Biochemical characterisation of purified human wild‐type p53 overexpressed in insect cells

Chalkley, Gillian E.; Knowles, Phillip P.; Whitehead, Philip; Coffer, Arnold I.

doi:10.1111/j.1432-1033.1994.tb18726.x

Cited by 14 publications

(11 citation statements)

References 45 publications

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“…1A, a band corresponding to the molecular weight of non‐ubiquitinated p53 could be detected in this assay. This band appeared even in the absence of ectopic His 6 ‐tagged ubiquitin and therefore could be due to non‐specific binding of p53 to the nickel‐agarose beads or to the ability of p53 to bind to nickel as reported by others [16,17].…”

Section: Resultsmentioning

confidence: 52%

Differences in the ubiquitination of p53 by Mdm2 and the HPV protein E6

Camus¹,

Higgins²,

Lane³

et al. 2003

FEBS Letters

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The human papillomavirus (HPV) protein E6 can promote the ubiquitination of the p53 tumour suppressor in vitro, providing an explanation for the ability of E6 to induce p53 degradation in vivo and contribute to the potential tumorigenic e¡ect of the virus. Instead, in non-infected cells, p53 levels are primarily destabilised by the ubiquitin E3 ligase activity of the Mdm2 protein. Here we have compared the e¡ects of E6 and Mdm2 on p53 ubiquitination in vivo. We show that whereas in the presence of Mdm2 proteasome inhibitors induce the accumulation of ubiquitinated forms of p53, this does not occur in the presence of E6. Accordingly, we con¢rm that the e¡ect of E6 and p53 is independent of the six C-terminal lysine residues in p53, which have previously been described to play an important role for e¡ective ubiquitination and degradation of 53 mediated by Mdm2. We also show that other yet unidenti¢ed residues in p53 are also susceptible to ubiquitination. These results indicate that E6 does not induce ubiquitination of p53 in the same way as Mdm2 in order to promote its degradation, suggesting important di¡erences between the Mdm2 and E6 e¡ects on p53 degradation. ß

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Section: Resultsmentioning

confidence: 52%

Differences in the ubiquitination of p53 by Mdm2 and the HPV protein E6

Camus¹,

Higgins²,

Lane³

et al. 2003

FEBS Letters

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“…The average radii Ru of the unaggregated protein, evaluated from the τ1s, range from 8 to 12 nm(Figure 4b), substantially greater than the ca. 4 nm radius expected for a p53 tetramer of molecular weight 175 kg mol -1 58. Consonantly with the exaggerated radius, the considerable polydispersity µτ 2 ≈ 0.35 -0.25(Figure 4c) indicates the dominance of highorder oligomers over monomeric, dimeric, and tetrameric p53 units.14,59 To further explore how temperature manipulates the state of the unaggregated protein, we measured average protein molecular weight Mw using static light scattering at 15, 24, and 37 o C(Figure 4d,e).…”

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confidence: 94%

Mesoscopic Liquid Clusters Represent a Distinct Condensate of Mutant p53

Yang

Saeedi

Davtyan

et al. 2020

Preprint

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The oncogenic properties of mutant p53 have been ascribed to destabilization of the p53 conformation, followed by aggregation into insoluble fibrils. Here we combine immunofluorescent 3D confocal microscopy of breast cancer cells expressing the p53 mutant Arg248Gln (R248Q) with light scattering from solutions of the purified protein and molecular simulations to probe the mechanisms that govern phase behaviors of the mutant across multiple length scales, from cellular to molecular. We establish that p53 R248Q forms mesoscopic proteinrich clusters, an anomalous liquid phase with several unique properties. We demonstrate that the clusters host and facilitate the nucleation of amyloid fibrils. The distinct characteristics of the clusters of R248Q and wild-type p53 and theoretical models indicate that p53 condensation into clusters is driven by the structural destabilization of the core domain and not by interactions of its extensive disordered region. Two-step nucleation of mutant p53 amyloids suggests means to IntroductionThe wild type p53 protein is a potent cancer suppressor, which is inactivated in almost every tumor, either through mutations in the TP53 gene (in 50% or more of human cancers) or deregulation of its associated pathways. 1-6 By contrast, p53 mutants emerge as potent cancer promoters because they exert a dominant-negative (DN) effect on the wild-type variant and also display oncogenic gain-of-function (GOF) properties by inhibiting other cancer suppressors. 1 Several mechanisms of cancer promotion by mutant p53 have been discussed. [6][7][8][9][10][11][12] It was recently suggested that the structural destabilization of the p53 mutants and the associated aggregation into insoluble, degradation resistant structures, designated fibrils, may play a decisive role in their oncogenicity. 2,[13][14][15] Concurrently, fibril suppression, for instance, by stabilization of the mutant p53 conformation, has been identified as a general way to fight cancer. 2,16 A parallel development identified a new form of biological organization, liquid-liquid phase separation, which constitutes several common membraneless organelles such as nucleoli, Cajal bodies, and P granules. [17][18][19][20][21] Many proteins forming dense liquid condensates in live cells incorporate significant disordered regions and condensation at the low concentrations of these proteins in the nucleoplasm and cytoplasm has been attributed to stronger intermolecular attraction due to the unstructured segments. 19,20 Furthermore, it is well-appreciated that protein dense liquids can serve as precursors and facilitators of higher order organization such as ordering into crystals 22-25 and the formation of polymers of sickle hemoglobin. 26,27 Theoretical analyses and recent experiments suggest that a similar precursor mechanism would apply to the nucleation of amyloid fibrils. [28][29][30][31][32][33]

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“…The dependence of complex formation on the presence of Zn 2ϩ was further established by titrating EDTA into the reaction. Since it has been shown that other divalent cations may bind to p53, the influence of other such ions (Ca 2ϩ , Cd 2ϩ , Co 2ϩ , Cu 2ϩ , Mg 2ϩ , Mn 2ϩ , and Ni 2ϩ ) on the formation of TAg-p53 complexes in this ELISA was investigated in the same manner as for Zn 2ϩ ; all ion solutions were prepared from the chloride salt (30).…”

Section: Methodsmentioning

confidence: 99%

Modification of an N-terminal Regulatory Domain of T Antigen Restores p53-T Antigen Complex Formation in the Absence of an Essential Metal Ion Cofactor

Kernohan

Hupp

Lane

1996

Journal of Biological Chemistry

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We have discovered that the ability of the tumor suppressor protein p53 to bind to the viral large T antigen (TAg) oncogene product is regulated by divalent cations. Both proteins were purified from an insect cell line infected with the appropriate baculovirus expression vector. In a two-site capture enzyme-linked immunosorbent assay, complex formation between the purified proteins is strictly dependent on the addition of specific concentrations of divalent metal ions, notably zinc, copper, cadmium, cobalt, manganese, and nickel. In the presence of zinc the pattern of proteolytic fragments obtained when TAg was subjected to proteolysis by endoproteinase Glu-C (V8) was strikingly different, supporting the idea that a conformational change in TAg associated with ion binding is required for it to complex with p53. Monoclonal antibody analysis provides supporting evidence for a conformational change. When TAg was captured onto an enzyme-linked immunosorbent assay plate coated with PAb 419 as opposed to many other anti-TAg antibodies, complex formation was completely independent of the presence of additional divalent cations. Our results suggest that the ability of p53 and TAg to form a stable complex in vitro is dependent upon a regulatory domain residing in the N terminus of TAg, zinc ions or the binding of a specific monoclonal antibody (PAb 419) provoking a conformational change in TAg that facilitates and supports complex formation.

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Biochemical characterisation of purified human wild‐type p53 overexpressed in insect cells

Cited by 14 publications

References 45 publications

Differences in the ubiquitination of p53 by Mdm2 and the HPV protein E6

Differences in the ubiquitination of p53 by Mdm2 and the HPV protein E6

Mesoscopic Liquid Clusters Represent a Distinct Condensate of Mutant p53

Modification of an N-terminal Regulatory Domain of T Antigen Restores p53-T Antigen Complex Formation in the Absence of an Essential Metal Ion Cofactor

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