Transcriptional activation functions in BRCA2.
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Amino Acid Sequence↗
Biomedical subjects
J Milner
Publications and source records attributed to J Milner.
Specific p53-DNA complexes contain an mdm2-related protein.
The mdm2 gene encodes a family of proteins, a subset of which bind p53 and negatively regulate its function as a transcription factor. We now show that an anti-mdm-2 monoclonal antibody, 2A10, recognises a protein present in rabbit reticulocyte lysate which binds murine p53 translated in vitro. Deletion of p53 residues 10-35, which encompass the mdm-2 binding site, abolished binding of this 2A10-reactive protein. Binding was also dependent upon p53 protein conformation and may require nascent p53 polypeptide since binding was lost following conformational shifting of the temperature-sensitive mutant A135V. Previous studies have shown that mdm-2-p53 complexes fail to exhibit detectable sequence-specific DNA binding. However, our present results demonstrate that p53 in complex with an mdm-2-related protein in vitro retained sequence-specific DNA binding capacity. Non-transformed (but not transformed) 3T3 cells were also found to express a similar 2A10-reactive protein, detectable by gel shift analysis of cellular p53 in complex with a specific DNA target. Mdm-2 in rabbit reticulocyte lysate and in normal, non-transformed 3T3 cells may represent constitutively expressed protein. Our results raise the possibility that constitutive mdm-2 may enhance and/or suppress functions of p53 as yet unidentified.
3T3 Cells↗
Proteolysis by calpains: a possible contribution to degradation of p53.
p53 is a short-lived transcription factor that is frequently mutated in tumor cells. Work by several laboratories has already shown that the ubiquitin-proteasome pathway can largely account for p53 destruction, at least under specific experimental conditions. We report here that, in vitro, wild-type p53 is a sensitive substrate for milli- and microcalpain, which are abundant and ubiquitous cytoplasmic proteases. Degradation was dependent on p53 protein conformation. Mutants of p53 with altered tertiary structure displayed a wide range of susceptibility to calpains, some of them being largely resistant to degradation and others being more sensitive. This result suggests that the different mutants tested here adopt slightly different conformations to which calpains are sensitive but that cannot be discriminated by using monoclonal antibodies such as PAb1620 and PAb240. Inhibition of calpains by using the physiological inhibitor calpastatin leads to an elevation of p53 steady-state levels in cells expressing wild-type p53. Conversely, activation of calpains by calcium ionophore led to a reduction of p53 in mammalian cells, and the effect was blocked by cell-permeant calpain inhibitors. Cotransfection of p53-null cell lines with p53 and calpastatin expression vectors resulted in an increase in p53-dependent transcription activity. Taken together, these data support the idea that calpains may also contribute to the regulation of wild-type p53 protein levels in vivo.
Animals↗
Interaction with damaged DNA induces selective proteolytic cleavage of p53 to yield 40 kDa and 35 kDa fragments competent for sequence-specific DNA binding.
The p53 protein binds sites of primary DNA damage via its C-terminus. This interaction in some way activates sequence-specific binding (via the central core domain) and transactivation of p53 target genes. We now show that interaction with non-specific DNA, but not specific DNA targets, induces selective proteolysis of p53 to give a 40 kDa fragment, comprising the core plus C-terminus, and a 35 kDa conformationally intact core domain. Proteolytic cleavage was limited and yielded roughly equivalent proportions of full length p53 and the 40 kDa and 35 kDa fragments. Significantly, both 40 kDa and 35 kDa products were activated for sequence-specific DNA binding. Similar p53-related products were induced by exposure of cells to DNA damage. We propose that some functions of p53 can be activated by proteolytic processing and that this may be important in the cellular response to DNA damage.
3T3 Cells↗
Needs of disabled children and their families.
In the new NHS those who provide services for disabled children need to measure and demonstrate their effectiveness, but there are no easily available outcome measures for use by child development centres and teams. The development of an alternative approach, using a series of statements of good practice, is described. Parents of children with cerebral palsy were asked to participate in semistructured interviews, to ascertain the value and relevance of these quality statements. Parents were most concerned about the standard of news breaking and early follow up, the sharing of information, and the supply and repair of equipment. The findings were used to modify the quality checklist and it is proposed that this should form the basis of a "charter for disabled children and their families'.
Attitude to Health↗
Specific DNA binding by different classes of human p53 mutants.
The p53 protein is a multifunctional transcription factor which orchestrates cellular responses to DNA damage, so helping to conserve genomic stability. It may also regulate genes involved in intercellular signalling, such as thrombospondin, a negative regulator of angiogenesis and metastatic spread. Activation of p53 target genes requires sequence-specific DNA binding, a function which maps to the central core of the protein. Missense point mutations within this domain inactivate p53 tumour suppressor function and involve either (i) DNA contact residues, or (ii) residues important for conformational structure. Using in vitro techniques we have analysed seven DNA contact mutants and 17 structural mutants known to occur in cancer. We show that DNA contact mutants can be carried into specific DNA interaction when co-expressed with wild type protein. For structural mutants, 9/17 retained DNA binding capacity and, with one exception, DNA binding correlated with conformational flexibility of the mutant protein. The exception was Asp281, which appeared essential for DNA interaction, probably due to its ability to form salt bridges with DNA contact residues Arg273 and Arg280. We suggest that different classes of p53 mutant may prove amenable to different strategies for restoration of wild type tumour suppressor function as means of anti-cancer therapy.
Amino Acid Sequence↗
p53 in complex with DNA is resistant to ubiquitin-dependent proteolysis in the presence of HPV-16 E6.
The tumour suppressor p53 is a transcription factor with high affinity for specific DNA target sequences. Wild type p53 has a very short half life in normal cells but the protein shows transient accumulation in response to DNA damage, accompanied by up-regulation of target genes such as p21 and induction of growth arrest in G1 of the cell cycle. The rapid turnover of p53 may involve the ubiquitin-dependent proteolytic pathway. In order to investigate p53 turnover we have employed an in vitro system with rabbit reticulocyte lysate, in which ubiquitin-dependent degradation of p53 is mediated by the oncoprotein E6 of human papilloma virus type 16 (HPV-16). Using this system we have previously shown that E6-mediated degradation is preferential for p53 in the 1620+ conformation (reactive with the monoclonal antibody PAb1620). p53-1620+ is a pre-requisite for specific DNA binding and we have now asked if p53 in complex with DNA remains susceptible to ubiquitin-dependent proteolysis in the presence of E6. Our results indicate that p53-DNA complexes are resistant to degradation, whereas the 'free' protein is completely degraded within 20 min. Moreover, E6 did not complex with p53-DNA, possibly due to masking of sites recognised either by E6 or by the E6-associated protein (E6-AP) which facilitates E6-p53 interaction. Preincubation with E6 inhibited the DNA binding capacity of p53 and this effect could be explained, at least in part, by ubiquitination of the p53 protein.
Base Sequence↗
Structural and kinetic analysis of p53-DNA complexes and comparison of human and murine p53.
Sequence-specific DNA binding by p53 is dependent upon protein conformation. The 1620+ form correlates with wild type p53 suppressor function and is a prerequisite for binding to the DNA consensus p53-CON in vitro. It has been reported that murine p53 changes conformation on interaction with high affinity DNA target sequences and in the present study we have analysed p53-DNA complexes using conformation-specific monoclonal antibodies against p53. For murine p53 (mp53) we show (i) the 1620+ form is retained and stabilised in complex with DNA, and (ii) the complexes are dissociated by the PAb1620 monoclonal antibody. In contrast, PAb1620 did not detect nor dissociate human p53-DNA complexes nor did it interfere with complex formation. In competition experiments murine p53 replaced human p53 (hp53) in p53-DNA complexes and this correlated with the greater lability observed for hp53-DNA complexes at a given temperature. Mixed human-murine p53 oligomers were competent for DNA binding with an estimated affinity around 5 x 10(-10) M, similar to that observed for either human or murine p53 alone. The potential significance of these observations is discussed in relation p53 function in vivo.
Animals↗
Modulation by copper of p53 conformation and sequence-specific DNA binding: role for Cu(II)/Cu(I) redox mechanism.
The tumor suppressor protein p53 is a metal-binding transcription factor whose conformation and function are altered by mutation in cancers. Using murine p53 translated in vitro, we report here that concentrations of copper within the physiological range (< 30 microM) alter the conformation of wild-type p53 and inhibit sequence-specific DNA-binding. Direct binding of copper to p53 in the form of Cu(I) was demonstrated by Electron Spin Resonance using a purified recombinant protein containing residues 1-343 of murine wild-type p53 fused to E. coli maltose binding protein. Moreover, protection against the effect of Cu(II) sulfate was achieved by the Cu(I)-specific chelator bathocuproinedisulfonic acid but not by scavengers of reactive oxygen species, suggesting that alteration of p53 by copper depends upon a Cu(II)/Cu(I) redox mechanism, but does not require the production of reactive oxygen species. Thus copper at physiological concentrations can interact with wild-type p53 and affect its DNA-binding capacity.
Animals↗
Flexibility: the key to p53 function?
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Models, Molecular↗
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↗