Abstract
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.
Highlights
The tumour-suppressor protein p53 plays a role in the control of cell proliferation, differentiation and survival after DNA damage. p53 is a sequence-specific transcriptional regulator which transactivates genes such as GADD45 (Kastan et al, 1992), MDM2 (Momand et al, 1992) and WAF-l (El-Deiry et al, 1993)
Specific DNA binding is restricted to oligomeric forms of p53 which adopt a specific tertiary structure characterised by reactivity with a monoclonal antibody recognising a conformation-dependent epitope, PAb 1620
Sequence-specific DNA binding was assayed by electromobility shift assay (EMSA) using as target the double-stranded oligonucleotide 5'-GGGCATGTCCGGGCATGTCC-3' (p53 CON; Funk et al, 1992) as described previously (Hainaut and Milner, 1993b)
Summary
The tumour-suppressor protein p53 plays a role in the control of cell proliferation, differentiation and survival after DNA damage (for review see Levine et al, 1994). p53 is a sequence-specific transcriptional regulator which transactivates genes such as GADD45 (Kastan et al, 1992), MDM2 (Momand et al, 1992) and WAF-l (El-Deiry et al, 1993). We have shown that chelating and oxidising agents disrupt the PAb 1620 + conformation of p53 and inhibit specific DNA binding (Hainaut and Milner, 1993a,b).
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