Flexibility: the key to p53 function?
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Models, Molecular↗
Biomedical subjects
J Milner
Publications and source records attributed to J Milner.
Temperature sensitivity for conformation is an intrinsic property of wild-type p53.
The tumour-suppressor protein p53 is a metal-binding transcription factor with sequence-specific DNA-binding capacity. In cancer, mutation of p53 disrupts protein conformation with consequent loss of DNA binding and associated tumour-suppressor function. In vitro, the conformation and DNA-binding activity of wild-type p53 are subject to redox modulation and are abrogated by exposure to metal chelators. In the present study, we have used the chelator 1, 10-phenanthroline (OP) to probe the effect of temperature on the conformational stability of p53 translated in vitro. Whereas low temperature (30 degrees C) stabilised wild-type p53 conformation and protected against chelation, high temperature (41 degrees C) promoted destabilisation and enhanced chelation, indicating that temperature influences the folding of wild-type p53. Destabilisation of p53 tertiary structure induced protein aggregation through hydrophobic interactions, consistent with the notion that wild-type p53 contains a hydrophobic core which may become exposed by metal chelation. These results indicate that temperature sensitivity for conformation is an intrinsic property of wild-type p53 and suggests that small changes in temperature may directly affect p53 function.
3T3 Cells↗
DNA damage, p53 and anticancer therapies.
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Animals↗
The growth of Malawian preschool children from different socioeconomic groups.
OBJECTIVE: The growth of Malawian preschool children from different socioeconomic groups was examined to determine the relevance of the NCHS/WHO growth reference data for assessing child nutritional status in Malawi. DESIGN: The study involved a comparison of anthropometric data from three cross-sectional surveys of preschool children over 24 months of age. SETTING: Malawi, Central Africa. SUBJECTS: Anthropometric measurements were taken on high income Malawian children (n = 380) during a census of affluent preschools in the country's three major urban centres. Comparative data were obtained from two existing sample surveys of low income urban children (n = 225) and rural village children (n = 667). RESULTS: The distribution of weight-for-age Z-scores (HAZ) for the high income children 24-35 months of age closely resembled the NCHS/WHO child reference population (mean HAZ = -0.21; SD = 1.05). After this age HAZ decreased to a mean value of -0.58 between 60 and 71 months. Large differences in growth were observed between children from different socioeconomic groups. Regression analysis showed that at 24 months the high income children were, on average, 6.6 cm taller than the low income urban children (P < 0.001), and 9.2 cm taller than the rural children (P < 0.001). By 59 months of age these differences increased to 9.6 cm and 11.1 cm, respectively. CONCLUSIONS: Although some questions remain regarding the growth potential of Malawian children, the results suggest the NCHS/WHO reference data are relevant for Malawi and the high levels of child stunting found in the country are due to environmental not genetic factors.
Body Height↗
Comparative study of the enzyme activities of Borrelia burgdorferi and other non-intestinal and intestinal spirochaetes.
Comparative analysis of the enzymatic profiles of 58 spirochaetal isolates clearly differentiated borrelias from leptospires, serpulinas and a treponeme. Strains of both Borrelia burgdorferi and Borrelia hermsii characteristically produced significant amounts of leucine arylamidase. This enzyme activity was not unique to borrelias but was also detected amongst pathogenic and non-pathogenic leptospira serovars. This fact, however, did not hamper a correct differentiation of borrelias from these spirochaetes, because leptospires possessed unique enzyme profiles. The API ZYM system could not differentiate the human strains of B. burgdorferi from those isolated from ticks, or from B. hermsii. Treponema phagedenis could be differentiated from all the other spirochaetes by the production of alpha-fucosidase. Our results confirm and extend previous studies indicating that human and animal intestinal spirochaetes have many common enzyme activities. All strains produced reactions of maximum intensity when tested for the presence of beta-galactosidase activity. However the avian strains lacked esterase (C4) which was present in human and swine intestinal spirochaetes. All strains of Serpulina hyodysenteriae, and Serpulina innocens as well as the human intestinal spirochaete strain HRM-14 showed alpha and beta glucosidase activity. Both enzyme activities were absent or insignificant in most other intestinal spirochaetes examined: 25 different human strains, non-pathogenic swine strain M1 and the avian strain 4742. However, swine strain LL3 and avian strain 1380 showed some beta-glucosidase activity.
Animals↗
Increased salt concentration reversibly destabilizes p53 quaternary structure and sequence-specific DNA binding.
Growth suppression by p53 correlates with sequence-specific DNA binding and is determined by tertiary and quaternary protein structures. Exposure to 300 mM NaCl did not affect p53 tertiary structure, but dissociated high-molecular-mass complexes with concomitant loss of specific DNA binding. Both effects were reversible. We conclude that high salt can reversibly destabilize the quaternary structure of p53 that is most efficient for sequence-specific DNA binding.
Animals↗
Forms and functions of p53.
There is evidence that wild-type p53 can both promote and suppress cell proliferation and these opposing functions correlate with alternative conformations of the p53 protein. In the light of more recent evidence I now propose that wild-type p53 can adopt at least three different forms, each of which correlates with a defined function in cell growth control. The three forms are most simply defined by reactivity with two monoclonal antibodies, PAb421 and PAb1620. One form (421 degrees/1620+) suppresses cell growth and maintains quiescence, and a second form (421+/1620 degrees) activates and promotes cell proliferation. The third form (421+/1620+) acts as a differential sensor for incoming positive and negative growth regulatory signals and can be converted either to the suppressor or to the promoter form for cell growth. Regulation of p53 tertiary structure involves redox modulation and phosphorylation, and offers a novel rationale for anti-cancer therapy.
Animals↗
Specific DNA binding by p53 is independent of mutation at serine 389, the casein kinase II site.
The best understood function of p53 is that of cell growth suppression and this is likely to involve sequence-specific DNA binding and modulation of gene expression. Casein kinase II phosphorylates the C-terminal serine of p53 (residue 389 for murine p53) and mutation of this site abolishes p53 growth suppressor function. DNA binding by purified p53 is 'activated' by casein kinase II, suggesting that the carboxyl terminus of p53 represents a critical regulatory domain for sequence-specific DNA binding and hence for growth suppressor function. In the present study we have substituted serine 389 with either aspartic acid (mimics phosphoserine and partially conserves p53 suppressor function) or with alanine, a non-phosphorylable residue which abolishes suppressor function (Milne et al., 1992; Nucleic Acids Research 20, 5565-5570). When expressed in vitro p53ala389 and p53asp389 were both indistinguishable from wild type p53 on the basis of size fractionation and immunoreactivity with PAb421, PAb246 and PAb1620. Both mutants also exhibited specific binding for the DNA consensus p53-CON. Since p53ala389 retains the ability to bind DNA and yet is known to lack growth suppressor function we conclude that phosphorylation by casein kinase II is important for p53 growth suppressor function via a mechanism which is ancillary to p53 sequence-specific DNA binding.
Animals↗
Analysis of p53 quaternary structure in relation to sequence-specific DNA binding.
Quaternary interactions of p53 influence its tertiary structure which, in turn, is critical for sequence-specific DNA binding and tumour suppressor function. Given its regulatory potential we have sought to define the quaternary structure of p53 involved in sequence-specific DNA binding. Double stranded DNA [5'-GGACATGCCCGGGCATGTCC-3'; Funk et al. (1992) Mol. Cell. Biol., 12, 2866-2871] was used to test p53 binding capacity in vitro. The p53 protein was translated in vitro and size fractionated prior to the DNA binding reaction. Two independent DNA binding assays were employed. The first detected electromobility shift of 32P-labelled DNA and was carried out in the presence of PAb421, which stabilises and supershifts p53-DNA complexes. The second detected 35S-labelled p53 bound to biotinylated target DNA in the absence of PAb421. Sequence-specific DNA binding was found to be a property of full length, oligomeric p53. Greatest binding activity involved tetramers and/or higher molecular weight forms of p53, minimal binding was observed for dimers. This size profile was unaffected by PAb421 and it therefore seems unlikely that PAb421 dissociates high molecular weight forms of p53 into dimers. We conclude that high molecular weight forms of p53 are the most effective structures for sequence-specific DNA binding in vitro; these structures may represent tetramers and/or heterogeneous complexes of p53 with other proteins.
Animals↗
Redox modulation of p53 conformation and sequence-specific DNA binding in vitro.
The p53 protein is a transcription factor, the function of which is abrogated by oncogenic mutations which affect a flexible domain in the central portion of p53, altering its reactivity with conformation-specific antibodies. Here we show that both conformation and sequence-specific DNA binding of p53 translated in vitro can be modulated by metal chelators and oxidizing agents. Oxidation disrupted wild-type p53 conformation and inhibited DNA binding. Conversely, reduction favored folding of p53 into the wild-type form and restored DNA binding. Redox regulation of p53 protein conformation could represent an important mechanism for the control of p53 function.
Amino Acid Sequence↗
A structural role for metal ions in the "wild-type" conformation of the tumor suppressor protein p53.
In human tumors, many different point mutations of the p53 gene knock out suppressor function and induce the p53 polypeptide to adopt an immunologically distinct, "mutant" conformation. Here we show that exposure to the metal chelator 1,10-phenanthroline induces wild-type p53 to adopt the mutant conformation and that this process is reversible. Conversion to mutant phenotype also occurs after exposure to (a) an organic mercurial reagent targeting cysteinyl residues and (b) low concentrations of mercury or cadmium. We propose that binding of metal ions, most probably zinc, to conserved cysteinyl residues stabilizes the tertiary structure of wild-type p53.
Amino Acid Sequence↗
Tight DNA binding and oligomerization are dispensable for the ability of p53 to transactivate target genes and suppress transformation.
The p53 tumor suppressor protein can bind tightly to specific sequence elements in the DNA and induce the transactivation of genes harboring such p53 binding sites. Various lines of evidence suggest that p53 binds to its target site as an oligomer. To test whether oligomerization is essential for the biological and biochemical activities of p53, we deleted a major part of the dimerization domain of mouse wild-type p53. The resultant protein, termed p53wt delta SS, was shown to be incapable of forming detectable homo-oligomers in vitro and is, therefore, likely to be predominantly if not exclusively monomeric. In agreement with the accepted model, p53wt delta SS indeed failed to exhibit measurable DNA binding in vitro. Surprisingly, though, it was still capable of suppressing oncogene-mediated transformation and of transactivating in vivo a target gene containing p53 binding sites. These findings indicate that dimerization-defective p53 is biologically active and may engage in productive sequence-specific DNA interactions in vivo. Furthermore, p53 dimerization probably leads to cooperative binding to specific DNA sequences.
Amino Acid Sequence↗
Targeting and degradation of p53 by E6 of human papillomavirus type 16 is preferential for the 1620+ p53 conformation.
E6-mediated degradation of p53 is believed to play a role in the transformation of cells by high-risk types of human papillomavirus. In order to explore the structural requirements for targeting of p53 we have compared E6-mediated degradation of variant p53 forms expressed in vitro. Complete degradation was observed in samples containing monomers, dimers and higher molecular weight structures of wild-type p53, indicating that E6 targets all quaternary forms of wild-type p53. Wild-type human and murine p53s reactive with PAb 1620 (which recognizes a conformation-dependent epitope) were degraded when incubated with E6. Mutant p53 proteins were variably resistant to E6-mediated degradation, and this correlated with PAb 1620 reactivity. Thus, mutants hp53Val-154, hp53Val-266 and hp53Pro-273 (1620 degrees) were completely resistant to degradation, whereas hp53Ile-247 and hp53Trp-248 (1620+) were degraded. Mutants hp53Leu-273 and mp53Val-135, which are temperature sensitive for conformation, were completely degraded in the 1620+ form but degradation resistant in the 1620 degrees form. Although the PAb 1620+ conformation appeared important for recognition of p53 by E6, the epitope itself is unlikely to be the actual recognition target since the PAb 1620 monoclonal antibody failed to protect against E6-mediated degradation.
Epitopes↗
Partially transformed T3T3 cells express high levels of mutant p53 in the 'wild-type' immunoreactive form with defective oligomerization.
High levels of wild-type p53 suppress transformed growth of many cell lines and yet murine T3T3 cells shown partially transformed growth despite high endogenous levels of phenotypically 'wild-type' p53. On sequencing T3T3 p53 was found to encode missense mutations at codons 230 and 287 and, although endogenous T3T3 p53 is 'wild type', the protein adopted the mutant phenotype when expressed in vitro. Size fractionation of T3T3 cell lysate indicated monomeric p53 possibly in complex with a low molecular weight protein. When expressed in vitro T3T3 p53 formed dimers and higher order structures. Thus T3T3 cells appear (i) to drive endogenous mutant p53 to adopt conformational epitopes characteristic of the 'wild-type' protein, and (ii) to interfere with normal assembly of p53 quaternary structure. Phosphopeptide mapping of p53 from 3T3x cells, T3T3 cells and SV3T3 cells indicated reduced amino terminal phosphorylation of the mutant p53 phenotype. Alternative splicing of p53 was also detected in 3T3x cells; similar splicing occurs in wild-type p53 (Han & Kulesz-Martin, 1992; Nucl. Acids Res., 20, 1979-1981) and a possible regulatory function is discussed.
3T3 Cells↗
Interaction of heat-shock protein 70 with p53 translated in vitro: evidence for interaction with dimeric p53 and for a role in the regulation of p53 conformation.
In intact cells, hsp70 proteins selectively complex with mutant p53. We report here that rabbit reticulocyte lysate contains hsp70 which selectively complexes with the mutant p53 translated in vitro. Hsp70 complexes with dimers and possibly monomers of p53 in a manner that requires the terminal 28 amino acids of p53. Using murine p53Val135, which is temperature-sensitive for phenotype, we demonstrate that p53-hsp70 complexes can occur after post-translational switching from wild-type to mutant p53 phenotype. Moreover, the temperature-induced switch of full-length p53Val135 from wild-type to mutant phenotype is ATP-independent, whereas the switch from mutant to wild-type form requires ATP hydrolysis and involves hsp70. These results imply that hsp70 is involved in the regulation of p53 conformation.
Adenosine Triphosphate↗
Inhibition of the serine/threonine protein phosphatases PP1 and PP2A in lymphocytes: effect on mRNA levels for interleukin-2, IL-2R alpha, krox-24, p53, hsc70 and cyclophilin.
Lymphocyte activation requires signal transduction mediated by reversible phosphorylation. Changing profiles of phosphorylated intermediates relate to the progressive series of transduction pathways in cells moving from G0 to G1, and thereafter through the cell cycle. We have previously shown that transient inhibition of the serine/threonine protein phosphatases PP1 and PP2A by okadaic acid enhances early mitogenic stimulation. Thus target proteins of PP1/PP2A may be involved in regulation of early mitogenic signalling, with the phosphorylated form(s) being associated with signal enhancement. Later, pathways require dephosphorylation of these proteins, since continuous treatment with okadaic acid blocks lymphocyte progression through the cell cycle. Delayed addition of okadaic acid showed that this blockade occurs between 8 and 24 hr. Here we have furthered these observations to the level of gene induction by measuring messenger RNA (mRNA) levels for the following proteins: interleukin-2 (IL-2) and IL-2R alpha; p53, a tumour suppressor protein; the transcription factor krox-24; and two mediators of protein folding, namely cyclophilin and the heat-shock protein hsc70. An external standard was used to quantitate the mRNA levels per cell. We found that 24 hr exposure to okadaic acid has a general suppressive effect on concanavalin A (Con A)-stimulated gene induction. However, at 4 hr okadaic acid enhanced IL-2 mRNA levels induced by Con A. Moreover, in unstimulated lymphocytes, okadaic acid caused the induction of krox-24, indicating a role for PP1 and PP2A in the regulation of this gene in resting cells.
Amino Acid Isomerases↗
Correlation between the conformational phenotype of p53 and its subcellular location.
In order to obtain insight into the parameters determining the subcellular localization of mutant and wild-type forms of p53, we analysed the subcellular distribution of p53 in four Balb/c mouse-derived cell lines ranging in their cellular phenotypes from normal (3T3), via minimal transformant (T3T3), to maximally transformed (3T3tx, Meth A). Epitope mapping showed the p53 proteins in 3T3 and in T3T3 cells to be in a wild-type conformation, as they reacted with PAb246, whereas p53 in 3T3tx and in Meth A cells were PAb246 negative and thus displayed a mutant conformation. Despite its reactivity with PAb246, p53 in T3T3 cells had an extended half-life and accumulated to abnormally high levels. We show that the conformationally wild-type p53 in 3T3 and T3T3 cells predominantly localized to the cell nucleus, with about half of it being tightly associated with nuclear structures. In contrast, approximately 60% of mutant p53 in 3T3tx and Meth A cells localized to the cytoplasm, the rest residing in the cell nucleus; all the nuclear p53 in these cells appeared to be structurally bound. The cytoplasmic location of mutant p53 in 3T3tx and Meth A cells was not seen by immunofluorescence microscopic analysis, and required cell fractionation for its detection. Both cytoplasmic and nuclear p53 of the mutant phenotype bound to hsc proteins with a similar stoichiometry, suggesting that hsc binding is not directly related to the subcellular distribution of these proteins. We suggest that the conformational phenotype of p53 is a major determinant of its subcellular location.
Animals↗
Temperature-sensitive mutants of p53 associated with human carcinoma of the lung.
We have compared the effects of specific point mutations on the tertiary and quaternary structure of the human p53 protein. Eight mutants, each derived from primary resected tissues of lung carcinomas, were expressed in vitro under strictly defined conditions, such that the only known variant was the point mutation present in each p53 mRNA. All the mutations were located in highly conserved domains. The tertiary structure of each mutant protein was investigated by reactivity with anti-p53 monoclonal antibodies directed against conformation-dependent epitopes. Quaternary structure was examined by gel filtration. Although all the mutant proteins exhibited abnormal tertiary structures, their quaternary structures appeared similar to wild type, the one exception being p53-tyr135, which contains tyrosine in place of cysteine at residue 135. The conformational phenotype of mutant human p53 was found to be dependent upon (i) the locus of the mutation and (ii) the nature of the amino acid substitution: two different substitutions at residue 273 yielded two mutants with differing structural properties. We have discovered three mutants of human p53 that are temperature sensitive for conformation; one is mutated at codon 273, a 'hotspot' for p53 mutation in human cancer.
Carcinoma↗