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Mutant p53 Aggregates into Prion-like Amyloid Oligomers and Fibrils

IMPLICATIONS FOR CANCER*
  • Ana P.D. Ano Bom
    Footnotes
    Affiliations
    Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-902, Brazil

    Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-902, Brazil
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  • Luciana P. Rangel
    Footnotes
    Affiliations
    Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-902, Brazil

    Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-902, Brazil
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  • Danielly C.F. Costa
    Affiliations
    Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-902, Brazil

    Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-902, Brazil
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  • Guilherme A.P. de Oliveira
    Affiliations
    Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-902, Brazil

    Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-902, Brazil
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  • Daniel Sanches
    Affiliations
    Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-902, Brazil

    Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-902, Brazil
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  • Carolina A. Braga
    Affiliations
    Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-902, Brazil

    Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-902, Brazil
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  • Lisandra M. Gava
    Affiliations
    Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-902, Brazil

    Instituto de Biologia, Universidade Estadual de Campinas, Instituto de Biologia, 13084-862 Campinas, SP, Brazil
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  • Carlos H.I. Ramos
    Affiliations
    Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-902, Brazil

    Instituto de Química, Universidade Estadual de Campinas, Instituto de Biologia, 13084-862 Campinas, SP, Brazil
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  • Ana O.T. Cepeda
    Affiliations
    Instituto de Biologia, Universidade Estadual de Campinas, Instituto de Biologia, 13084-862 Campinas, SP, Brazil
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  • Ana C. Stumbo
    Affiliations
    Departamento de Histologia e Embriologia, IBRAG, Universidade do Estado do Rio de Janeiro, RJ 20550-013 Rio de Janeiro, Brazil
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  • Claudia V. De Moura Gallo
    Affiliations
    Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-902, Brazil

    Departamento de Genética, IBRAG, Universidade do Estado do Rio de Janeiro, RJ 20550-013 Rio de Janeiro, Brazil
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  • Yraima Cordeiro
    Affiliations
    Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-902, Brazil

    Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-902, Brazil
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  • Jerson L. Silva
    Correspondence
    To whom correspondence should be addressed: Universidade Federal do Rio de Janeiro, Instituto de Bioquímica Médica, Bloco E Sala 10, Cidade Universitária, 21941–590, Rio de Janeiro, RJ, Brazil. Tel.: 55-21-25626756; Fax: 55-21-38814155
    Affiliations
    Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-902, Brazil

    Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-902, Brazil
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  • Author Footnotes
    * This work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), the Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem (INBEB), and the Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) of Brazil.
    This article contains supplemental Table S1 and Figs. S1 and S2.
    1 Both authors contributed equally to this work.
Open AccessPublished:June 19, 2012DOI:https://doi.org/10.1074/jbc.M112.340638
      Over 50% of all human cancers lose p53 function. To evaluate the role of aggregation in cancer, we asked whether wild-type (WT) p53 and the hot-spot mutant R248Q could aggregate as amyloids under physiological conditions and whether the mutant could seed aggregation of the wild-type form. The central domains (p53C) of both constructs aggregated into a mixture of oligomers and fibrils. R248Q had a greater tendency to aggregate than WT p53. Full-length p53 aggregated into amyloid-like species that bound thioflavin T. The amyloid nature of the aggregates was demonstrated using x-ray diffraction, electron microscopy, FTIR, dynamic light scattering, cell viabilility assay, and anti-amyloid immunoassay. The x-ray diffraction pattern of the fibrillar aggregates was consistent with the typical conformation of cross β-sheet amyloid fibers with reflexions of 4.7 Å and 10 Å. A seed of R248Q p53C amyloid oligomers and fibrils accelerated the aggregation of WT p53C, a behavior typical of a prion. The R248Q mutant co-localized with amyloid-like species in a breast cancer sample, which further supported its prion-like effect. A tumor cell line containing mutant p53 also revealed massive aggregation of p53 in the nucleus. We conclude that aggregation of p53 into a mixture of oligomers and fibrils sequestrates the native protein into an inactive conformation that is typical of a prionoid. This prion-like behavior of oncogenic p53 mutants provides an explanation for the negative dominance effect and may serve as a potential target for cancer therapy.

      Introduction

      Cancer is a leading cause of death worldwide. According to the World Health Organization (WHO), deaths from cancer will reach 11 million annually by 2030. Biomedical research has provided a great deal of information about cancer, but the molecular mechanisms that lead to cancer remain poorly understood. The role of the tumor suppressor protein p53 is regarded as vital, as p53 is a nuclear phosphoprotein that induces cell cycle arrest and apoptosis in response to cellular stress, particularly DNA damage. Moreover, mutations in the p53 gene (TP53) are strongly associated with increased susceptibility to cancer (
      • Vousden K.H.
      • Lane D.P.
      p53 in health and disease.
      ).
      p53 is a tetrameric flexible protein containing 393 residues. The N-terminal activation domain is able to interact with a number of proteins, whereas the C-terminal domain is responsible for tetramerization. The central region or core domain (p53C) of the protein constitutes the sequence-specific DNA-binding region (
      • Joerger A.C.
      • Fersht A.R.
      Structural biology of the tumor suppressor p53.
      ) and is the segment most involved in mutant-related tumors.
      Previously, we showed that the core domain of p53 forms β-sheet-rich fibrillar aggregates under mild denaturing conditions (hydrostatic pressure) (
      • Ishimaru D.
      • Andrade L.R.
      • Teixeira L.S.
      • Quesado P.A.
      • Maiolino L.M.
      • Lopez P.M.
      • Cordeiro Y.
      • Costa L.T.
      • Heckl W.M.
      • Weissmüller G.
      • Foguel D.
      • Silva J.L.
      Fibrillar aggregates of the tumor suppressor p53 core domain.
      ,
      • Silva J.L.
      • Vieira T.C.
      • Gomes M.P.
      • Ano Bom A.P.
      • Lima L.M.
      • Freitas M.S.
      • Ishimaru D.
      • Cordeiro Y.
      • Foguel D.
      Ligand binding and hydration in protein misfolding: insights from studies of prion and p53 tumor suppressor proteins.
      ). At that time, our group hypothesized that p53 aggregation could lead to cancer (
      • Ishimaru D.
      • Andrade L.R.
      • Teixeira L.S.
      • Quesado P.A.
      • Maiolino L.M.
      • Lopez P.M.
      • Cordeiro Y.
      • Costa L.T.
      • Heckl W.M.
      • Weissmüller G.
      • Foguel D.
      • Silva J.L.
      Fibrillar aggregates of the tumor suppressor p53 core domain.
      ). In support of this view, it has been reported that p53 regions other than the DNA-binding domain may undergo aggregation under physiological conditions (
      • Galea C.
      • Bowman P.
      • Kriwacki R.W.
      Disruption of an intermonomer salt bridge in the p53 tetramerization domain results in an increased propensity to form amyloid fibrils.
      ,
      • Higashimoto Y.
      • Asanomi Y.
      • Takakusagi S.
      • Lewis M.S.
      • Uosaki K.
      • Durell S.R.
      • Anderson C.W.
      • Appella E.
      • Sakaguchi K.
      Unfolding, aggregation, and amyloid formation by the tetramerization domain from mutant p53 associated with lung cancer.
      ). In addition, the largely unstructured N-terminal transactivation domain aggregates into fibrils when incubated at low pH (
      • Rigacci S.
      • Bucciantini M.
      • Relini A.
      • Pesce A.
      • Gliozzi A.
      • Berti A.
      • Stefani M.
      The (1–63) region of the p53 transactivation domain aggregates in vitro into cytotoxic amyloid assemblies.
      ). We found that small cognate DNAs stabilize the core domain and full-length p53 and have the potential to rescue aggregated, misfolded species (
      • Ishimaru D.
      • Ano Bom A.P.
      • Lima L.M.
      • Quesado P.A.
      • Oyama M.F.
      • de Moura Gallo C.V.
      • Cordeiro Y.
      • Silva J.L.
      Cognate DNA stabilizes the tumor suppressor p53 and prevents misfolding and aggregation.
      ). Recently, we demonstrated that mutant p53 co-localizes with amyloid-like protein aggregates in cancer biopsies (
      • Levy C.B.
      • Stumbo A.C.
      • Ano Bom A.P.
      • Portari E.A.
      • Cordeiro Y.
      • Silva J.L.
      • De Moura-Gallo C.V.
      Co-localization of mutant p53 and amyloid-like protein aggregates in breast tumors.
      ). Finally, aggregated mutant p53 induces co-aggregation of wild-type p53 and its paralogs p63 and p73 (
      • Xu J.
      • Reumers J.
      • Couceiro J.R.
      • De Smet F.
      • Gallardo R.
      • Rudyak S.
      • Cornelis A.
      • Rozenski J.
      • Zwolinska A.
      • Marine J.C.
      • Lambrechts D.
      • Suh Y.A.
      • Rousseau F.
      • Schymkowitz J.
      Gain of function of mutant p53 by coaggregation with multiple tumor suppressors.
      ). Therefore, the hypothesis that p53 aggregation may participate in some cancers similarly to the situation in Alzheimer and Parkinson disease (
      • Chiti F.
      • Dobson C.M.
      Protein misfolding, functional amyloid, and human disease.
      ,
      • Pastore A.
      • Temussi P.A.
      The two faces of Janus: functional interactions and protein aggregation.
      ) has attracted increasing attention (
      • Ishimaru D.
      • Andrade L.R.
      • Teixeira L.S.
      • Quesado P.A.
      • Maiolino L.M.
      • Lopez P.M.
      • Cordeiro Y.
      • Costa L.T.
      • Heckl W.M.
      • Weissmüller G.
      • Foguel D.
      • Silva J.L.
      Fibrillar aggregates of the tumor suppressor p53 core domain.
      ,
      • Silva J.L.
      • Vieira T.C.
      • Gomes M.P.
      • Ano Bom A.P.
      • Lima L.M.
      • Freitas M.S.
      • Ishimaru D.
      • Cordeiro Y.
      • Foguel D.
      Ligand binding and hydration in protein misfolding: insights from studies of prion and p53 tumor suppressor proteins.
      ,
      • Xu J.
      • Reumers J.
      • Couceiro J.R.
      • De Smet F.
      • Gallardo R.
      • Rudyak S.
      • Cornelis A.
      • Rozenski J.
      • Zwolinska A.
      • Marine J.C.
      • Lambrechts D.
      • Suh Y.A.
      • Rousseau F.
      • Schymkowitz J.
      Gain of function of mutant p53 by coaggregation with multiple tumor suppressors.
      ).
      Although p53 aggregation has been shown to occur (
      • Ishimaru D.
      • Andrade L.R.
      • Teixeira L.S.
      • Quesado P.A.
      • Maiolino L.M.
      • Lopez P.M.
      • Cordeiro Y.
      • Costa L.T.
      • Heckl W.M.
      • Weissmüller G.
      • Foguel D.
      • Silva J.L.
      Fibrillar aggregates of the tumor suppressor p53 core domain.
      ,
      • Silva J.L.
      • Vieira T.C.
      • Gomes M.P.
      • Ano Bom A.P.
      • Lima L.M.
      • Freitas M.S.
      • Ishimaru D.
      • Cordeiro Y.
      • Foguel D.
      Ligand binding and hydration in protein misfolding: insights from studies of prion and p53 tumor suppressor proteins.
      ,
      • Galea C.
      • Bowman P.
      • Kriwacki R.W.
      Disruption of an intermonomer salt bridge in the p53 tetramerization domain results in an increased propensity to form amyloid fibrils.
      ,
      • Higashimoto Y.
      • Asanomi Y.
      • Takakusagi S.
      • Lewis M.S.
      • Uosaki K.
      • Durell S.R.
      • Anderson C.W.
      • Appella E.
      • Sakaguchi K.
      Unfolding, aggregation, and amyloid formation by the tetramerization domain from mutant p53 associated with lung cancer.
      ,
      • Rigacci S.
      • Bucciantini M.
      • Relini A.
      • Pesce A.
      • Gliozzi A.
      • Berti A.
      • Stefani M.
      The (1–63) region of the p53 transactivation domain aggregates in vitro into cytotoxic amyloid assemblies.
      ,
      • Ishimaru D.
      • Ano Bom A.P.
      • Lima L.M.
      • Quesado P.A.
      • Oyama M.F.
      • de Moura Gallo C.V.
      • Cordeiro Y.
      • Silva J.L.
      Cognate DNA stabilizes the tumor suppressor p53 and prevents misfolding and aggregation.
      ,
      • Levy C.B.
      • Stumbo A.C.
      • Ano Bom A.P.
      • Portari E.A.
      • Cordeiro Y.
      • Silva J.L.
      • De Moura-Gallo C.V.
      Co-localization of mutant p53 and amyloid-like protein aggregates in breast tumors.
      ,
      • Xu J.
      • Reumers J.
      • Couceiro J.R.
      • De Smet F.
      • Gallardo R.
      • Rudyak S.
      • Cornelis A.
      • Rozenski J.
      • Zwolinska A.
      • Marine J.C.
      • Lambrechts D.
      • Suh Y.A.
      • Rousseau F.
      • Schymkowitz J.
      Gain of function of mutant p53 by coaggregation with multiple tumor suppressors.
      ,
      • Butler J.S.
      • Loh S.N.
      Structure, function, and aggregation of the zinc-free form of the p53 DNA binding domain.
      ), we know little about the molecular mechanisms involved and its potential relevance to the tumorigenic process (
      • Antony H
      • Wiegmans A.P.
      • Wei M.Q.
      • Chernoff Y.O.
      • Khanna K.K.
      • Munn A.L.
      Potential roles for prions and protein-only inheritance in cancer.
      ). The most frequent p53 mutation found in cancers is the hot-spot mutation R248Q, which renders the protein unable to bind cognate DNA and exert its proper functions (
      • Hollstein M.
      • Sidransky D.
      • Vogelstein B.
      • Harris C.C.
      p53 mutations in human cancers.
      ,
      • Olivier M.
      • Hollstein M.
      • Hainaut P.
      TP53 mutations in human cancers: origins, consequences, and clinical use.
      ). Here, we asked whether wild-type p53 and R248Q could form aggregates under physiological conditions, what the properties of these aggregates would be and whether mutant p53 aggregates could seed aggregation of the wild-type isoform. The morphology, structure, kinetics, and toxicity of the aggregates were characterized at pH 7.2 and 5.0 for the first time using electron microscopy, dot-blots, x-ray diffraction, FTIR, and LIVE/DEAD viability assays. We found that full-length p53 undergoes amyloid aggregation in a pattern similar to that of the p53 core domain. In addition, we performed immunofluorescence co-localization assays and found positive amyloid aggregation in a tumor sample bearing the R248Q mutant and in breast cancer tumoral cell lines. This result corroborates our in vitro finding that R248Q has a higher propensity to aggregate than wild-type p53. Most interestingly, a mixture of amyloid oligomers and fibrils of R248Q were found to seed the aggregation of wild-type p53 in a prion-like fashion. This prion-like aggregation behavior would explain the negative dominance of mutant p53, and this knowledge may help in developing new therapeutic strategies to prevent or control cancer progression.

      DISCUSSION

      In this report, we described p53 aggregation into amyloid structures at pH values of 7.2 and 5.0, which occur naturally in the cellular environment. In addition, we show for the first time that full-length p53 is prone to aggregate at 37 °C under physiological conditions. Remarkably, R248Q amyloid oligomers and fibrils were able to seed the aggregation of WT p53, which is a behavior typical of prions. We also detected p53 aggregates in breast cancer biopsy samples expressing the somatic mutation R248Q and in the nuclei of tumoral cells expressing the R280K mutation. Our study also provides the first description of the p53 core domain amyloid pattern based on x-ray diffraction and labeling with anti-oligomer A11 antibody under mild conditions. Moreover, the heterogeneous character of p53 aggregation was shown by transmission electron microscopy. Each of these approaches provided valuable information regarding the nature of the p53 fibrils and amyloid oligomers.
      The accumulation of p53 is related to a loss-of-function of this protein and has been observed in various cancers including neuroblastoma, retinoblastoma, breast and colon cancers (
      • Moll U.M.
      • LaQuaglia M.
      • Bénard J.
      • Riou G.
      Wild-type p53 protein undergoes cytoplasmic sequestration in undifferentiated neuroblastomas but not in differentiated tumors.
      ,
      • Elledge R.M.
      • Clark G.M.
      • Fuqua S.A.
      • Yu Y.Y.
      • Allred D.C.
      p53 protein accumulation detected by five different antibodies: relationship to prognosis and heat shock protein 70 in breast cancer.
      ,
      • Moll U.M.
      • Valea F.
      • Chumas J.
      Role of p53 alteration in primary peritoneal carcinoma.
      ). A better understanding of how aggregates form and their nature is crucial to fully dissect this mechanism, which is potentially related to cancer.
      We performed three procedures to obtain p53 aggregates. In all cases, we found that p53C aggregation was greater at pH 7.2 than at pH 5.0 (Figs. 1A and 5). TEM analysis showed that the 37T, HT, and HP aggregates formed fibrillar and amorphous aggregates at both pH values evaluated (Figs. 1B and 5D). Moreover, the CD and FTIR data demonstrate that the aggregate forms of WT and mutant p53C possess more β-sheet content than the soluble species, and significantly bind to Congo Red (Figs. 2 and 5, C and D), which is typical of amyloid fibril formation (
      • Levy C.B.
      • Stumbo A.C.
      • Ano Bom A.P.
      • Portari E.A.
      • Cordeiro Y.
      • Silva J.L.
      • De Moura-Gallo C.V.
      Co-localization of mutant p53 and amyloid-like protein aggregates in breast tumors.
      ).
      The x-ray diffraction data exhibited an amyloid pattern for both WT p53C and R248Q samples subjected to high pressure and high temperature (Fig. 6, B and C and supplemental Fig. S2). These diffraction patterns revealed characteristic amyloid reflections (4.7 Å and 10 Å) due to the spacing in the regular repetitions of cross-β structures (
      • Sunde M.
      • Serpell L.C.
      • Bartlam M.
      • Fraser P.E.
      • Pepys M.B.
      • Blake C.C.
      Common core structure of amyloid fibrils by synchrotron X-ray diffraction.
      ). Another region of p53 has also been described to aggregate into amyloid structures when incubated at pH 3.0 (
      • Rigacci S.
      • Bucciantini M.
      • Relini A.
      • Pesce A.
      • Gliozzi A.
      • Berti A.
      • Stefani M.
      The (1–63) region of the p53 transactivation domain aggregates in vitro into cytotoxic amyloid assemblies.
      ).
      Oligomers, which occur within the pathway to fibril formation, have been described as the toxic species in amyloid diseases (
      • Lambert M.P.
      • Barlow A.K.
      • Chromy B.A.
      • Edwards C.
      • Freed R.
      • Liosatos M.
      • Morgan T.E.
      • Rozovsky I.
      • Trommer B.
      • Viola K.L.
      • Wals P.
      • Zhang C.
      • Finch C.E.
      • Krafft G.A.
      • Klein W.L.
      Diffusible, nonfibrillar ligands derived from Abeta1–42 are potent central nervous system neurotoxins.
      ,
      • Kayed R.
      • Head E.
      • Thompson J.L.
      • McIntire T.M.
      • Milton S.C.
      • Cotman C.W.
      • Glabe C.G.
      Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis.
      ). To evaluate the amyloid oligomers of p53C, we used the anti-oligomer antibody A11 (
      • Glabe C.G.
      Conformation-dependent antibodies target diseases of protein misfolding.
      ) and found that only the HT and HP aggregates were labeled by the antibody at pH 7.2 (Fig. 6A). It is possible that only the aggregates that formed at pH 7.2 contained a significant population of amyloid oligomeric precursors following the period chosen for incubation.
      Moreover, we found that p53C aggregates induced cell death, as other amyloidogenic proteins. Cell death occurred in the presence of fibrillar or amyloid oligomers (WT and R248Q mutant p53) (Fig. 7). Previous studies using an MTT reduction assay indicated that aggregates of WT p53C (obtained at pH 7.2) could cause cellular dysfunction in cultured macrophages (
      • Ishimaru D.
      • Andrade L.R.
      • Teixeira L.S.
      • Quesado P.A.
      • Maiolino L.M.
      • Lopez P.M.
      • Cordeiro Y.
      • Costa L.T.
      • Heckl W.M.
      • Weissmüller G.
      • Foguel D.
      • Silva J.L.
      Fibrillar aggregates of the tumor suppressor p53 core domain.
      ). N-terminal aggregates of p53 have also been shown to be cytotoxic (
      • Rigacci S.
      • Bucciantini M.
      • Relini A.
      • Pesce A.
      • Gliozzi A.
      • Berti A.
      • Stefani M.
      The (1–63) region of the p53 transactivation domain aggregates in vitro into cytotoxic amyloid assemblies.
      ). This behavior of p53 aggregates is also similar to that observed for aggregates of mammalian prion proteins (
      • Antony H
      • Wiegmans A.P.
      • Wei M.Q.
      • Chernoff Y.O.
      • Khanna K.K.
      • Munn A.L.
      Potential roles for prions and protein-only inheritance in cancer.
      ,
      • Gomes M.P.
      • Millen T.A.
      • Ferreira P.S.
      • e Silva N.L.
      • Vieira T.C.
      • Almeida M.S.
      • Silva J.L.
      • Cordeiro Y.
      Prion protein complexed to N2a cellular RNAs through its N-terminal domain forms aggregates and is toxic to murine neuroblastoma cells.
      ).
      Mutant p53 proteins often accumulate at extremely high levels in tumors (
      • Gottifredi V.
      • Prives C.
      Molecular biology. Getting p53 out of the nucleus.
      ). In fact, immunohistochemical analyses of p53 in tumors have detected mutant p53 produced by gene missense mutations, which are related to poor cancer prognoses (
      • Soussi T.
      • Béroud C.
      Assessing TP53 status in human tumours to evaluate clinical outcome.
      ). In addition, in a subset of tumors, inactive wild-type p53 is retained in the cytoplasm and impairs the transcription factor activity of the active p53 species (
      • Moll U.M.
      • LaQuaglia M.
      • Bénard J.
      • Riou G.
      Wild-type p53 protein undergoes cytoplasmic sequestration in undifferentiated neuroblastomas but not in differentiated tumors.
      ,
      • Elledge R.M.
      • Clark G.M.
      • Fuqua S.A.
      • Yu Y.Y.
      • Allred D.C.
      p53 protein accumulation detected by five different antibodies: relationship to prognosis and heat shock protein 70 in breast cancer.
      ,
      • Moll U.M.
      • Valea F.
      • Chumas J.
      Role of p53 alteration in primary peritoneal carcinoma.
      ).
      To determine whether full-length p53 would also undergo aggregation, we evaluated the p53 status in diseased tissues. Aggregates of p53 were detected in breast cancer tissue samples using an immunofluorescence co-localization assay (Fig. 8). Most interestingly, we identified the mutant R248Q protein in the amyloid-like aggregated state in a breast cancer sample expressing this hot-spot mutant (Fig. 8). In our previous work (
      • Levy C.B.
      • Stumbo A.C.
      • Ano Bom A.P.
      • Portari E.A.
      • Cordeiro Y.
      • Silva J.L.
      • De Moura-Gallo C.V.
      Co-localization of mutant p53 and amyloid-like protein aggregates in breast tumors.
      ), we demonstrated that the R273H mutant had a high propensity to form amyloid-like aggregates, whereas the hot-spot mutant R175H co-localized with amyloid-like species in very few cells. These observations suggest that p53 aggregation may be dependent on mutation type. Our in vitro and ex vivo results suggest that the mutant R248Q is prone to aggregate in tumors. Moreover, the high degree of co-localization between p53 and the aggregates also indicates that in vivo cells expressing the mutant isoform lead to the co-aggregation of the wild-type isoform, which further supports the evidence for the prion-like action of these proteins.
      The significantly higher aggregation propensity of mutant p53 was confirmed by the co-localization of full-length p53 and aggregates in tumoral cell lines (Fig. 9). Whereas there was a very faint labeling of p53 aggregates in the wild-type p53 cell line (MCF-7), there was significant labeling in the nuclei of aggregates of mutant p53 in MDA-MB 231 cells.
      Our results strongly suggest a correlation between p53 mutation and p53 aggregation in cells. We propose that the buildup and further aggregation of mutants into ordered species is caused by an inhibition of the degradation process, which may be due to defects in MDM2 protein expression or p53 ubiquitination (
      • Soussi T.
      • Béroud C.
      Assessing TP53 status in human tumours to evaluate clinical outcome.
      ,
      • Chowdary D.R.
      • Dermody J.J.
      • Jha K.K.
      • Ozer H.L.
      Accumulation of p53 in a mutant cell line defective in the ubiquitin pathway.
      ,
      • Chen L.
      • Lu W.
      • Agrawal S.
      • Zhou W.
      • Zhang R.
      Ubiquitous induction of p53 in tumor cells by antisense inhibition of MDM2 expression.
      ), as both processes are known to be involved in p53 clearance. In addition, altered cellular trafficking of p53 could lead to abnormal accumulation of p53 in the nucleus or cytoplasm, which would prevent the protein from exerting its normal functions (for a review, see Refs.
      • Gottifredi V.
      • Prives C.
      Molecular biology. Getting p53 out of the nucleus.
      and
      • Goh A.M.
      • Coffill C.R.
      • Lane D.P.
      The role of mutant p53 in human cancer.
      ). This seemed to be the case for the results obtained with tumoral cell lines harboring the R280K mutant of p53, where massive p53 aggregation in the nuclei was found (Fig. 9).
      It has been suggested that higher concentrations of p53 mutants promote a negative dominance mechanism (
      • Joerger A.C.
      • Rajagopalan S.
      • Natan E.
      • Veprintsev D.B.
      • Robinson C.V.
      • Fersht A.R.
      Structural evolution of p53, p63, and p73: implication for heterotetramer formation.
      ). According to one hypothesis, wild-type p53 molecules, which are present at a lower concentration, may form heterotetramers with mutants to result in a reduced p53 affinity for DNA (
      • Nicholls C.D.
      • McLure K.G.
      • Shields M.A.
      • Lee P.W.
      Biogenesis of p53 involves cotranslational dimerization of monomers and post-translational dimerization of dimers. Implications on the dominant negative effect.
      ). Our group has previously proposed an alternative hypothesis for the negative dominance effect, in which wild-type p53 at lower concentrations would be incorporated into aggregates containing the mutant species (
      • Ishimaru D.
      • Andrade L.R.
      • Teixeira L.S.
      • Quesado P.A.
      • Maiolino L.M.
      • Lopez P.M.
      • Cordeiro Y.
      • Costa L.T.
      • Heckl W.M.
      • Weissmüller G.
      • Foguel D.
      • Silva J.L.
      Fibrillar aggregates of the tumor suppressor p53 core domain.
      ,
      • Silva J.L.
      • Vieira T.C.
      • Gomes M.P.
      • Ano Bom A.P.
      • Lima L.M.
      • Freitas M.S.
      • Ishimaru D.
      • Cordeiro Y.
      • Foguel D.
      Ligand binding and hydration in protein misfolding: insights from studies of prion and p53 tumor suppressor proteins.
      ).
      The presence of a misfolded conformation would sequester the correctly folded form, thus suppressing function. This view is consistent with a prion-like mechanism, where the pathogenic species acts as an altered molecular chaperone to induce the correctly folded native protein to acquire the misfolded conformation, thereby increasing aggregation. In the case of p53, mutant forms may be even more susceptible to aggregation, which would amplify this process (Fig. 10). Finally, we propose that aggregation of p53 may act as a sink to sequestrate the native protein into the inactive conformation via a mechanism typical of a prionoid (
      • Aguzzi A.
      • Rajendran L.
      The transcellular spread of cytosolic amyloids, prions, and prionoids.
      ,
      • Frost B.
      • Diamond M.I.
      Prion-like mechanisms in neurodegenerative diseases.
      ).
      Figure thumbnail gr10
      FIGURE 10Schematic model for the prionoid conversion and negative dominance of mutant p53. The native conformations of WT and R248Q p53 are represented as green and orange molecules, respectively. The misfolded conformation of either molecule is represented in purple. According to the model, the prion-like character responsible for the negative dominance effect would occur in the oligomers.
      The observation that WT and mutant p53 forms aggregate as amyloids, which are associated with the negative dominant effect, adds an amyloid characteristic to cancer. In a recent review article, Antony et al. (
      • Antony H
      • Wiegmans A.P.
      • Wei M.Q.
      • Chernoff Y.O.
      • Khanna K.K.
      • Munn A.L.
      Potential roles for prions and protein-only inheritance in cancer.
      ) discussed the potential role of prions and protein-only inheritance in cancer. They argue how somatic inheritance in mammalian cells (including p53) may contribute to cancer phenotypes, and these authors also stress how the involvement of prion-like mechanisms in cancer can lead to novel therapeutic targets.
      Fig. 10 presents a schematic diagram for how misfolded p53 could divert native protein into aggregates and how the mutant form, with its greater propensity for aggregation, would lead to a negative dominance effect. This diagram has evolved from a previous proposal (
      • Ishimaru D.
      • Andrade L.R.
      • Teixeira L.S.
      • Quesado P.A.
      • Maiolino L.M.
      • Lopez P.M.
      • Cordeiro Y.
      • Costa L.T.
      • Heckl W.M.
      • Weissmüller G.
      • Foguel D.
      • Silva J.L.
      Fibrillar aggregates of the tumor suppressor p53 core domain.
      ) by incorporating the results demonstrating the seeding potential of R248Q aggregates and our findings that both wild-type and mutant p53 form heterogeneous mixtures of amyloid oligomers and amyloid fibrils. Heterooligomerization is more likely to occur in smaller aggregates, and the formation of fibrils leads the system toward fewer reversible species. Thus, in contrast to previous proposals (
      • Ishimaru D.
      • Andrade L.R.
      • Teixeira L.S.
      • Quesado P.A.
      • Maiolino L.M.
      • Lopez P.M.
      • Cordeiro Y.
      • Costa L.T.
      • Heckl W.M.
      • Weissmüller G.
      • Foguel D.
      • Silva J.L.
      Fibrillar aggregates of the tumor suppressor p53 core domain.
      ,
      • Xu J.
      • Reumers J.
      • Couceiro J.R.
      • De Smet F.
      • Gallardo R.
      • Rudyak S.
      • Cornelis A.
      • Rozenski J.
      • Zwolinska A.
      • Marine J.C.
      • Lambrechts D.
      • Suh Y.A.
      • Rousseau F.
      • Schymkowitz J.
      Gain of function of mutant p53 by coaggregation with multiple tumor suppressors.
      ), the heterogeneous character of the amyloid aggregates is the key feature that leads to the negative dominance effect, not only the tendency to form fibrils. This feature also likely explains why the anti-oligomer antibody bound a significant amount of targets in tumor tissues containing the R248Q mutation.
      At low pH, there was less formation of fibrillar p53 species. Aggregation at acidic pH may occur in some cellular compartments, such as lysosomes, endosomes, and proteasomes, which are associated with protein translocation and degradation. It has been reported that p53 adopts a molten-globule state at low pH (
      • Bom A.P.
      • Freitas M.S.
      • Moreira F.S.
      • Ferraz D.
      • Sanches D.
      • Gomes A.M.
      • Valente A.P.
      • Cordeiro Y.
      • Silva J.L.
      The p53 core domain is a molten globule at low pH: functional implications of a partially unfolded structure.
      ) and would therefore have a lower tendency toward aggregation. It is noteworthy that the molten-globule state of the p53 DNA-binding domain is the client conformation for interaction with the chaperone Hsp90 (
      • Park S.J.
      • Borin B.N.
      • Martinez-Yamout M.A.
      • Dyson H.J.
      The client protein p53 adopts a molten globule-like state in the presence of Hsp90.
      ).
      The prionoid character of mutant p53 is also a potential target for therapeutic action. Aptameric nucleic acids and glycosaminoglycans have been evaluated as drug candidates against mammalian prions (
      • Kocisko D.A.
      • Vaillant A.
      • Lee K.S.
      • Arnold K.M.
      • Bertholet N.
      • Race R.E.
      • Olsen E.A.
      • Juteau J.M.
      • Caughey B.
      Potent antiscrapie activities of degenerate phosphorothioate oligonucleotides.
      ,
      • Caughey B.
      • Caughey W.S.
      • Kocisko D.A.
      • Lee K.S.
      • Silveira J.R.
      • Morrey J.D.
      Prions and transmissible spongiform encephalopathy (TSE) chemotherapeutics: A common mechanism for anti-TSE compounds?.
      ,
      • Vieira T.C.
      • Reynaldo D.P.
      • Gomes M.P.
      • Almeida M.S.
      • Cordeiro Y.
      • Silva J.L.
      Heparin binding by murine recombinant prion protein leads to transient aggregation and formation of RNA-resistant species.
      ) and therefore represent strong candidates to tackle p53 prion-like aggregation. Moreover, complementary studies may reveal the biological and clinical importance of p53 aggregates and help to develop new strategies for intervening against aggregation formation.

      Acknowledgments

      We thank Martha M. Sorenson for carefully reading the manuscript and providing helpful suggestions. We also thank Mariana P. B. Gomes for suggestions and aid in the drawing of Fig. 10.

      REFERENCES

        • Vousden K.H.
        • Lane D.P.
        p53 in health and disease.
        Nat. Rev. Mol. Cell Biol. 2007; 8: 275-283
        • Joerger A.C.
        • Fersht A.R.
        Structural biology of the tumor suppressor p53.
        Annu. Rev. Biochem. 2008; 77: 557-582
        • Ishimaru D.
        • Andrade L.R.
        • Teixeira L.S.
        • Quesado P.A.
        • Maiolino L.M.
        • Lopez P.M.
        • Cordeiro Y.
        • Costa L.T.
        • Heckl W.M.
        • Weissmüller G.
        • Foguel D.
        • Silva J.L.
        Fibrillar aggregates of the tumor suppressor p53 core domain.
        Biochemistry. 2003; 42: 9022-9027
        • Silva J.L.
        • Vieira T.C.
        • Gomes M.P.
        • Ano Bom A.P.
        • Lima L.M.
        • Freitas M.S.
        • Ishimaru D.
        • Cordeiro Y.
        • Foguel D.
        Ligand binding and hydration in protein misfolding: insights from studies of prion and p53 tumor suppressor proteins.
        Acc. Chem. Res. 2010; 43: 271-279
        • Galea C.
        • Bowman P.
        • Kriwacki R.W.
        Disruption of an intermonomer salt bridge in the p53 tetramerization domain results in an increased propensity to form amyloid fibrils.
        Prot. Sci. 2005; 14: 2993-3003
        • Higashimoto Y.
        • Asanomi Y.
        • Takakusagi S.
        • Lewis M.S.
        • Uosaki K.
        • Durell S.R.
        • Anderson C.W.
        • Appella E.
        • Sakaguchi K.
        Unfolding, aggregation, and amyloid formation by the tetramerization domain from mutant p53 associated with lung cancer.
        Biochemistry. 2006; 45: 1608-1619
        • Rigacci S.
        • Bucciantini M.
        • Relini A.
        • Pesce A.
        • Gliozzi A.
        • Berti A.
        • Stefani M.
        The (1–63) region of the p53 transactivation domain aggregates in vitro into cytotoxic amyloid assemblies.
        Biophys. J. 2008; 94: 3635-3646
        • Ishimaru D.
        • Ano Bom A.P.
        • Lima L.M.
        • Quesado P.A.
        • Oyama M.F.
        • de Moura Gallo C.V.
        • Cordeiro Y.
        • Silva J.L.
        Cognate DNA stabilizes the tumor suppressor p53 and prevents misfolding and aggregation.
        Biochemistry. 2009; 48: 6126-6135
        • Levy C.B.
        • Stumbo A.C.
        • Ano Bom A.P.
        • Portari E.A.
        • Cordeiro Y.
        • Silva J.L.
        • De Moura-Gallo C.V.
        Co-localization of mutant p53 and amyloid-like protein aggregates in breast tumors.
        Int. J. Biochem. Cell Biol. 2011; 43: 60-64
        • Xu J.
        • Reumers J.
        • Couceiro J.R.
        • De Smet F.
        • Gallardo R.
        • Rudyak S.
        • Cornelis A.
        • Rozenski J.
        • Zwolinska A.
        • Marine J.C.
        • Lambrechts D.
        • Suh Y.A.
        • Rousseau F.
        • Schymkowitz J.
        Gain of function of mutant p53 by coaggregation with multiple tumor suppressors.
        Nat. Chem. Biol. 2011; 7: 285-295
        • Chiti F.
        • Dobson C.M.
        Protein misfolding, functional amyloid, and human disease.
        Annu. Rev. Biochem. 2006; 75: 333-366
        • Pastore A.
        • Temussi P.A.
        The two faces of Janus: functional interactions and protein aggregation.
        Curr. Opin. Struct. Biol. 2012; 22: 30-37
        • Butler J.S.
        • Loh S.N.
        Structure, function, and aggregation of the zinc-free form of the p53 DNA binding domain.
        Biochemistry. 2003; 42: 2396-2403
        • Antony H
        • Wiegmans A.P.
        • Wei M.Q.
        • Chernoff Y.O.
        • Khanna K.K.
        • Munn A.L.
        Potential roles for prions and protein-only inheritance in cancer.
        Cancer Metastasis Rev. 2011; 31: 1-19
        • Hollstein M.
        • Sidransky D.
        • Vogelstein B.
        • Harris C.C.
        p53 mutations in human cancers.
        Science. 1991; 253: 49-53
        • Olivier M.
        • Hollstein M.
        • Hainaut P.
        TP53 mutations in human cancers: origins, consequences, and clinical use.
        Cold Spring Harb. Perspect. Biol. 2010; 2: a001008
        • Cordeiro Y.
        • Kraineva J.
        • Gomes M.P.
        • Lopes M.H.
        • Martins V.R.
        • Lima L.M.
        • Foguel D.
        • Winter R.
        • Silva J.L.
        The amino-terminal PrP domain is crucial to modulate prion misfolding and aggregation.
        Biophys. J. 2005; 89: 2667-2676
        • Cordeiro Y.
        • Kraineva J.
        • Ravindra R.
        • Lima L.M.
        • Gomes M.P.
        • Foguel D.
        • Winter R.
        • Silva J.L.
        Hydration and packing effects on prion folding and β-sheet conversion. High pressure spectroscopy and pressure perturbation calorimetry studies.
        J. Biol. Chem. 2004; 279: 32354-33259
        • Glabe C.G.
        Conformation-dependent antibodies target diseases of protein misfolding.
        Trends Biochem. Sci. 2004; 29: 542-547
        • Lai Z.
        • Colón W.
        • Kelly J.W.
        The acid-mediated denaturation pathway of transthyretin yields a conformational intermediate that can self-assemble into amyloid.
        Biochemistry. 1996; 35: 6470-6482
        • Howie A.J.
        • Brewer D.B.
        • Howell D.
        • Jones A.P.
        Physical basis of colors seen in Congo red-stained amyloid in polarized light.
        Lab. Invest. 2008; 88: 232-242
        • Moll U.M.
        • LaQuaglia M.
        • Bénard J.
        • Riou G.
        Wild-type p53 protein undergoes cytoplasmic sequestration in undifferentiated neuroblastomas but not in differentiated tumors.
        Proc. Natl. Acad. Sci. U.S.A. 1995; 92: 4407-4411
        • Ostermeyer A.G.
        • Runko E.
        • Winkfield B.
        • Ahn B.
        • Moll U.M.
        Cytoplasmically sequestered wild-type p53 protein in neuroblastoma is relocated to the nucleus by a C-terminal peptide.
        Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 15190-15194
        • Bom A.P.
        • Freitas M.S.
        • Moreira F.S.
        • Ferraz D.
        • Sanches D.
        • Gomes A.M.
        • Valente A.P.
        • Cordeiro Y.
        • Silva J.L.
        The p53 core domain is a molten globule at low pH: functional implications of a partially unfolded structure.
        J. Biol. Chem. 2010; 285: 2857-2866
        • Gerweck L.E.
        Tumor pH: implications for treatment and novel drug design.
        Semin. Radiat. Oncol. 1998; 8: 176-182
        • Gomes M.P.
        • Millen T.A.
        • Ferreira P.S.
        • e Silva N.L.
        • Vieira T.C.
        • Almeida M.S.
        • Silva J.L.
        • Cordeiro Y.
        Prion protein complexed to N2a cellular RNAs through its N-terminal domain forms aggregates and is toxic to murine neuroblastoma cells.
        J. Biol. Chem. 2008; 283: 19616-19625
        • Ishimaru D.
        • Lima L.M.
        • Maia L.F.
        • Lopez P.M.
        • Ano Bom A.P.
        • Valente A.P.
        • Silva J.L.
        Reversible aggregation plays a crucial role on the folding landscape of p53 core domain.
        Biophys. J. 2004; 87: 2691-2700
        • Sunde M.
        • Serpell L.C.
        • Bartlam M.
        • Fraser P.E.
        • Pepys M.B.
        • Blake C.C.
        Common core structure of amyloid fibrils by synchrotron X-ray diffraction.
        J. Mol. Biol. 1997; 273: 729-739
        • Novitskaya V.
        • Bocharova O.V.
        • Bronstein I.
        • Baskakov I.V.
        Amyloid fibrils of mammalian prion protein are highly toxic to cultured cells and primary neurons.
        J. Biol. Chem. 2006; 281: 13828-13836
        • Vieira M.N.
        • Forny-Germano L.
        • Saraiva L.M.
        • Sebollela A.
        • Martinez A.M.
        • Houzel J.C.
        • De Felice F.G.
        • Ferreira S.T.
        Soluble oligomers from a non-disease related protein mimic Aβ-induced tau hyperphosphorylation and neurodegeneration.
        J. Neurochem. 2007; 103: 736-748
        • Gomes M.P.
        • Cordeiro Y.
        • Silva J.L.
        The peculiar interaction between mammalian prion protein and RNA.
        Prion. 2008; 2: 64-66
        • Elledge R.M.
        • Clark G.M.
        • Fuqua S.A.
        • Yu Y.Y.
        • Allred D.C.
        p53 protein accumulation detected by five different antibodies: relationship to prognosis and heat shock protein 70 in breast cancer.
        Cancer Res. 1994; 54: 3752-3757
        • Moll U.M.
        • Valea F.
        • Chumas J.
        Role of p53 alteration in primary peritoneal carcinoma.
        Int. J. Gynecol. Pathol. 1997; 16: 156-162
        • Lambert M.P.
        • Barlow A.K.
        • Chromy B.A.
        • Edwards C.
        • Freed R.
        • Liosatos M.
        • Morgan T.E.
        • Rozovsky I.
        • Trommer B.
        • Viola K.L.
        • Wals P.
        • Zhang C.
        • Finch C.E.
        • Krafft G.A.
        • Klein W.L.
        Diffusible, nonfibrillar ligands derived from Abeta1–42 are potent central nervous system neurotoxins.
        Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 6448-6453
        • Kayed R.
        • Head E.
        • Thompson J.L.
        • McIntire T.M.
        • Milton S.C.
        • Cotman C.W.
        • Glabe C.G.
        Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis.
        Science. 2003; 300: 486-489
        • Gottifredi V.
        • Prives C.
        Molecular biology. Getting p53 out of the nucleus.
        Science. 2001; 292: 1851-1852
        • Soussi T.
        • Béroud C.
        Assessing TP53 status in human tumours to evaluate clinical outcome.
        Nat. Rev. Cancer. 2001; 1: 233-240
        • Chowdary D.R.
        • Dermody J.J.
        • Jha K.K.
        • Ozer H.L.
        Accumulation of p53 in a mutant cell line defective in the ubiquitin pathway.
        Mol. Cell Biol. 1994; 14: 1997-2003
        • Chen L.
        • Lu W.
        • Agrawal S.
        • Zhou W.
        • Zhang R.
        Ubiquitous induction of p53 in tumor cells by antisense inhibition of MDM2 expression.
        Mol. Med. 1999; 5: 21-34
        • Goh A.M.
        • Coffill C.R.
        • Lane D.P.
        The role of mutant p53 in human cancer.
        J. Pathol. 2011; 223: 116-126
        • Joerger A.C.
        • Rajagopalan S.
        • Natan E.
        • Veprintsev D.B.
        • Robinson C.V.
        • Fersht A.R.
        Structural evolution of p53, p63, and p73: implication for heterotetramer formation.
        Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 17705-17710
        • Nicholls C.D.
        • McLure K.G.
        • Shields M.A.
        • Lee P.W.
        Biogenesis of p53 involves cotranslational dimerization of monomers and post-translational dimerization of dimers. Implications on the dominant negative effect.
        J. Biol. Chem. 2002; 277: 12937-12945
        • Aguzzi A.
        • Rajendran L.
        The transcellular spread of cytosolic amyloids, prions, and prionoids.
        Neuron. 2009; 64: 783-790
        • Frost B.
        • Diamond M.I.
        Prion-like mechanisms in neurodegenerative diseases.
        Nat. Rev. Neurosci. 2010; 11: 155-159
        • Park S.J.
        • Borin B.N.
        • Martinez-Yamout M.A.
        • Dyson H.J.
        The client protein p53 adopts a molten globule-like state in the presence of Hsp90.
        Nat. Struct. Mol. Biol. 2011; 18: 537-541
        • Kocisko D.A.
        • Vaillant A.
        • Lee K.S.
        • Arnold K.M.
        • Bertholet N.
        • Race R.E.
        • Olsen E.A.
        • Juteau J.M.
        • Caughey B.
        Potent antiscrapie activities of degenerate phosphorothioate oligonucleotides.
        Antimicrob. Agents Chemother. 2006; 50: 1034-1044
        • Caughey B.
        • Caughey W.S.
        • Kocisko D.A.
        • Lee K.S.
        • Silveira J.R.
        • Morrey J.D.
        Prions and transmissible spongiform encephalopathy (TSE) chemotherapeutics: A common mechanism for anti-TSE compounds?.
        Acc. Chem. Res. 2006; 39: 646-653
        • Vieira T.C.
        • Reynaldo D.P.
        • Gomes M.P.
        • Almeida M.S.
        • Cordeiro Y.
        • Silva J.L.
        Heparin binding by murine recombinant prion protein leads to transient aggregation and formation of RNA-resistant species.
        J. Am. Chem. Soc. 2010; 133: 334-344