studied in many tumor entities and successfully used to monitor treatment and follow up. Studies of ctDNA in DLBCL so far have mainly focused on tracking mutations in peripheral blood initially detected by next-generation sequencing (NGS) of tumor tissue from one lymphoma manifestation site. This approach, however, cannot capture the mutational heterogeneity of different tumor sites in its entirety. In this case report, we present repetitive targeted next-generation sequencing combined with digital PCR out of peripheral blood of a patient with DLBCL relapse. By combining both detection methods, we were able to detect a new dominant clone of ctDNA correlating with the development of secondary therapy-related acute myeloid leukemia (t-AML) during the course of observation. Conclusively, our case report reinforces the diagnostic importance of ctDNA in DLBCL as well as the importance of repeated ctDNA sequencing combined with focused digital PCR assays to display the dynamic mutational landscape during the clinical course.
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Affiliation(s)
- Irina A. Kerle
- Department of Medicine III, Klinikum Rechts der Isar, School of Medicine, Technical University of Munich, 81675 Munich, Germany; (I.A.K.); (P.B.); (K.S.G.); (F.B.)
- Center for Personalized Oncology, National Center for Tumor Diseases (NCT) Dresden and University Hospital Carl Gustav Carus Dresden at TU Dresden, 01307 Dresden, Germany
| | - Ludwig Jägerhuber
- Institute of Clinical Chemistry and Pathobiochemistry, Klinikum Rechts der Isar, School of Medicine, Technical University of Munich, 81675 Munich, Germany; (L.J.); (R.S.); (R.R.); (J.R.)
| | - Ramona Secci
- Institute of Clinical Chemistry and Pathobiochemistry, Klinikum Rechts der Isar, School of Medicine, Technical University of Munich, 81675 Munich, Germany; (L.J.); (R.S.); (R.R.); (J.R.)
| | - Nicole Pfarr
- Institute of Pathology, School of Medicine, Technical University of Munich, 81675 Munich, Germany; (N.P.); (W.W.)
| | - Philipp Blüm
- Department of Medicine III, Klinikum Rechts der Isar, School of Medicine, Technical University of Munich, 81675 Munich, Germany; (I.A.K.); (P.B.); (K.S.G.); (F.B.)
| | - Romina Roesch
- Institute of Clinical Chemistry and Pathobiochemistry, Klinikum Rechts der Isar, School of Medicine, Technical University of Munich, 81675 Munich, Germany; (L.J.); (R.S.); (R.R.); (J.R.)
| | - Katharina S. Götze
- Department of Medicine III, Klinikum Rechts der Isar, School of Medicine, Technical University of Munich, 81675 Munich, Germany; (I.A.K.); (P.B.); (K.S.G.); (F.B.)
- German Cancer Consortium (DKTK), Partner Site Munich, 81675 Munich, Germany
- German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany
| | - Wilko Weichert
- Institute of Pathology, School of Medicine, Technical University of Munich, 81675 Munich, Germany; (N.P.); (W.W.)
- German Cancer Consortium (DKTK), Partner Site Munich, 81675 Munich, Germany
- German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany
| | - Florian Bassermann
- Department of Medicine III, Klinikum Rechts der Isar, School of Medicine, Technical University of Munich, 81675 Munich, Germany; (I.A.K.); (P.B.); (K.S.G.); (F.B.)
- German Cancer Consortium (DKTK), Partner Site Munich, 81675 Munich, Germany
- German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany
- TranslaTUM, Center for Translational Cancer Research, Technical University of Munich, 81675 Munich, Germany
| | - Jürgen Ruland
- Institute of Clinical Chemistry and Pathobiochemistry, Klinikum Rechts der Isar, School of Medicine, Technical University of Munich, 81675 Munich, Germany; (L.J.); (R.S.); (R.R.); (J.R.)
- German Cancer Consortium (DKTK), Partner Site Munich, 81675 Munich, Germany
- German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany
- TranslaTUM, Center for Translational Cancer Research, Technical University of Munich, 81675 Munich, Germany
| | - Christof Winter
- Institute of Clinical Chemistry and Pathobiochemistry, Klinikum Rechts der Isar, School of Medicine, Technical University of Munich, 81675 Munich, Germany; (L.J.); (R.S.); (R.R.); (J.R.)
- German Cancer Consortium (DKTK), Partner Site Munich, 81675 Munich, Germany
- German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany
- TranslaTUM, Center for Translational Cancer Research, Technical University of Munich, 81675 Munich, Germany
- Correspondence:
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9
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Gomes AS, Ramos H, Inga A, Sousa E, Saraiva L. Structural and Drug Targeting Insights on Mutant p53. Cancers (Basel) 2021; 13:3344. [PMID: 34283062 PMCID: PMC8268744 DOI: 10.3390/cancers13133344] [Citation(s) in RCA: 34] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2021] [Revised: 06/29/2021] [Accepted: 06/29/2021] [Indexed: 12/20/2022] Open
Abstract
p53 is a transcription factor with a pivotal role in cell homeostasis and fate. Its impairment is a major event in tumor onset and development. In fact, about half of human cancers bear TP53 mutations that not only halt the normal function of p53, but also may acquire oncogenic gain of functions that favor tumorigenesis. Although considered undruggable for a long time, evidence has proven the capability of many compounds to restore a wild-type (wt)-like function to mutant p53 (mutp53). However, they have not reached the clinic to date. Structural studies have strongly contributed to the knowledge about p53 structure, stability, dynamics, function, and regulation. Importantly, they have afforded relevant insights into wt and mutp53 pharmacology at molecular levels, fostering the design and development of p53-targeted anticancer therapies. Herein, we provide an integrated view of mutp53 regulation, particularly focusing on mutp53 structural traits and on targeting agents capable of its reactivation, including their biological, biochemical and biophysical features. With this, we expect to pave the way for the development of improved small molecules that may advance precision cancer therapy by targeting p53.
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Affiliation(s)
- Ana Sara Gomes
- LAQV/REQUIMTE, Laboratório de Microbiologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, 4050-313 Porto, Portugal; (A.S.G.); (H.R.)
| | - Helena Ramos
- LAQV/REQUIMTE, Laboratório de Microbiologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, 4050-313 Porto, Portugal; (A.S.G.); (H.R.)
| | - Alberto Inga
- Laboratory of Transcriptional Networks, Department CIBIO, University of Trento, Via Sommarive 9, 38123 Trento, Italy;
| | - Emília Sousa
- Laboratory of Organic and Pharmaceutical Chemistry, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal;
- CIIMAR-Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Novo Edifício do Terminal de Cruzeiros do Porto de Leixões, Avenida General Norton de Matos, S/N, 4450-208 Matosinhos, Portugal
| | - Lucília Saraiva
- LAQV/REQUIMTE, Laboratório de Microbiologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, 4050-313 Porto, Portugal; (A.S.G.); (H.R.)
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10
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Li P, Hao Z, Zeng F. Tumor suppressor stars in yeast G1/S transition. Curr Genet 2020; 67:207-212. [PMID: 33175222 DOI: 10.1007/s00294-020-01126-3] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2020] [Revised: 10/22/2020] [Accepted: 10/28/2020] [Indexed: 12/11/2022]
Abstract
Yeast is one of the best-understood biological systems for genetic research. Over the last 40 years, geneticists have striven to search for homologues of tumor suppressors in yeast to simplify cancer research. The star tumor suppressor p21, downstream target of p53, is one of the primary factors on the START point through negatively regulating CycD/E-CDK, the yeast counterpart Cln3-Cdk1. Not like yeast Whi5 that was identified as the analog of the retinoblastoma tumor suppressor protein (Rb) and hence promoted to uncover the mechanism of its cancer suppression, homologue of p21 had not been found in yeast. Our lab identified Cip1 in budding yeast as a novel negative regulator of G1-Cdk1 and proposed that Cip1 is an analog of human p21. Recently, we demonstrated a dual repressive function of Cip1 on START timing via the redundant Cln3 and Ccr4 pathways. This work in yeast may help clarify the complex regulation in human p53-p21 signaling cascade. In this review, we will discuss the yeast paralogs of star tumor suppressors in the control of G1/S transition and present the new findings in this field.
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Affiliation(s)
- Pan Li
- College of Life Sciences, Hebei Agricultural University, Baoding, 071001, Hebei, China
| | - Zhimin Hao
- College of Life Sciences, Hebei Agricultural University, Baoding, 071001, Hebei, China
| | - Fanli Zeng
- College of Life Sciences, Hebei Agricultural University, Baoding, 071001, Hebei, China.
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11
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Rochman ND, Wolf YI, Koonin EV. Deep phylogeny of cancer drivers and compensatory mutations. Commun Biol 2020; 3:551. [PMID: 33009502 PMCID: PMC7532533 DOI: 10.1038/s42003-020-01276-7] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2020] [Accepted: 09/03/2020] [Indexed: 12/14/2022] Open
Abstract
Driver mutations (DM) are the genetic impetus for most cancers. The DM are assumed to be deleterious in species evolution, being eliminated by purifying selection unless compensated by other mutations. We present deep phylogenies for 84 cancer driver genes and investigate the prevalence of 434 DM across gene-species trees. The DM are rare in species evolution, and 181 are completely absent, validating their negative fitness effect. The DM are more common in unicellular than in multicellular eukaryotes, suggesting a link between these mutations and cell proliferation control. 18 DM appear as the ancestral state in one or more major clades, including 3 among mammals. We identify within-gene, compensatory mutations for 98 DM and infer likely interactions between the DM and compensatory sites in protein structures. These findings elucidate the evolutionary status of DM and are expected to advance the understanding of the functions and evolution of oncogenes and tumor suppressors. Rochman et al. present deep phylogenies for 84 cancer driver genes and examine the prevalence of driver mutations across gene-species trees. Their results show that driver mutations are rare in species evolution and give insight into the evolution of driver mutations and oncogenes.
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Affiliation(s)
- Nash D Rochman
- National Center for Biotechnology Information, National Library of Medicine, Bethesda, MD, 20894, USA
| | - Yuri I Wolf
- National Center for Biotechnology Information, National Library of Medicine, Bethesda, MD, 20894, USA
| | - Eugene V Koonin
- National Center for Biotechnology Information, National Library of Medicine, Bethesda, MD, 20894, USA.
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12
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Bromley D, Daggett V. Tumorigenic p53 mutants undergo common structural disruptions including conversion to α-sheet structure. Protein Sci 2020; 29:1983-1999. [PMID: 32715544 DOI: 10.1002/pro.3921] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2019] [Revised: 05/15/2020] [Accepted: 07/17/2020] [Indexed: 12/28/2022]
Abstract
The p53 protein is a commonly studied cancer target because of its role in tumor suppression. Unfortunately, it is susceptible to mutation-associated loss of function; approximately 50% of cancers are associated with mutations to p53, the majority of which are located in the central DNA-binding domain. Here, we report molecular dynamics simulations of wild-type (WT) p53 and 20 different mutants, including a stabilized pseudo-WT mutant. Our findings indicate that p53 mutants tend to exacerbate latent structural-disruption tendencies, or vulnerabilities, already present in the WT protein, suggesting that it may be possible to develop cancer therapies by targeting a relatively small set of structural-disruption motifs rather than a multitude of effects specific to each mutant. In addition, α-sheet secondary structure formed in almost all of the proteins. α-Sheet has been hypothesized and recently demonstrated to play a role in amyloidogenesis, and its presence in the reported p53 simulations coincides with the recent re-consideration of cancer as an amyloid disease.
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Affiliation(s)
- Dennis Bromley
- Division of Biomedical and Health Informatics, Department of Biomedical Informatics and Medical Education, University of Washington, Seattle, Washington, USA
| | - Valerie Daggett
- Division of Biomedical and Health Informatics, Department of Biomedical Informatics and Medical Education, University of Washington, Seattle, Washington, USA.,Department of Bioengineering, University of Washington, Seattle, Washington, USA
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13
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Ding Y, Xue H, Ding X, Zhao Y, Zhao Z, Wang D, Wu J. On the complexity measures of mutation hotspots in human TP53 protein. CHAOS (WOODBURY, N.Y.) 2020; 30:073118. [PMID: 32752620 DOI: 10.1063/1.5143584] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/27/2019] [Accepted: 06/15/2020] [Indexed: 06/11/2023]
Abstract
The role of sequence complexity in 23 051 somatic missense mutations including 73 well-known mutation hotspots across 22 major cancers was studied in human TP53 proteins. A role for sequence complexity in TP53 protein mutations is suggested since (i) the mutation rate significantly increases in low amino acid pair bias complexity; (ii) probability distribution complexity increases following single point substitution mutations and strikingly increases after mutation at the mutation hotspots including six detectable hotspot mutations (R175, G245, R248, R249, R273, and R282); and (iii) the degree of increase in distribution complexity is significantly correlated with the frequency of missense mutations (r = -0.5758, P < 0.0001) across 20 major types of solid tumors. These results are consistent with the hypothesis that amino acid pair bias and distribution probability may be used as novel measures for protein sequence complexity, and the degree of complexity is related to its susceptibility to mutation, as such, it may be used as a predictor for modeling protein mutations in human cancers.
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Affiliation(s)
- Yan Ding
- Department of Pediatrics, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA
| | - Hongsheng Xue
- Institute for Translational Medicine, The Affiliated Zhongshan Hospital of Dalian University, Dalian 116001, China
| | - Xinjia Ding
- Department of Urology, The Second Affiliated Hospital of Dalian Medical University, Dalian 116023, China
| | - Yuqing Zhao
- Department of Urology, The Second Affiliated Hospital of Dalian Medical University, Dalian 116023, China
| | - Zhilong Zhao
- Institute for Translational Medicine, The Affiliated Zhongshan Hospital of Dalian University, Dalian 116001, China
| | - Dazhi Wang
- Department of Pediatrics, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA
| | - Jianlin Wu
- Institute for Translational Medicine, The Affiliated Zhongshan Hospital of Dalian University, Dalian 116001, China
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14
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Loh SN. Follow the Mutations: Toward Class-Specific, Small-Molecule Reactivation of p53. Biomolecules 2020; 10:biom10020303. [PMID: 32075132 PMCID: PMC7072143 DOI: 10.3390/biom10020303] [Citation(s) in RCA: 23] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2020] [Revised: 02/09/2020] [Accepted: 02/11/2020] [Indexed: 12/17/2022] Open
Abstract
The mutational landscape of p53 in cancer is unusual among tumor suppressors because most of the alterations are of the missense type and localize to a single domain: the ~220 amino acid DNA-binding domain. Nearly all of these mutations produce the common effect of reducing p53’s ability to interact with DNA and activate transcription. Despite this seemingly simple phenotype, no mutant p53-targeted drugs are available to treat cancer patients. One of the main reasons for this is that the mutations exert their effects via multiple mechanisms—loss of DNA contacts, reduction in zinc-binding affinity, and lowering of thermodynamic stability—each of which involves a distinct type of physical impairment. This review discusses how this knowledge is informing current efforts to develop small molecules that repair these defects and restore function to mutant p53. Categorizing the spectrum of p53 mutations into discrete classes based on their inactivation mechanisms is the initial step toward personalized cancer therapy based on p53 allele status.
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Affiliation(s)
- Stewart N Loh
- Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, Syracuse, NY 13210, USA
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15
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Pradhan MR, Siau JW, Kannan S, Nguyen MN, Ouaray Z, Kwoh CK, Lane DP, Ghadessy F, Verma CS. Simulations of mutant p53 DNA binding domains reveal a novel druggable pocket. Nucleic Acids Res 2019; 47:1637-1652. [PMID: 30649466 PMCID: PMC6393305 DOI: 10.1093/nar/gky1314] [Citation(s) in RCA: 40] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2018] [Revised: 11/25/2018] [Accepted: 01/09/2019] [Indexed: 01/01/2023] Open
Abstract
The DNA binding domain (DBD) of the tumor suppressor p53 is the site of several oncogenic mutations. A subset of these mutations lowers the unfolding temperature of the DBD. Unfolding leads to the exposure of a hydrophobic β-strand and nucleates aggregation which results in pathologies through loss of function and dominant negative/gain of function effects. Inspired by the hypothesis that structural changes that are associated with events initiating unfolding in DBD are likely to present opportunities for inhibition, we investigate the dynamics of the wild type (WT) and some aggregating mutants through extensive all atom explicit solvent MD simulations. Simulations reveal differential conformational sampling between the WT and the mutants of a turn region (S6-S7) that is contiguous to a known aggregation-prone region (APR). The conformational properties of the S6-S7 turn appear to be modulated by a network of interacting residues. We speculate that changes that take place in this network as a result of the mutational stress result in the events that destabilize the DBD and initiate unfolding. These perturbations also result in the emergence of a novel pocket that appears to have druggable characteristics. FDA approved drugs are computationally screened against this pocket.
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Affiliation(s)
- Mohan R Pradhan
- Bioinformatics Institute, A*STAR (Agency for Science, Technology and Research), 30 Biopolis Street, #07-01 Matrix, Singapore 138671.,School of Computer Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798
| | - Jia Wei Siau
- p53 Laboratory, A*STAR (Agency for Science, Technology and Research), 8A Biomedical Grove, #06-04/05, Neuros/Immunos, Singapore 138648
| | - Srinivasaraghavan Kannan
- Bioinformatics Institute, A*STAR (Agency for Science, Technology and Research), 30 Biopolis Street, #07-01 Matrix, Singapore 138671
| | - Minh N Nguyen
- Bioinformatics Institute, A*STAR (Agency for Science, Technology and Research), 30 Biopolis Street, #07-01 Matrix, Singapore 138671
| | - Zohra Ouaray
- Bioinformatics Institute, A*STAR (Agency for Science, Technology and Research), 30 Biopolis Street, #07-01 Matrix, Singapore 138671.,School of Chemistry, University of Southampton, SO17 1BJ, United Kingdom
| | - Chee Keong Kwoh
- School of Computer Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798
| | - David P Lane
- p53 Laboratory, A*STAR (Agency for Science, Technology and Research), 8A Biomedical Grove, #06-04/05, Neuros/Immunos, Singapore 138648
| | - Farid Ghadessy
- p53 Laboratory, A*STAR (Agency for Science, Technology and Research), 8A Biomedical Grove, #06-04/05, Neuros/Immunos, Singapore 138648
| | - Chandra S Verma
- Bioinformatics Institute, A*STAR (Agency for Science, Technology and Research), 30 Biopolis Street, #07-01 Matrix, Singapore 138671.,Department of Biological sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543.,School of Biological sciences, Nanyang Technological University, 50 Nanyang Drive, Singapore 637551
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16
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Chou A, Froio D, Nagrial AM, Parkin A, Murphy KJ, Chin VT, Wohl D, Steinmann A, Stark R, Drury A, Walters SN, Vennin C, Burgess A, Pinese M, Chantrill LA, Cowley MJ, Molloy TJ, Australian Pancreatic Cancer Genome Initiative (APGI), Waddell N, Johns A, Grimmond SM, Chang DK, Biankin AV, Sansom OJ, Morton JP, Grey ST, Cox TR, Turchini J, Samra J, Clarke SJ, Timpson P, Gill AJ, Pajic M. Tailored first-line and second-line CDK4-targeting treatment combinations in mouse models of pancreatic cancer. Gut 2018; 67:2142-2155. [PMID: 29080858 PMCID: PMC6241608 DOI: 10.1136/gutjnl-2017-315144] [Citation(s) in RCA: 90] [Impact Index Per Article: 12.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/27/2017] [Revised: 09/27/2017] [Accepted: 09/28/2017] [Indexed: 01/05/2023]
Abstract
OBJECTIVE Extensive molecular heterogeneity of pancreatic ductal adenocarcinoma (PDA), few effective therapies and high mortality make this disease a prime model for advancing development of tailored therapies. The p16-cyclin D-cyclin-dependent kinase 4/6-retinoblastoma (RB) protein (CDK4) pathway, regulator of cell proliferation, is deregulated in PDA. Our aim was to develop a novel personalised treatment strategy for PDA based on targeting CDK4. DESIGN Sensitivity to potent CDK4/6 inhibitor PD-0332991 (palbociclib) was correlated to protein and genomic data in 19 primary patient-derived PDA lines to identify biomarkers of response. In vivo efficacy of PD-0332991 and combination therapies was determined in subcutaneous, intrasplenic and orthotopic tumour models derived from genome-sequenced patient specimens and genetically engineered model. Mechanistically, monotherapy and combination therapy were investigated in the context of tumour cell and extracellular matrix (ECM) signalling. Prognostic relevance of companion biomarker, RB protein, was evaluated and validated in independent PDA patient cohorts (>500 specimens). RESULTS Subtype-specific in vivo efficacy of PD-0332991-based therapy was for the first time observed at multiple stages of PDA progression: primary tumour growth, recurrence (second-line therapy) and metastatic setting and may potentially be guided by a simple biomarker (RB protein). PD-0332991 significantly disrupted surrounding ECM organisation, leading to increased quiescence, apoptosis, improved chemosensitivity, decreased invasion, metastatic spread and PDA progression in vivo. RB protein is prevalent in primary operable and metastatic PDA and may present a promising predictive biomarker to guide this therapeutic approach. CONCLUSION This study demonstrates the promise of CDK4 inhibition in PDA over standard therapy when applied in a molecular subtype-specific context.
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Affiliation(s)
- Angela Chou
- The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Sydney, New South Wales, Australia
- Faculty of Medicine, St Vincent’s Clinical School, University of NSW, Sydney, New South Wales, Australia
- Department of Anatomical Pathology, SYDPATH, Darlinghurst, Australia
| | - Danielle Froio
- The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Sydney, New South Wales, Australia
| | - Adnan M Nagrial
- The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Sydney, New South Wales, Australia
- Crown Princess Mary Cancer Centre, Westmead Hospital, Sydney, New South Wales, Australia
| | - Ashleigh Parkin
- The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Sydney, New South Wales, Australia
| | - Kendelle J Murphy
- The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Sydney, New South Wales, Australia
| | - Venessa T Chin
- The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Sydney, New South Wales, Australia
| | - Dalia Wohl
- The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Sydney, New South Wales, Australia
| | - Angela Steinmann
- The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Sydney, New South Wales, Australia
| | - Rhys Stark
- The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Sydney, New South Wales, Australia
| | - Alison Drury
- The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Sydney, New South Wales, Australia
| | - Stacey N Walters
- The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Sydney, New South Wales, Australia
| | - Claire Vennin
- The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Sydney, New South Wales, Australia
| | - Andrew Burgess
- The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Sydney, New South Wales, Australia
- Faculty of Medicine, St Vincent’s Clinical School, University of NSW, Sydney, New South Wales, Australia
| | - Mark Pinese
- The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Sydney, New South Wales, Australia
| | - Lorraine A Chantrill
- The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Sydney, New South Wales, Australia
- St. Vincent’s Hospital, Darlinghurst, Australia
| | - Mark J Cowley
- The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Sydney, New South Wales, Australia
| | - Timothy J Molloy
- St Vincent’s Centre for Applied Medical Research, Darlinghurst, New South Wales, Australia
| | | | - Nicola Waddell
- Department of Genetics and Computational Biology, QIMR Berghofer Medical Research Institute, Queensland, Australia
| | - Amber Johns
- The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Sydney, New South Wales, Australia
| | | | - David K Chang
- Wolfson Wohl Cancer Research Centre, Institute of Cancer Sciences, University of Glasgow, Glasgow, UK
- West of Scotland Pancreatic Unit, Glasgow Royal Infirmary, Glasgow, UK
| | - Andrew V Biankin
- Wolfson Wohl Cancer Research Centre, Institute of Cancer Sciences, University of Glasgow, Glasgow, UK
- West of Scotland Pancreatic Unit, Glasgow Royal Infirmary, Glasgow, UK
| | - Owen J Sansom
- Department of Surgery, Cancer Research UK, Beatson Institute, Institute of Cancer Sciences, University of Glasgow, Glasgow, UK
| | - Jennifer P Morton
- Department of Surgery, Cancer Research UK, Beatson Institute, Institute of Cancer Sciences, University of Glasgow, Glasgow, UK
| | - Shane T Grey
- The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Sydney, New South Wales, Australia
- Faculty of Medicine, St Vincent’s Clinical School, University of NSW, Sydney, New South Wales, Australia
| | - Thomas R Cox
- The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Sydney, New South Wales, Australia
| | - John Turchini
- Sydney Medical School, University of Sydney, Sydney, New South Wales, Australia
- Department of Anatomical Pathology, Royal North Shore Hospital, Sydney, New South Wales, Australia
- Cancer Diagnosis and Pathology Research Group, Kolling Institute of Medical Research, New South Wales, Australia
| | - Jaswinder Samra
- Department of Surgery, Royal North Shore Hospital, Sydney, New South Wales, Australia
| | - Stephen J Clarke
- Sydney Medical School, University of Sydney, Sydney, New South Wales, Australia
| | - Paul Timpson
- The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Sydney, New South Wales, Australia
- Faculty of Medicine, St Vincent’s Clinical School, University of NSW, Sydney, New South Wales, Australia
| | - Anthony J Gill
- The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Sydney, New South Wales, Australia
- Sydney Medical School, University of Sydney, Sydney, New South Wales, Australia
- Department of Anatomical Pathology, Royal North Shore Hospital, Sydney, New South Wales, Australia
- Cancer Diagnosis and Pathology Research Group, Kolling Institute of Medical Research, New South Wales, Australia
| | - Marina Pajic
- The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Sydney, New South Wales, Australia
- Faculty of Medicine, St Vincent’s Clinical School, University of NSW, Sydney, New South Wales, Australia
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17
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APR-246 reactivates mutant p53 by targeting cysteines 124 and 277. Cell Death Dis 2018; 9:439. [PMID: 29670092 PMCID: PMC5906465 DOI: 10.1038/s41419-018-0463-7] [Citation(s) in RCA: 180] [Impact Index Per Article: 25.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2017] [Revised: 02/24/2018] [Accepted: 02/28/2018] [Indexed: 12/14/2022]
Abstract
The TP53 tumor suppressor gene is frequently inactivated in human tumors by missense mutations in the DNA binding domain. TP53 mutations lead to protein unfolding, decreased thermostability and loss of DNA binding and transcription factor function. Pharmacological targeting of mutant p53 to restore its tumor suppressor function is a promising strategy for cancer therapy. The mutant p53 reactivating compound APR-246 (PRIMA-1Met) has been successfully tested in a phase I/IIa clinical trial. APR-246 is converted to the reactive electrophile methylene quinuclidinone (MQ), which binds covalently to p53 core domain. We identified cysteine 277 as a prime binding target for MQ in p53. Cys277 is also essential for MQ-mediated thermostabilization of wild-type, R175H and R273H mutant p53, while both Cys124 and Cys277 are required for APR-246-mediated functional restoration of R175H mutant p53 in living tumor cells. These findings may open opportunities for rational design of novel mutant p53-targeting compounds.
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18
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Tiberti M, Pandini A, Fraternali F, Fornili A. In silico identification of rescue sites by double force scanning. Bioinformatics 2018; 34:207-214. [PMID: 28961796 PMCID: PMC5860198 DOI: 10.1093/bioinformatics/btx515] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2017] [Revised: 06/23/2017] [Accepted: 08/10/2017] [Indexed: 01/03/2023] Open
Abstract
Motivation A deleterious amino acid change in a protein can be compensated by a second-site rescue mutation. These compensatory mechanisms can be mimicked by drugs. In particular, the location of rescue mutations can be used to identify protein regions that can be targeted by small molecules to reactivate a damaged mutant. Results We present the first general computational method to detect rescue sites. By mimicking the effect of mutations through the application of forces, the double force scanning (DFS) method identifies the second-site residues that make the protein structure most resilient to the effect of pathogenic mutations. We tested DFS predictions against two datasets containing experimentally validated and putative evolutionary-related rescue sites. A remarkably good agreement was found between predictions and experimental data. Indeed, almost half of the rescue sites in p53 was correctly predicted by DFS, with 65% of remaining sites in contact with DFS predictions. Similar results were found for other proteins in the evolutionary dataset. Availability and implementation The DFS code is available under GPL at https://fornililab.github.io/dfs/. Supplementary information Supplementary data are available at Bioinformatics online.
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Affiliation(s)
- Matteo Tiberti
- School of Biological and Chemical Sciences, Queen Mary University of London, London, UK
| | - Alessandro Pandini
- Department of Computer Science, College of Engineering, Design and Physical Sciences and Synthetic Biology Theme, Institute of Environment, Health and Societies, Brunel University London, Uxbridge, London, UK
| | - Franca Fraternali
- Randall Division of Cell and Molecular Biophysics, King‘s College London, London, UK
- The Francis Crick Institute, London, UK
- The Thomas Young Centre for Theory and Simulation of Materials, London, UK
| | - Arianna Fornili
- School of Biological and Chemical Sciences, Queen Mary University of London, London, UK
- The Thomas Young Centre for Theory and Simulation of Materials, London, UK
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19
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Shamalov K, Levy SN, Horovitz-Fried M, Cohen CJ. The mutational status of p53 can influence its recognition by human T-cells. Oncoimmunology 2017; 6:e1285990. [PMID: 28507791 PMCID: PMC5414872 DOI: 10.1080/2162402x.2017.1285990] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2016] [Revised: 01/09/2017] [Accepted: 01/18/2017] [Indexed: 10/20/2022] Open
Abstract
p53 was reported to be an attractive immunotherapy target because it is mutated in approximately half of human cancers, resulting in its inactivation and often accumulation in tumor cells. Peptides derived from p53 are presented by class I MHC molecules and may act as tumor-associated epitopes which could be targeted by p53-specific T cells. Interestingly, it was recently shown that there is a lack of significant correlation between p53 expression levels in tumors and their recognition by p53-TCR transduced T cells. To better understand the influence of the mutational status of p53 on its presentation by the MHC system and on T cell antitumor reactivity, we generated several mutant p53 constructs and expressed them in HLA-A2+/p53- cells. Upon co-culture with p53-specific T cells, we measured the specific recognition of p53-expressing target cells by means of cytokine secretion, marker upregulation and cytotoxicity, and in parallel determined p53 expression levels by intracellular staining. We also examined the relevance of antigen presentation components on p53 recognition and the impact of mutant p53 expression on cell-cycle dynamics. Our results show that selected p53 mutations altering protein stability can modulate p53 presentation to T cells, leading to a differential immune reactivity inversely correlated with measured p53 protein levels. Thus, p53 may behave differently than other classical tumor antigens and its mutational status should therefore be taken into account when elaborating immunotherapy treatments of cancer patients targeting p53.
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Affiliation(s)
- Katerina Shamalov
- The Laboratory of Tumor Immunology and Immunotherapy, The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel
| | - Shlomo N. Levy
- The Laboratory of Tumor Immunology and Immunotherapy, The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel
| | - Miryam Horovitz-Fried
- The Laboratory of Tumor Immunology and Immunotherapy, The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel
| | - Cyrille J. Cohen
- The Laboratory of Tumor Immunology and Immunotherapy, The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel
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20
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Structure and Function of p53-DNA Complexes with Inactivation and Rescue Mutations: A Molecular Dynamics Simulation Study. PLoS One 2015; 10:e0134638. [PMID: 26244575 PMCID: PMC4526489 DOI: 10.1371/journal.pone.0134638] [Citation(s) in RCA: 41] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2015] [Accepted: 07/10/2015] [Indexed: 01/10/2023] Open
Abstract
The tumor suppressor protein p53 can lose its function upon DNA-contact mutations (R273C and R273H) in the core DNA-binding domain. The activity can be restored by second-site suppressor or rescue mutations (R273C_T284R, R273H_T284R, and R273H_S240R). In this paper, we elucidate the structural and functional consequence of p53 proteins upon DNA-contact mutations and rescue mutations and the underlying mechanisms at the atomic level by means of molecular dynamics simulations. Furthermore, we also apply the docking approach to investigate the binding phenomena between the p53 protein and DNA upon DNA-contact mutations and rescue mutations. This study clearly illustrates that, due to DNA-contact mutants, the p53 structure loses its stability and becomes more rigid than the native protein. This structural loss might affect the p53-DNA interaction and leads to inhibition of the cancer suppression. Rescue mutants (R273C_T284R, R273H_T284R and R273H_S240R) can restore the functional activity of the p53 protein upon DNA-contact mutations and show a good interaction between the p53 protein and a DNA molecule, which may lead to reactivate the cancer suppression function. Understanding the effects of p53 cancer and rescue mutations at the molecular level will be helpful for designing drugs for p53 associated cancer diseases. These drugs should be designed so that they can help to inhibit the abnormal function of the p53 protein and to reactivate the p53 function (cell apoptosis) to treat human cancer.
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21
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Salehi F, Baronio R, Idrogo-Lam R, Vu H, Hall LV, Kaiser P, Lathrop RH. CHOPER filters enable rare mutation detection in complex mutagenesis populations by next-generation sequencing. PLoS One 2015; 10:e0116877. [PMID: 25692681 PMCID: PMC4333345 DOI: 10.1371/journal.pone.0116877] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2014] [Accepted: 12/08/2014] [Indexed: 01/12/2023] Open
Abstract
Next-generation sequencing (NGS) has revolutionized genetics and enabled the accurate identification of many genetic variants across many genomes. However, detection of biologically important low-frequency variants within genetically heterogeneous populations remains challenging, because they are difficult to distinguish from intrinsic NGS sequencing error rates. Approaches to overcome these limitations are essential to detect rare mutations in large cohorts, virus or microbial populations, mitochondria heteroplasmy, and other heterogeneous mixtures such as tumors. Modifications in library preparation can overcome some of these limitations, but are experimentally challenging and restricted to skilled biologists. This paper describes a novel quality filtering and base pruning pipeline, called Complex Heterogeneous Overlapped Paired-End Reads (CHOPER), designed to detect sequence variants in a complex population with high sequence similarity derived from All-Codon-Scanning (ACS) mutagenesis. A novel fast alignment algorithm, designed for the specified application, has O(n) time complexity. CHOPER was applied to a p53 cancer mutant reactivation study derived from ACS mutagenesis. Relative to error filtering based on Phred quality scores, CHOPER improved accuracy by about 13% while discarding only half as many bases. These results are a step toward extending the power of NGS to the analysis of genetically heterogeneous populations.
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Affiliation(s)
- Faezeh Salehi
- Department of Computer Science, University of California Irvine, Irvine, CA, 92697, United States of America
- Institute for Genomics and Bioinformatics, University of California Irvine, Irvine, CA, 92697, United States of America
- * E-mail: (FS); (PK)
| | - Roberta Baronio
- Department of Biological Chemistry, University of California Irvine, Irvine, CA, 92697, United States of America
- Institute for Genomics and Bioinformatics, University of California Irvine, Irvine, CA, 92697, United States of America
| | - Ryan Idrogo-Lam
- Department of Computer Science, University of California Irvine, Irvine, CA, 92697, United States of America
| | - Huy Vu
- Department of Computer Science, University of California Irvine, Irvine, CA, 92697, United States of America
| | - Linda V. Hall
- Department of Biological Chemistry, University of California Irvine, Irvine, CA, 92697, United States of America
- Institute for Genomics and Bioinformatics, University of California Irvine, Irvine, CA, 92697, United States of America
| | - Peter Kaiser
- Department of Biological Chemistry, University of California Irvine, Irvine, CA, 92697, United States of America
- Institute for Genomics and Bioinformatics, University of California Irvine, Irvine, CA, 92697, United States of America
- Chao Family Comprehensive Cancer Center, University of California Irvine, Irvine, CA, 92697, United States of America
- * E-mail: (FS); (PK)
| | - Richard H. Lathrop
- Department of Computer Science, University of California Irvine, Irvine, CA, 92697, United States of America
- Institute for Genomics and Bioinformatics, University of California Irvine, Irvine, CA, 92697, United States of America
- Chao Family Comprehensive Cancer Center, University of California Irvine, Irvine, CA, 92697, United States of America
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22
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Risso VA, Manssour-Triedo F, Delgado-Delgado A, Arco R, Barroso-delJesus A, Ingles-Prieto A, Godoy-Ruiz R, Gavira JA, Gaucher EA, Ibarra-Molero B, Sanchez-Ruiz JM. Mutational studies on resurrected ancestral proteins reveal conservation of site-specific amino acid preferences throughout evolutionary history. Mol Biol Evol 2014; 32:440-55. [PMID: 25392342 PMCID: PMC4298172 DOI: 10.1093/molbev/msu312] [Citation(s) in RCA: 59] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Abstract
Local protein interactions (“molecular context” effects) dictate amino acid replacements and can be described in terms of site-specific, energetic preferences for any different amino acid. It has been recently debated whether these preferences remain approximately constant during evolution or whether, due to coevolution of sites, they change strongly. Such research highlights an unresolved and fundamental issue with far-reaching implications for phylogenetic analysis and molecular evolution modeling. Here, we take advantage of the recent availability of phenotypically supported laboratory resurrections of Precambrian thioredoxins and β-lactamases to experimentally address the change of site-specific amino acid preferences over long geological timescales. Extensive mutational analyses support the notion that evolutionary adjustment to a new amino acid may occur, but to a large extent this is insufficient to erase the primitive preference for amino acid replacements. Generally, site-specific amino acid preferences appear to remain conserved throughout evolutionary history despite local sequence divergence. We show such preference conservation to be readily understandable in molecular terms and we provide crystallographic evidence for an intriguing structural-switch mechanism: Energetic preference for an ancestral amino acid in a modern protein can be linked to reorganization upon mutation to the ancestral local structure around the mutated site. Finally, we point out that site-specific preference conservation naturally leads to one plausible evolutionary explanation for the existence of intragenic global suppressor mutations.
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Affiliation(s)
- Valeria A Risso
- Departamento de Quimica Fisica, Facultad de Ciencias, Universidad de Granada, Granada, Spain
| | - Fadia Manssour-Triedo
- Departamento de Quimica Fisica, Facultad de Ciencias, Universidad de Granada, Granada, Spain
| | | | - Rocio Arco
- Departamento de Quimica Fisica, Facultad de Ciencias, Universidad de Granada, Granada, Spain
| | - Alicia Barroso-delJesus
- Unidad de Genómica, Instituto de Parasitología y Biomedicina López Neyra CSIC, PTS Granada, Granada, Spain
| | - Alvaro Ingles-Prieto
- Departamento de Quimica Fisica, Facultad de Ciencias, Universidad de Granada, Granada, Spain
| | - Raquel Godoy-Ruiz
- Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD
| | - Jose A Gavira
- Unidad de Genómica, Instituto de Parasitología y Biomedicina López Neyra CSIC, PTS Granada, Granada, Spain
| | - Eric A Gaucher
- Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD
| | - Beatriz Ibarra-Molero
- Departamento de Quimica Fisica, Facultad de Ciencias, Universidad de Granada, Granada, Spain
| | - Jose M Sanchez-Ruiz
- Departamento de Quimica Fisica, Facultad de Ciencias, Universidad de Granada, Granada, Spain
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23
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Bykov VJ, Wiman KG. Mutant p53 reactivation by small molecules makes its way to the clinic. FEBS Lett 2014; 588:2622-7. [DOI: 10.1016/j.febslet.2014.04.017] [Citation(s) in RCA: 111] [Impact Index Per Article: 10.1] [Reference Citation Analysis] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2014] [Revised: 04/14/2014] [Accepted: 04/14/2014] [Indexed: 01/22/2023]
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Abstract
The design of a broad-spectrum cancer drug would provide enormous clinical benefits to treat cancer patients. Most of cancerous cells have a mutation in the p53 gene that results in an inactive mutant p53 protein. For this reason, p53 is a prime target for the development of a broad-spectrum cancer drug. To provide the atomic information to rationally design a drug to recover p53 activity is the main goal of the structural studies on mutant p53. We review three mechanisms that influence p53 activity and provide information about how reactivation of mutant p53 can be achieved: stabilization of the active conformation of the DNA-binding domain of the protein, suppression of missense mutations in the DNA-binding domain by a second-site mutation, and increased transactivation.
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Affiliation(s)
- Hector Viadiu
- Instituto de Química, Universidad Nacional Autónoma de México (UNAM), Mexico City, D.F., Mexico,
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25
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Koulgi S, Achalere A, Sharma N, Sonavane U, Joshi R. QM-MM simulations on p53-DNA complex: a study of hot spot and rescue mutants. J Mol Model 2013; 19:5545-59. [DOI: 10.1007/s00894-013-2042-2] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/04/2013] [Accepted: 10/21/2013] [Indexed: 01/27/2023]
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26
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Eldar A, Rozenberg H, Diskin-Posner Y, Rohs R, Shakked Z. Structural studies of p53 inactivation by DNA-contact mutations and its rescue by suppressor mutations via alternative protein-DNA interactions. Nucleic Acids Res 2013; 41:8748-59. [PMID: 23863845 PMCID: PMC3794590 DOI: 10.1093/nar/gkt630] [Citation(s) in RCA: 59] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023] Open
Abstract
A p53 hot-spot mutation found frequently in human cancer is the replacement of R273 by histidine or cysteine residues resulting in p53 loss of function as a tumor suppressor. These mutants can be reactivated by the incorporation of second-site suppressor mutations. Here, we present high-resolution crystal structures of the p53 core domains of the cancer-related proteins, the rescued proteins and their complexes with DNA. The structures show that inactivation of p53 results from the incapacity of the mutated residues to form stabilizing interactions with the DNA backbone, and that reactivation is achieved through alternative interactions formed by the suppressor mutations. Detailed structural and computational analysis demonstrates that the rescued p53 complexes are not fully restored in terms of DNA structure and its interface with p53. Contrary to our previously studied wild-type (wt) p53-DNA complexes showing non-canonical Hoogsteen A/T base pairs of the DNA helix that lead to local minor-groove narrowing and enhanced electrostatic interactions with p53, the current structures display Watson-Crick base pairs associated with direct or water-mediated hydrogen bonds with p53 at the minor groove. These findings highlight the pivotal role played by R273 residues in supporting the unique geometry of the DNA target and its sequence-specific complex with p53.
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Affiliation(s)
- Amir Eldar
- Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel and Molecular and Computational Biology Program, University of Southern California, Los Angeles, CA 90089, USA
| | - Haim Rozenberg
- Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel and Molecular and Computational Biology Program, University of Southern California, Los Angeles, CA 90089, USA
| | - Yael Diskin-Posner
- Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel and Molecular and Computational Biology Program, University of Southern California, Los Angeles, CA 90089, USA
| | - Remo Rohs
- Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel and Molecular and Computational Biology Program, University of Southern California, Los Angeles, CA 90089, USA
| | - Zippora Shakked
- Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel and Molecular and Computational Biology Program, University of Southern California, Los Angeles, CA 90089, USA,*To whom correspondence should be addressed. Tel: +972 8 934 2672; Fax: +972 8 934 6278;
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27
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Wallentine BD, Wang Y, Tretyachenko-Ladokhina V, Tan M, Senear DF, Luecke H. Structures of oncogenic, suppressor and rescued p53 core-domain variants: mechanisms of mutant p53 rescue. ACTA CRYSTALLOGRAPHICA. SECTION D, BIOLOGICAL CRYSTALLOGRAPHY 2013; 69:2146-56. [PMID: 24100332 PMCID: PMC3792646 DOI: 10.1107/s0907444913020830] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/22/2013] [Accepted: 07/25/2013] [Indexed: 11/10/2022]
Abstract
To gain insights into the mechanisms by which certain second-site suppressor mutations rescue the function of a significant number of cancer mutations of the tumor suppressor protein p53, X-ray crystallographic structures of four p53 core-domain variants were determined. These include an oncogenic mutant, V157F, two single-site suppressor mutants, N235K and N239Y, and the rescued cancer mutant V157F/N235K/N239Y. The V157F mutation substitutes a smaller hydrophobic valine with a larger hydrophobic phenylalanine within strand S4 of the hydrophobic core. The structure of this cancer mutant shows no gross structural changes in the overall fold of the p53 core domain, only minor rearrangements of side chains within the hydrophobic core of the protein. Based on biochemical analysis, these small local perturbations induce instability in the protein, increasing the free energy by 3.6 kcal mol(-1) (15.1 kJ mol(-1)). Further biochemical evidence shows that each suppressor mutation, N235K or N239Y, acts individually to restore thermodynamic stability to V157F and that both together are more effective than either alone. All rescued mutants were found to have wild-type DNA-binding activity when assessed at a permissive temperature, thus pointing to thermodynamic stability as the critical underlying variable. Interestingly, thermodynamic analysis shows that while N239Y demonstrates stabilization of the wild-type p53 core domain, N235K does not. These observations suggest distinct structural mechanisms of rescue. A new salt bridge between Lys235 and Glu198, found in both the N235K and rescued cancer mutant structures, suggests a rescue mechanism that relies on stabilizing the β-sandwich scaffold. On the other hand, the substitution N239Y creates an advantageous hydrophobic contact between the aromatic ring of this tyrosine and the adjacent Leu137. Surprisingly, the rescued cancer mutant shows much larger structural deviations than the cancer mutant alone when compared with wild-type p53. These suppressor mutations appear to rescue p53 function by creating novel intradomain interactions that stabilize the core domain, allowing compensation for the destabilizing V157F mutation.
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Affiliation(s)
- Brad D. Wallentine
- Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA 92697, USA
| | - Ying Wang
- Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA 92697, USA
| | | | - Martha Tan
- Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA 92697, USA
| | - Donald F. Senear
- Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA 92697, USA
| | - Hartmut Luecke
- Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA 92697, USA
- Department of Physiology and Biophysics, University of California, Irvine, Irvine, CA 92697, USA
- Department of Computer Science, University of California, Irvine, Irvine, CA 92697, USA
- Center for Biomembrane Systems, University of California, Irvine, Irvine, CA 92697, USA
- Unidad de Biofisica (CSIC, UPV/EHU) and Departamento de Bioquimica, Universidad del Pais Vasco, 48940 Leioa, Spain
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28
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Ciribilli Y, Monti P, Bisio A, Nguyen HT, Ethayathulla AS, Ramos A, Foggetti G, Menichini P, Menendez D, Resnick MA, Viadiu H, Fronza G, Inga A. Transactivation specificity is conserved among p53 family proteins and depends on a response element sequence code. Nucleic Acids Res 2013; 41:8637-53. [PMID: 23892287 PMCID: PMC3794606 DOI: 10.1093/nar/gkt657] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
Structural and biochemical studies have demonstrated that p73, p63 and p53 recognize DNA with identical amino acids and similar binding affinity. Here, measuring transactivation activity for a large number of response elements (REs) in yeast and human cell lines, we show that p53 family proteins also have overlapping transactivation profiles. We identified mutations at conserved amino acids of loops L1 and L3 in the DNA-binding domain that tune the transactivation potential nearly equally in p73, p63 and p53. For example, the mutant S139F in p73 has higher transactivation potential towards selected REs, enhanced DNA-binding cooperativity in vitro and a flexible loop L1 as seen in the crystal structure of the protein–DNA complex. By studying, how variations in the RE sequence affect transactivation specificity, we discovered a RE-transactivation code that predicts enhanced transactivation; this correlation is stronger for promoters of genes associated with apoptosis.
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Affiliation(s)
- Yari Ciribilli
- Laboratory of Transcriptional Networks, Centre for Integrative Biology (CIBIO), University of Trento, TN, 38060 Italy, Molecular Mutagenesis and DNA Repair Unit, IRCSS Azienda Ospedaliera Universitaria San Martino-IST-Istituto Nazionale per la Ricerca sul Cancro, Genoa 16132, Italy, Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA, 92093, USA and Chromosome Stability Group, Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, NIEHS, NIH, RTP, NC, 27709, USA
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29
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Odell AF, Odell LR, Askham JM, Alogheli H, Ponnambalam S, Hollstein M. A novel p53 mutant found in iatrogenic urothelial cancers is dysfunctional and can be rescued by a second-site global suppressor mutation. J Biol Chem 2013; 288:16704-16714. [PMID: 23612969 DOI: 10.1074/jbc.m112.443168] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Exposure to herbal remedies containing the carcinogen aristolochic acid (AA) has been widespread in some regions of the world. Rare A→T TP53 mutations were recently discovered in AA-associated urothelial cancers. The near absence of these mutations among all other sequenced human tumors suggests that they could be biologically silent. There are no cell banks with established lines derived from human tumors with which to explore the influence of the novel mutants on p53 function and cellular behavior. To investigate their impact, we generated isogenic mutant clones by integrase-mediated cassette exchange at the p53 locus of platform (null) murine embryonic fibroblasts and kidney epithelial cells. Common tumor mutants (R248W, R273C) were compared with the AA-associated mutants N131Y, R249W, and Q104L. Assays of cell proliferation, migration, growth in soft agar, apoptosis, senescence, and gene expression revealed contrasting outcomes on cellular behavior following introduction of N131Y or Q104L. The N131Y mutant demonstrated a phenotype akin to common tumor mutants, whereas Q104L clone behavior resembled that of cells with wild-type p53. Wild-type p53 responses were restored in double-mutant cells harboring N131Y and N239Y, a second-site rescue mutation, suggesting that pharmaceutical reactivation of p53 function in tumors expressing N131Y could have therapeutic benefit. N131Y is likely to contribute directly to tumor phenotype and is a promising candidate biomarker of AA exposure and disease. Rare mutations thus do not necessarily point to sites where amino acid exchanges are phenotypically neutral. Encounter with mutagenic insults targeting cryptic sites can reveal specific signature hotspots.
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Affiliation(s)
- Adam F Odell
- Faculty of Medicine and Health, University of Leeds, Leeds LS2 9JT, United Kingdom.
| | - Luke R Odell
- Division of Organic Pharmaceutical Chemistry, Department of Medicinal Chemistry, Uppsala University, 75123 Uppsala, Sweden
| | - Jon M Askham
- Faculty of Medicine and Health, University of Leeds, Leeds LS2 9JT, United Kingdom
| | - Hiba Alogheli
- Division of Organic Pharmaceutical Chemistry, Department of Medicinal Chemistry, Uppsala University, 75123 Uppsala, Sweden
| | - Sreenivasan Ponnambalam
- School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom
| | - Monica Hollstein
- Faculty of Medicine and Health, University of Leeds, Leeds LS2 9JT, United Kingdom; Department C016, German Cancer Research Centre, 69120 Heidelberg, Germany.
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30
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Geetha Ramani R, Jacob SG. Prediction of P53 mutants (multiple sites) transcriptional activity based on structural (2D&3D) properties. PLoS One 2013; 8:e55401. [PMID: 23468845 PMCID: PMC3572112 DOI: 10.1371/journal.pone.0055401] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2012] [Accepted: 12/21/2012] [Indexed: 01/05/2023] Open
Abstract
Prediction of secondary site mutations that reinstate mutated p53 to normalcy has been the focus of intense research in the recent past owing to the fact that p53 mutants have been implicated in more than half of all human cancers and restoration of p53 causes tumor regression. However laboratory investigations are more often laborious and resource intensive but computational techniques could well surmount these drawbacks. In view of this, we formulated a novel approach utilizing computational techniques to predict the transcriptional activity of multiple site (one-site to five-site) p53 mutants. The optimal MCC obtained by the proposed approach on prediction of one-site, two-site, three-site, four-site and five-site mutants were 0.775,0.341,0.784,0.916 and 0.655 respectively, the highest reported thus far in literature. We have also demonstrated that 2D and 3D features generate higher prediction accuracy of p53 activity and our findings revealed the optimal results for prediction of p53 status, reported till date. We believe detection of the secondary site mutations that suppress tumor growth may facilitate better understanding of the relationship between p53 structure and function and further knowledge on the molecular mechanisms and biological activity of p53, a targeted source for cancer therapy. We expect that our prediction methods and reported results may provide useful insights on p53 functional mechanisms and generate more avenues for utilizing computational techniques in biological data analysis.
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Affiliation(s)
- R. Geetha Ramani
- Department of Information Science and Technology, College of Engineering, Guindy, Anna University, Chennai, Tamilnadu, India
| | - Shomona Gracia Jacob
- Faculty of Information and Communication Engineering, Anna University, Chennai, Tamilnadu, India
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31
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Wassman CD, Baronio R, Demir Ö, Wallentine BD, Chen CK, Hall LV, Salehi F, Lin DW, Chung BP, Wesley Hatfield G, Richard Chamberlin A, Luecke H, Lathrop RH, Kaiser P, Amaro RE. Computational identification of a transiently open L1/S3 pocket for reactivation of mutant p53. Nat Commun 2013; 4:1407. [PMID: 23360998 PMCID: PMC3562459 DOI: 10.1038/ncomms2361] [Citation(s) in RCA: 173] [Impact Index Per Article: 14.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2012] [Accepted: 12/06/2012] [Indexed: 12/22/2022] Open
Abstract
The tumour suppressor p53 is the most frequently mutated gene in human cancer. Reactivation of mutant p53 by small molecules is an exciting potential cancer therapy. Although several compounds restore wild-type function to mutant p53, their binding sites and mechanisms of action are elusive. Here computational methods identify a transiently open binding pocket between loop L1 and sheet S3 of the p53 core domain. Mutation of residue Cys124, located at the centre of the pocket, abolishes p53 reactivation of mutant R175H by PRIMA-1, a known reactivation compound. Ensemble-based virtual screening against this newly revealed pocket selects stictic acid as a potential p53 reactivation compound. In human osteosarcoma cells, stictic acid exhibits dose-dependent reactivation of p21 expression for mutant R175H more strongly than does PRIMA-1. These results indicate the L1/S3 pocket as a target for pharmaceutical reactivation of p53 mutants.
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Affiliation(s)
- Christopher D. Wassman
- Department of Computer Science, University of California, Irvine, Irvine, California 92697, USA
- Institute for Genomics and Bioinformatics, University of California, Irvine, Irvine, California 92697, USA
- These authors contributed equally to this work
- Present address: Google Inc., 1600 Amphitheatre Parkway Mountain View, California 94043, USA
| | - Roberta Baronio
- Institute for Genomics and Bioinformatics, University of California, Irvine, Irvine, California 92697, USA
- Department of Biological Chemistry, University of California, Irvine, Irvine, California 92697, USA
- These authors contributed equally to this work
| | - Özlem Demir
- Department of Pharmaceutical Sciences, University of California, Irvine, Irvine, California 92697, USA
- These authors contributed equally to this work
- Present addresses: Department of Chemistry and Biochemistry, University of California, San Diego; La Jolla, California 92093, USA
| | - Brad D. Wallentine
- Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, California 92697, USA
| | - Chiung-Kuang Chen
- Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, California 92697, USA
| | - Linda V. Hall
- Institute for Genomics and Bioinformatics, University of California, Irvine, Irvine, California 92697, USA
- Department of Biological Chemistry, University of California, Irvine, Irvine, California 92697, USA
| | - Faezeh Salehi
- Department of Computer Science, University of California, Irvine, Irvine, California 92697, USA
- Institute for Genomics and Bioinformatics, University of California, Irvine, Irvine, California 92697, USA
| | - Da-Wei Lin
- Department of Biological Chemistry, University of California, Irvine, Irvine, California 92697, USA
| | - Benjamin P. Chung
- Department of Biological Chemistry, University of California, Irvine, Irvine, California 92697, USA
| | - G. Wesley Hatfield
- Institute for Genomics and Bioinformatics, University of California, Irvine, Irvine, California 92697, USA
- Department of Microbiology and Molecular Genetics, University of California, Irvine, Irvine, California 92697, USA
- Department of Chemical Engineering and Materials Science, University of California, Irvine, Irvine, California 92697, USA
| | - A. Richard Chamberlin
- Department of Pharmaceutical Sciences, University of California, Irvine, Irvine, California 92697, USA
- Department of Chemistry, University of California, Irvine, Irvine, California 92697, USA
- Chao Family Comprehensive Cancer Center, University of California, Irvine, Irvine, California 92697, USA
| | - Hartmut Luecke
- Institute for Genomics and Bioinformatics, University of California, Irvine, Irvine, California 92697, USA
- Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, California 92697, USA
- Chao Family Comprehensive Cancer Center, University of California, Irvine, Irvine, California 92697, USA
- Department of Physiology and Biophysics, University of California, Irvine, Irvine, California 92697, USA
- Center for Biomembrane Systems, University of California, Irvine, Irvine, California 92697, USA
| | - Richard H. Lathrop
- Department of Computer Science, University of California, Irvine, Irvine, California 92697, USA
- Institute for Genomics and Bioinformatics, University of California, Irvine, Irvine, California 92697, USA
- Chao Family Comprehensive Cancer Center, University of California, Irvine, Irvine, California 92697, USA
- Department of Biomedical Engineering, University of California, Irvine, Irvine, California 92697, USA
| | - Peter Kaiser
- Institute for Genomics and Bioinformatics, University of California, Irvine, Irvine, California 92697, USA
- Department of Biological Chemistry, University of California, Irvine, Irvine, California 92697, USA
- Chao Family Comprehensive Cancer Center, University of California, Irvine, Irvine, California 92697, USA
| | - Rommie E. Amaro
- Department of Computer Science, University of California, Irvine, Irvine, California 92697, USA
- Department of Pharmaceutical Sciences, University of California, Irvine, Irvine, California 92697, USA
- Department of Chemistry, University of California, Irvine, Irvine, California 92697, USA
- Present addresses: Department of Chemistry and Biochemistry, University of California, San Diego; La Jolla, California 92093, USA
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32
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Araya CL, Fowler DM, Chen W, Muniez I, Kelly JW, Fields S. A fundamental protein property, thermodynamic stability, revealed solely from large-scale measurements of protein function. Proc Natl Acad Sci U S A 2012; 109:16858-63. [PMID: 23035249 PMCID: PMC3479514 DOI: 10.1073/pnas.1209751109] [Citation(s) in RCA: 170] [Impact Index Per Article: 13.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
Abstract
The ability of a protein to carry out a given function results from fundamental physicochemical properties that include the protein's structure, mechanism of action, and thermodynamic stability. Traditional approaches to study these properties have typically required the direct measurement of the property of interest, oftentimes a laborious undertaking. Although protein properties can be probed by mutagenesis, this approach has been limited by its low throughput. Recent technological developments have enabled the rapid quantification of a protein's function, such as binding to a ligand, for numerous variants of that protein. Here, we measure the ability of 47,000 variants of a WW domain to bind to a peptide ligand and use these functional measurements to identify stabilizing mutations without directly assaying stability. Our approach is rooted in the well-established concept that protein function is closely related to stability. Protein function is generally reduced by destabilizing mutations, but this decrease can be rescued by stabilizing mutations. Based on this observation, we introduce partner potentiation, a metric that uses this rescue ability to identify stabilizing mutations, and identify 15 candidate stabilizing mutations in the WW domain. We tested six candidates by thermal denaturation and found two highly stabilizing mutations, one more stabilizing than any previously known mutation. Thus, physicochemical properties such as stability are latent within these large-scale protein functional data and can be revealed by systematic analysis. This approach should allow other protein properties to be discovered.
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Affiliation(s)
| | - Douglas M. Fowler
- Departments of Genome Sciences and
- The Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195; and
| | | | | | - Jeffery W. Kelly
- Departments of Chemistry and
- Molecular and Experimental Medicine and
- Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037
| | - Stanley Fields
- Departments of Genome Sciences and
- Medicine and
- The Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195; and
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Abstract
There is now strong evidence that mutation not only abrogates p53 tumor-suppressive functions, but in some instances can also endow mutant proteins with novel activities. Such neomorphic p53 proteins are capable of dramatically altering tumor cell behavior, primarily through their interactions with other cellular proteins and regulation of cancer cell transcriptional programs. Different missense mutations in p53 may confer unique activities and thereby offer insight into the mutagenic events that drive tumor progression. Here we review mechanisms by which mutant p53 exerts its cellular effects, with a particular focus on the burgeoning mutant p53 transcriptome, and discuss the biological and clinical consequences of mutant p53 gain of function.
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Pereira C, Coutinho I, Soares J, Bessa C, Leão M, Saraiva L. New insights into cancer-related proteins provided by the yeast model. FEBS J 2012; 279:697-712. [PMID: 22239976 DOI: 10.1111/j.1742-4658.2012.08477.x] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023]
Abstract
Cancer is a devastating disease with a profound impact on society. In recent years, yeast has provided a valuable contribution with respect to uncovering the molecular mechanisms underlying this disease, allowing the identification of new targets and novel therapeutic opportunities. Indeed, several attributes make yeast an ideal model system for the study of human diseases. It combines a high level of conservation between its cellular processes and those of mammalian cells, with advantages such as a short generation time, ease of genetic manipulation and a wealth of experimental tools for genome- and proteome-wide analyses. Additionally, the heterologous expression of disease-causing proteins in yeast has been successfully used to gain an understanding of the functions of these proteins and also to provide clues about the mechanisms of disease progression. Yeast research performed in recent years has demonstrated the tremendous potential of this model system, especially with the validation of findings obtained with yeast in more physiologically relevant models. The present review covers the major aspects of the most recent developments in the yeast research area with respect to cancer. It summarizes our current knowledge on yeast as a cellular model for investigating the molecular mechanisms of action of the major cancer-related proteins that, even without yeast orthologues, still recapitulate in yeast some of the key aspects of this cellular pathology. Moreover, the most recent contributions of yeast genetics and high-throughput screening technologies that aim to identify some of the potential causes underpinning this disorder, as well as discover new therapeutic agents, are discussed.
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Affiliation(s)
- Clara Pereira
- REQUIMTE, Department of Biological Sciences, Laboratory of Microbiology, University of Porto, Portugal
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35
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Ensemble-based computational approach discriminates functional activity of p53 cancer and rescue mutants. PLoS Comput Biol 2011; 7:e1002238. [PMID: 22028641 PMCID: PMC3197647 DOI: 10.1371/journal.pcbi.1002238] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2011] [Accepted: 09/05/2011] [Indexed: 11/19/2022] Open
Abstract
The tumor suppressor protein p53 can lose its function upon single-point missense mutations in the core DNA-binding domain (“cancer mutants”). Activity can be restored by second-site suppressor mutations (“rescue mutants”). This paper relates the functional activity of p53 cancer and rescue mutants to their overall molecular dynamics (MD), without focusing on local structural details. A novel global measure of protein flexibility for the p53 core DNA-binding domain, the number of clusters at a certain RMSD cutoff, was computed by clustering over 0.7 µs of explicitly solvated all-atom MD simulations. For wild-type p53 and a sample of p53 cancer or rescue mutants, the number of clusters was a good predictor of in vivo p53 functional activity in cell-based assays. This number-of-clusters (NOC) metric was strongly correlated (r2 = 0.77) with reported values of experimentally measured ΔΔG protein thermodynamic stability. Interpreting the number of clusters as a measure of protein flexibility: (i) p53 cancer mutants were more flexible than wild-type protein, (ii) second-site rescue mutations decreased the flexibility of cancer mutants, and (iii) negative controls of non-rescue second-site mutants did not. This new method reflects the overall stability of the p53 core domain and can discriminate which second-site mutations restore activity to p53 cancer mutants. p53 is a tumor suppressor protein that controls a central apoptotic pathway (programmed cell death). Thus, it is the most-mutated gene in human cancers. Due to the marginal stability of p53, a single mutation can abolish p53 function (“cancer mutants”), while a second mutation (or several) can restore it (“rescue mutants”). Restoring p53 function is a promising therapeutic goal that has been strongly supported by recent experimental results on mice. Understanding of the effects of p53 cancer and rescue mutations would be helpful for designing drugs that are able to achieve the same goal. The challenge is that cancer and rescue mutations are distributed widely in the protein, and experimental testing of all possible combinations of mutations is not feasible. This paper describes a simple computational metric that reflects the overall stability of the p53 core domain and can discriminate which second-site mutations restore activity to p53 cancer mutants.
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36
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Baronio R, Danziger SA, Hall LV, Salmon K, Hatfield GW, Lathrop RH, Kaiser P. All-codon scanning identifies p53 cancer rescue mutations. Nucleic Acids Res 2010; 38:7079-88. [PMID: 20581117 PMCID: PMC2978351 DOI: 10.1093/nar/gkq571] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
In vitro scanning mutagenesis strategies are valuable tools to identify critical residues in proteins and to generate proteins with modified properties. We describe the fast and simple All-Codon Scanning (ACS) strategy that creates a defined gene library wherein each individual codon within a specific target region is changed into all possible codons with only a single codon change per mutagenesis product. ACS is based on a multiplexed overlapping mutagenesis primer design that saturates only the targeted gene region with single codon changes. We have used ACS to produce single amino-acid changes in small and large regions of the human tumor suppressor protein p53 to identify single amino-acid substitutions that can restore activity to inactive p53 found in human cancers. Single-tube reactions were used to saturate defined 30-nt regions with all possible codon changes. The same technique was used in 20 parallel reactions to scan the 600-bp fragment encoding the entire p53 core domain. Identification of several novel p53 cancer rescue mutations demonstrated the utility of the ACS approach. ACS is a fast, simple and versatile method, which is useful for protein structure–function analyses and protein design or evolution problems.
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Affiliation(s)
- Roberta Baronio
- Institute for Genomics and Bioinformatics, Department of Biomedical Engineering, University of California, Irvine, CA 92697, USA
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37
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Current strategies to target p53 in cancer. Biochem Pharmacol 2010; 80:724-30. [PMID: 20450892 DOI: 10.1016/j.bcp.2010.04.031] [Citation(s) in RCA: 122] [Impact Index Per Article: 8.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2010] [Revised: 04/26/2010] [Accepted: 04/27/2010] [Indexed: 01/27/2023]
Abstract
Tumor suppressor p53 is a transcription factor that guards the genome stability and normal cell growth. Stresses like DNA damage, oncogenic assault will turn on p53 function which leads to cell cycle arrest for DNA repair, senescence for permanent growth arrest or apoptosis for programmed cell death. At the late stage of cancer progression, p53 is hijacked in all forms of tumors either trapped in the negative regulator such as MDM2/viral proteins or directly mutated/deleted. Re-introduction of a functional p53 alone has been proven to induce tumor regression robustly. Also, an active p53 pathway is essential for effective chemo- or radio-therapy. The emerging cyclotherapy in which p53 acts as a chemoprotectant of normal tissues further expands the utility of p53 activators. Functionally, it is unquestionable that drugging p53 will render tumor-specific intervention. One direct method is to deliver the functional wild-type (wt) p53 to tumors via gene therapy. The small molecule strategies consist of activation of p53 family member such as p73, manipulating p53 posttranslational modulators to increase wt p53 protein levels, protein-protein interaction inhibitors to free wt p53 from MDM2 or viral protein, and restoring p53 function to mutant p53 by direct modulation of its conformation. Although most of the current pre-clinical leads are in microM range and need further optimization, the success in proving that small molecules can reactivate p53 marks the beginning of the clinical development of p53-based cancer therapy.
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38
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Mutants of the tumour suppressor p53 L1 loop as second-site suppressors for restoring DNA binding to oncogenic p53 mutations: structural and biochemical insights. Biochem J 2010; 427:225-36. [DOI: 10.1042/bj20091888] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023]
Abstract
To assess the potential of mutations from the L1 loop of the tumour suppressor p53 as second-site suppressors, the effect of H115N and S116M on the p53 ‘hot spot’ mutations has been investigated using the double-mutant approach. The effects of these two mutants on the p53 hot spots in terms of thermal stability and DNA binding were evaluated. The results show that: (i) the p53 mutants H115N and S116M are thermally more stable than wild-type p53; (ii) H115N but not S116M is capable of rescuing the DNA binding of one of the most frequent p53 mutants in cancer, R248Q, as shown by binding of R248Q/H115N to gadd45 (the promoter of a gene involved in cell-cycle arrest); (iii) the double mutant R248Q/H115N is more stable than wild-type p53; (iv) the effect of H115N as a second-site suppressor to restore DNA-binding activity is specific to R248Q, but not to R248W; (v) molecular-dynamics simulations indicate that R248Q/H115N has a conformation similar to wild-type p53, which is distinct from that of R248Q. These findings could be exploited in designing strategies for cancer therapy to identify molecules that could mimic the effect of H115N in restoring function to oncogenic p53 mutants.
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39
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Su Q, Xing D, Zhou X. Magnetic beads based rolling circle amplification-electrochemiluminescence assay for highly sensitive detection of point mutation. Biosens Bioelectron 2009; 25:1615-21. [PMID: 20034781 DOI: 10.1016/j.bios.2009.11.025] [Citation(s) in RCA: 58] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2009] [Revised: 11/24/2009] [Accepted: 11/25/2009] [Indexed: 01/16/2023]
Abstract
The identification of point mutations is particularly essential in the fields of medical diagnosis and prognosis of many pathogenic and genetic diseases. In this study, an rolling circle amplification (RCA) based electrochemiluminescence (ECL) assay for highly sensitive point mutation detection was developed. In the assay, an allele-discriminating padlock probe was designed for targeting the sequence in the p53 oncogene locus. A circular template generated by enzymatic ligation upon the recognition of a point mutation (CGT to CAT) on the oncogene could be amplified isothermally by Phi 29 DNA polymerase. The elongated products, containing hundreds of copies of the circular DNA template sequence, were hybridized with Ru(bpy)(3)(2+) (TBR)-tagged probes and then captured onto streptavidin-coated paramagnetic beads. The resulting products were analyzed by magnetic bead based ECL platform. As low as 2 amol of mutated strands was detected by this assay, which could be attributed to the high amplification efficiency of Phi 29 DNA polymerase and current magnetic bead based ECL detection platform. In addition, the positive mutation detection was achieved with a wild-type to mutant ratio of 10000:1, due to the high fidelity of DNA ligase in differentiating mismatched bases at the ligation site. It is demonstrated that this proposed method provides a highly sensitive and specific approach for point mutation detection.
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Affiliation(s)
- Qiang Su
- MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, China
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40
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TP53 mutation profile of esophageal squamous cell carcinomas of patients from Southeastern Brazil. Mutat Res 2009; 696:10-5. [PMID: 19944185 DOI: 10.1016/j.mrgentox.2009.11.005] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2009] [Revised: 11/10/2009] [Accepted: 11/13/2009] [Indexed: 11/24/2022]
Abstract
Esophageal cancer (EC) is among the 10 most common and fatal malignacies in the world, presenting a marked geographic variation in incidence rates between and within different countries. The TP53 tumor suppressor gene is highly mutated in esophageal tumors and its mutation pattern can offer clues to the etiopathology of the tumor. As Brazil presents one of the highest incidence areas in the West, a deeper knowledge of the molecular mechanisms related to EC development in the Brazilian population is needed. We analyzed the mutation profile of 110 esophageal squamous cell carcinomas (ESCC) of patients from Southeastern Brazil (Rio de Janeiro and São Paulo) and collected data regarding alcohol intake and tobacco smoking. We detected 41 mutations in tumor samples from 38 patients. There was no association between mutation frequency and tobacco smoking or alcohol drinking. The most frequently mutated codons were 179, 214, 220 and 248. Codons 179, 220 and 248 are hot-spots for ESCC, but codon 214 presents only 0.7% of the mutations registered in the IARC database. The mutation profile revealed a high percentage of mutations at A:T base pairs (34.1%) followed by deletions (17.1%). We concluded that the mutation profile detected in this study is different from that of patients from Southern Brazil but very similar to that previously seen in French patients, being characterized by a high frequency of mutations at A:T base pairs, which may be associated with acetaldehyde, the metabolic product of ethanol.
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41
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Abstract
The tumor suppressor protein p53 is a transcription factor that plays a key role in the prevention of cancer development. In response to oncogenic or other stresses, the p53 protein is activated and regulates the expression of a variety of target genes, resulting in cell cycle arrest, senescence, or apoptosis. Mutation of the p53 gene is the most common genetic alteration in human cancer, affecting more than 50% of human tumors. Most of these mutations inactivate the DNA-binding domain of the protein. In this chapter, we describe the structure of the wild-type p53 protein and present structural and functional data that provide the molecular basis for understanding the effects of common cancer mutations. Further, we assess novel therapeutic strategies that aim to rescue the function of p53 cancer mutants.
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Affiliation(s)
- Andreas C Joerger
- MRC Centre for Protein Engineering, Cambridge CB2 2QH, United Kingdom
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42
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Tan YH, Chen YM, Ye X, Lu Q, Tretyachenko-Ladokhina V, Yang W, Senear DF, Luo R. Molecular mechanisms of functional rescue mediated by P53 tumor suppressor mutations. Biophys Chem 2009; 145:37-44. [PMID: 19748724 DOI: 10.1016/j.bpc.2009.08.008] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2009] [Revised: 08/18/2009] [Accepted: 08/23/2009] [Indexed: 11/30/2022]
Abstract
We have utilized both molecular dynamics simulations and solution biophysical measurements to investigate the rescue mechanism of mutation N235K, which plays a key role in the recently identified global suppressor motif of K235/Y239/R240 in the human p53 DNA-binding domain (DBD). Previous genetic analysis indicates that N235K alone rescues five out of six destabilized cancer mutants. However, the solution biophysical measurement shows that N235K generates only a slight increase to the stability of DBD, implying a rescue mechanism that is not a simple additive contribution to thermodynamic stability. Our molecular simulations show that the N235K substitution generates two non-native salt bridges with residues D186 and E198. We find that the nonnative salt bridges, D186-K235 and E198-K235, and a native salt bridge, E171-R249, are mutually exclusive, thus resulting in only a marginal increase in stability as compared to the wild type protein. When a destabilized V157F is paired with N235K, the native salt bridge E171-R249 is retained. In this context, the non-native salt bridges, D186-K235 and E198-K235, produce a net increase in stability as compared to V157F alone. A similar rescue mechanism may explain how N235K stabilize other highly unstable beta-sandwich cancer mutants.
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Affiliation(s)
- Yu-Hong Tan
- Department of Molecular Biology and Biochemistry, University of California, Irvine, California, 92697, USA
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43
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Suad O, Rozenberg H, Brosh R, Diskin-Posner Y, Kessler N, Shimon LJW, Frolow F, Liran A, Rotter V, Shakked Z. Structural basis of restoring sequence-specific DNA binding and transactivation to mutant p53 by suppressor mutations. J Mol Biol 2008; 385:249-65. [PMID: 18996393 DOI: 10.1016/j.jmb.2008.10.063] [Citation(s) in RCA: 50] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2008] [Revised: 10/19/2008] [Accepted: 10/23/2008] [Indexed: 11/27/2022]
Abstract
The tumor suppressor protein p53 is mutated in more than 50% of invasive cancers. About 30% of the mutations are found in six major "hot spot" codons located in its DNA binding core domain. To gain structural insight into the deleterious effects of such mutations and their rescue by suppressor mutations, we determined the crystal structures of the p53 core domain incorporating the hot spot mutation R249S, the core domain incorporating R249S and a second-site suppressor mutation H168R (referred to as the double mutant R249S/H168R) and its sequence-specific complex with DNA and of the triple mutant R249S/H168R/T123A. The structural studies were accompanied by transactivation and apoptosis experiments. The crystal structures show that the region at the vicinity of the mutation site in the R249S mutant displays a range of conformations [wild-type (wt) and several mutant-type conformations] due to the loss of stabilizing interactions mediated by R249 in the wt protein. As a consequence, the protein surface that is critical to the formation of functional p53-DNA complexes, through protein-protein and protein-DNA interactions, is largely distorted in the mutant conformations, thus explaining the protein's "loss of function" as a transcription factor. The structure of this region is restored in both R249S/H168R and R249S/H168R/T123A and is further stabilized in the complex of R249S/H168R with DNA. Our functional data show that the introduction of H168R as a second-site suppressor mutation partially restores the transactivation capacity of the protein and that this effect is further amplified by the addition of a third-site mutation T123A. These findings together with previously reported data on wt and mutant p53 provide a structural framework for understanding p53 dysfunction as a result of oncogenic mutations and its rescue by suppressor mutations and for a potential drug design aimed at restoring wt activity to aberrant p53 proteins.
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Affiliation(s)
- Oded Suad
- Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel
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44
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Predicting positive p53 cancer rescue regions using Most Informative Positive (MIP) active learning. PLoS Comput Biol 2008; 5:e1000498. [PMID: 19756158 PMCID: PMC2742196 DOI: 10.1371/journal.pcbi.1000498] [Citation(s) in RCA: 46] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/21/2009] [Accepted: 08/04/2009] [Indexed: 11/19/2022] Open
Abstract
Many protein engineering problems involve finding mutations that produce proteins
with a particular function. Computational active learning is an attractive
approach to discover desired biological activities. Traditional active learning
techniques have been optimized to iteratively improve classifier accuracy, not
to quickly discover biologically significant results. We report here a novel
active learning technique, Most Informative Positive (MIP), which is tailored to
biological problems because it seeks novel and informative positive results. MIP
active learning differs from traditional active learning methods in two ways:
(1) it preferentially seeks Positive (functionally active) examples; and (2) it
may be effectively extended to select gene regions suitable for high throughput
combinatorial mutagenesis. We applied MIP to discover mutations in the tumor
suppressor protein p53 that reactivate mutated p53 found in human cancers. This
is an important biomedical goal because p53 mutants have been
implicated in half of all human cancers, and restoring active p53 in tumors
leads to tumor regression. MIP found Positive (cancer rescue) p53 mutants
in silico using 33% fewer experiments than
traditional non-MIP active learning, with only a minor decrease in classifier
accuracy. Applying MIP to in vivo experimentation yielded
immediate Positive results. Ten different p53 mutations found in human cancers
were paired in silico with all possible single amino acid
rescue mutations, from which MIP was used to select a Positive Region predicted
to be enriched for p53 cancer rescue mutants. In vivo assays
showed that the predicted Positive Region: (1) had significantly more
(p<0.01) new strong cancer rescue mutants than control regions (Negative,
and non-MIP active learning); (2) had slightly more new strong cancer rescue
mutants than an Expert region selected for purely biological considerations; and
(3) rescued for the first time the previously unrescuable p53 cancer mutant
P152L. Engineering proteins to acquire or enhance a particular useful function is at the
core of many biomedical problems. This paper presents Most Informative Positive
(MIP) active learning, a novel integrated computational/biological approach
designed to help guide biological discovery of novel and informative positive
mutants. A classifier, together with modeled structure-based features, helps
guide biological experiments and so accelerates protein engineering studies. MIP
reduces the number of expensive biological experiments needed to achieve novel
and informative positive results. We used the MIP method to discover novel p53
cancer rescue mutants. p53 is a tumor suppressor protein, and destructive p53
mutations have been implicated in half of all human cancers. Second-site cancer
rescue mutations restore p53 activity and eventually may facilitate rational
design of better cancer drugs. This paper shows that, even in the first round of
in vivo experiments, MIP significantly increased the discovery rate of novel and
informative positive mutants.
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45
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Abstract
The tumor suppressor protein p53 induces or represses the expression of a variety of target genes involved in cell cycle control, senescence, and apoptosis in response to oncogenic or other cellular stress signals. It exerts its function as guardian of the genome through an intricate interplay of independently folded and intrinsically disordered functional domains. In this review, we provide insights into the structural complexity of p53, the molecular mechanisms of its inactivation in cancer, and therapeutic strategies for the pharmacological rescue of p53 function in tumors. p53 emerges as a paradigm for a more general understanding of the structural organization of modular proteins and the effects of disease-causing mutations.
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Affiliation(s)
- Andreas C Joerger
- Medical Research Council Centre for Protein Engineering, Cambridge, United Kingdom.
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46
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Cheok CF, Lane DP. New developments in small molecules targeting p53 pathways in anticancer therapy. Drug Dev Res 2008. [DOI: 10.1002/ddr.20261] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
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47
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DNA topology influences p53 sequence-specific DNA binding through structural transitions within the target sites. Biochem J 2008; 412:57-63. [PMID: 18271758 DOI: 10.1042/bj20071648] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
The tumour suppressor protein p53 is one of the most important factors regulating cell proliferation, differentiation and programmed cell death in response to a variety of cellular stress signals. P53 is a nuclear phosphoprotein and its biochemical function is closely associated with its ability to bind DNA in a sequence-specific manner and operate as a transcription factor. Using a competition assay, we investigated the effect of DNA topology on the DNA binding of human wild-type p53 protein. We prepared sets of topoisomers of plasmid DNA with and without p53 target sequences, differing in their internal symmetry. Binding of p53 to DNA increased with increasing negative superhelix density (-sigma). At -sigma < or = 0.03, the relative effect of DNA supercoiling on protein-DNA binding was similar for DNA containing both symmetrical and non-symmetrical target sites. On the other hand, at higher -sigma, target sites with a perfect inverted repeat sequence exhibited a more significant enhancement of p53 binding as a result of increasing levels of negative DNA supercoiling. For -sigma = 0.07, an approx. 3-fold additional increase in binding was observed for a symmetrical target site compared with a non-symmetrical target site. The p53 target sequences possessing the inverted repeat symmetry were shown to form a cruciform structure in sufficiently negative supercoiled DNA. We show that formation of cruciforms in DNA topoisomers at -sigma > or = 0.05 correlates with the extra enhancement of p53-DNA binding.
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48
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Golubovskaya VM, Finch R, Kweh F, Massoll NA, Campbell-Thompson M, Wallace MR, Cance WG. p53 regulates FAK expression in human tumor cells. Mol Carcinog 2008; 47:373-82. [PMID: 17999388 PMCID: PMC4562219 DOI: 10.1002/mc.20395] [Citation(s) in RCA: 78] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
Attenuation of the p53 protein is one of the most common abnormalities in human tumors. Another important marker of tumorigenesis is focal adhesion kinase (FAK), a 125-kDa tyrosine kinase that is overexpressed at the mRNA and protein levels in a variety of human tumors. FAK is a critical regulator of adhesion, motility, metastasis, and survival signaling. We have characterized the FAK promoter and demonstrated that p53 can inhibit the FAK promoter activity in vitro. In the present study, we showed that p53 can bind the FAK promoter-chromatin region in vivo by chromatin immunoprecipitation (ChIP) assay. Furthermore, we demonstrated down-regulation of FAK mRNA and protein levels by adenoviral overexpression of p53. We introduced plasmids with different mutations in the DNA-binding domain of p53 (R175H, p53 R248W and R273H) into HCTp53(-/-) cells and showed that these mutations of p53 did not bind FAK promoter and did not inhibit FAK promoter activity, unlike wild type p53. We analyzed primary breast and colon cancers for p53 mutations and FAK expression, and showed that FAK expression was increased in tumors containing mutations of p53 compared to tumors with wild type p53. In addition, tumor-derived missense mutations in the DNA-binding domain (R282, R249, and V173) also led to increased FAK promoter activity. Thus, the present data show that p53 can regulate FAK expression during tumorigenesis.
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49
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Yousef AF, Xu GW, Mendez M, Brandl CJ, Mymryk JS. Coactivator requirements for p53-dependent transcription in the yeast Saccharomyces cerevisiae. Int J Cancer 2008; 122:942-6. [PMID: 17957787 DOI: 10.1002/ijc.23174] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
p53 is a sequence-specific DNA-binding transcription factor and key regulator of cell cycle arrest and apoptosis. p53 is mutated in most human cancers and these mutations generally impair its ability to activate transcription. When expressed in Saccharomyces cerevisiae, p53 acts as a strong transcriptional activator allowing yeast to be used as a model system to study the effects of p53 mutations on activity. However, little is known about the exact mechanisms by which p53 functions in yeast. Using 76 mutant yeast strains, we have evaluated the effect of deleting components of the ADA, COMPASS, INO80, ISW1, Mediator, RSC, SAGA, SAS, SLIK, SWI/SNF, and SWR1 transcriptional regulatory complexes on p53-dependent transcription. In addition, we examined the role of histone H2B ubiquitylation by Rad6/Bre1 on p53 activation. Overall, our analysis indicates that there are several remarkable similarities between p53-dependent transcription in yeast and mammalian cells, suggesting that yeast can serve as a valid model system for at least some aspects of p53 function.
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Affiliation(s)
- Ahmed F Yousef
- Department of Microbiology and Immunology, University of Western Ontario, London, ON, Canada
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Camps M, Herman A, Loh E, Loeb LA. Genetic constraints on protein evolution. Crit Rev Biochem Mol Biol 2008; 42:313-26. [PMID: 17917869 DOI: 10.1080/10409230701597642] [Citation(s) in RCA: 90] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
Evolution requires the generation and optimization of new traits ("adaptation") and involves the selection of mutations that improve cellular function. These mutations were assumed to arise by selection of neutral mutations present at all times in the population. Here we review recent evidence that indicates that deleterious mutations are more frequent in the population than previously recognized and that these mutations play a significant role in protein evolution through continuous positive selection. Positively selected mutations include adaptive mutations, i.e. mutations that directly affect enzymatic function, and compensatory mutations, which suppress the pleiotropic effects of adaptive mutations. Compensatory mutations are by far the most frequent of the two and would allow potentially adaptive but deleterious mutations to persist long enough in the population to be positively selected during episodes of adaptation. Compensatory mutations are, by definition, context-dependent and thus constrain the paths available for evolution. This provides a mechanistic basis for the examples of highly constrained evolutionary landscapes and parallel evolution reported in natural and experimental populations. The present review article describes these recent advances in the field of protein evolution and discusses their implications for understanding the genetic basis of disease and for protein engineering in vitro.
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Affiliation(s)
- Manel Camps
- Department of Environmental Toxicology, University of California at Santa Cruz, Santa Cruz, California 95064, USA.
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