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

Nikolova, Penka V.; Wong, Kam‐Bo; DeDecker, Brian S.; Henckel, Julia; Fersht, Alan R.

doi:10.1093/emboj/19.3.370

Cited by 161 publications

(158 citation statements)

References 34 publications

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“…N239Y increases the thermodynamic stability of the p53 core domain by about 1.5 kcal mol Ϫ1 , which is able to restore wild type stability in G245S/N239Y (42). We observed that the N239Y mutation led to higher transcriptional and apoptotic activity in mammalian cells.…”

Section: Comparison Of P53 Levels and The Role Of Thermodynamicmentioning

confidence: 64%

Effects of Stability on the Biological Function of p53

Khoo¹,

Mayer

Fersht

2009

Journal of Biological Chemistry

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The core domain of the tumor suppressor p53 has low thermodynamic stability, and many oncogenic mutations cause it to denature rapidly at body temperature. We made a series of core domain mutants that are significantly less or more stable than wild type to investigate effects of stability on the transcriptional activity and levels of native full-length p53 in H1299 mammalian cells. The levels of transcriptionally inactive native protein with inactivating mutations in the N-terminal transactivation domain correlated strongly with stability. The levels of transcriptionally active proteins, however, depended on both their stability and the transcriptional activity that leads to the feedback loop of proteolytic degradation via transcription of E3 ligases. A very highly stabilized quadruple mutant and an even more stable hexamutant were more active than wild-type p53 in terms of Bax transcription and apoptotic activity, and reached higher levels than wild type in cells. The increased activity did not result from increased overall stability but was due to a single known suppressor mutation, N239Y. It is possible that the low intrinsic stability of p53 is a means of keeping its level low in the cell by spontaneous denaturation, by a route additional to that of proteolytic degradation via E3 ligase pathways. Denatured p53 does accumulate in cells, and there are pathways for the proteolysis of denatured proteins.

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Section: Comparison Of P53 Levels and The Role Of Thermodynamicmentioning

confidence: 64%

Effects of Stability on the Biological Function of p53

Khoo¹,

Mayer

Fersht

2009

Journal of Biological Chemistry

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“…The mutations N239Y and N268D have been reported to restore transcriptional activity in a subset of cancer mutants, including G245S (36). Double-mutant cycles suggest that they act as global stability suppressors (37). As such, they mimic the effects of a hypothetical generic small-molecule drug.…”

Section: T-p53c-y220cmentioning

confidence: 99%

“…A prime example is the mutant R249S. Studies on second-site suppressor mutations have shown that full restoration of DNA-binding activity is found only in the presence of a mutation (H168R) that specifically reverses the structural changes induced by the oncogenic mutation (16,36,37).…”

Section: T-p53c-y220cmentioning

confidence: 99%

Structural basis for understanding oncogenic p53 mutations and designing rescue drugs

Joerger

Ang

Fersht³

2006

Proc. Natl. Acad. Sci. U.S.A.

270

319

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The DNA-binding domain of the tumor suppressor p53 is inactivated by mutation in Ϸ50% of human cancers. We have solved high-resolution crystal structures of several oncogenic mutants to investigate the structural basis of inactivation and provide information for designing drugs that may rescue inactivated mutants. We found a variety of structural consequences upon mutation: (i) the removal of an essential contact with DNA, (ii) creation of large, water-accessible crevices or hydrophobic internal cavities with no other structural changes but with a large loss of thermodynamic stability, (iii) distortion of the DNA-binding surface, and (iv) alterations to surfaces not directly involved in DNA binding but involved in domain-domain interactions on binding as a tetramer. These findings explain differences in functional properties and associated phenotypes (e.g., temperature sensitivity). Some mutants have the potential of being rescued by a generic stabilizing drug. In addition, a mutation-induced crevice is a potential target site for a mutant-selective stabilizing drug.cancer ͉ crystal ͉ structure ͉ drug design ͉ polymorphism T he tumor suppressor protein p53 is a 393-aa transcription factor that regulates the cell cycle and plays a key role in the prevention of cancer development. In response to oncogenic and other stresses, p53 induces the transcription of a number of target genes, resulting in cell-cycle arrest, senescence, or apoptosis (1, 2). In Ϸ50% of human cancers, p53 is inactivated as a result of missense mutation in the p53 gene (3, 4).The multifunctionality of p53 is reflected in the complexity of its structure. Each chain in the p53 tetramer is composed of several domains. There are well defined DNA-binding and tetramerization domains and highly mobile, largely unstructured regions (5-9). Most p53 cancer mutations are located in the DNA-binding core domain of the protein (3). This domain has been structurally characterized in complex with its cognate DNA by x-ray crystallography (5, 10, 11) and in its free form in solution by NMR (12). It consists of a central ␤-sandwich that serves as a basic scaffold for the DNA-binding surface. The DNA-binding surface is composed of two large loops (L2 and L3) that are stabilized by a zinc ion and a loop-sheet-helix motif. Together, these structural elements form an extended surface that makes specific contacts with the various p53 response elements. The six amino acid residues that are most frequently mutated in human cancer are located in or close to the DNA-binding surface (compare release R10 of the TP53 mutation database at www-p53.iarc.fr) (3). These residues have been classified as ''contact'' (Arg-248 and Arg-273) or ''structural'' (Arg-175, Gly-245, Arg-249, and Arg-282) residues, depending on whether they directly contact DNA or play a role in maintaining the structural integrity of the DNA-binding surface (Fig. 1) (5).Urea denaturation studies have shown that the contact mutation R273H has no effect on the thermodynamic stability of the core domain, wher...

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“…Unlike p63 and p73, human p53 DBD is extremely susceptible to mutational inactivation that lowers its thermodynamic stability (35). A second site suppressor mutation in p53, N268D, restores DNA binding activity by increasing the thermodynamic stability in some oncogenic mutants (35)(36)(37). The amino acid asparagine (Asn-268) in human p53 is not conserved and is substituted by aspartic acid in rat and mouse p53 and by arginine in chicken, Xenopus, and other species (38).…”

Section: Sch529074 Binds To P53 Core and Acts As A Chaperone-mentioning

confidence: 99%

SCH529074, a Small Molecule Activator of Mutant p53, Which Binds p53 DNA Binding Domain (DBD), Restores Growth-suppressive Function to Mutant p53 and Interrupts HDM2-mediated Ubiquitination of Wild Type p53

Demma¹,

Maxwell²,

Ramos³

et al. 2010

Journal of Biological Chemistry

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Abrogation of p53 function occurs in almost all human cancers, with more than 50% of cancers harboring inactivating mutations in p53 itself. Mutation of p53 is indicative of highly aggressive cancers and poor prognosis. The vast majority of mutations in p53 occur in its core DNA binding domain (DBD) and result in inactivation of p53 by reducing its thermodynamic stability at physiological temperature. Here, we report a small molecule, SCH529074, that binds specifically to the p53 DBD in a saturable manner with an affinity of 1-2 M. Binding restores wild type function to many oncogenic mutant forms of p53. This small molecule reactivates mutant p53 by acting as a chaperone, in a manner similar to that previously reported for the peptide CDB3. Binding of SCH529074 to the p53 DBD is specifically displaced by an oligonucleotide with a sequence derived from the p53-response element. In addition to reactivating mutant p53, SCH529074 binding inhibits ubiquitination of p53 by HDM2. We have also developed a novel variant of p53 by changing a single amino acid in the core domain of p53 (N268R), which abolishes binding of SCH529074. This amino acid change also inhibits HDM2-mediated ubiquitination of p53. Our novel findings indicate that through its interaction with p53 DBD, SCH529074 restores DNA binding activity to mutant p53 and inhibits HDM2-mediated ubiquitination.

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

Cited by 161 publications

References 34 publications

Effects of Stability on the Biological Function of p53

Effects of Stability on the Biological Function of p53

Structural basis for understanding oncogenic p53 mutations and designing rescue drugs

SCH529074, a Small Molecule Activator of Mutant p53, Which Binds p53 DNA Binding Domain (DBD), Restores Growth-suppressive Function to Mutant p53 and Interrupts HDM2-mediated Ubiquitination of Wild Type p53

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