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Yeast As a Chassis for Developing Functional Assays to Study Human P53
Published on: August 4, 2019
A cellular protein activates the sequence-specific DNA-binding of p53 by interacting with the central conserved
1Department of Thoracic and Cardiovascular Surgery, University of Texas M.D. Anderson Cancer Center, Houston 77030, USA.
Abstract:
Mutational inactivation of the p53 gene product is one of the most common genetic aberations so far identified in human cancers. The p53 protein suppresses the transformed phenotype by transactivation or repression of genes involved in cell growth control. Missense mutations in the p53 protein coding sequence observed in human cancers are clustered within a central conserved (conformational) domain spanning amino acid residues 100-300 of a total of 393. Using the conformational domain of p53 fused with protein A, we have shown that the p53 conformational domain possesses Zn+2-dependent, sequence-specific DNA-binding activity. In addition to binding DNA, this domain interacts with at least five cellular proteins ranging in sizes from 30K to 90K M(r) and with the SV40 large T antigen viral oncoprotein. We investigated these cellular proteins for their modulatory effects on the sequence-specific DNA binding activity of full-length wild-type p53. A mixture of p53 conformational domain-binding proteins in bulk enhanced the DNA-binding activity of p53 greater than two-fold. Selective elution of the p53-binding proteins from the p53 hybrid protein by using a sequential step-wise NaCl gradient implicated one protein of 35K M(r) as contributing to a greater than four-fold activation of p53 DNA-binding activity. A p53 conformational domain protein containing a tumor-derived mutation at amino acid 175 failed to associate with the 35K M(r) protein. We propose that proteins interacting with the conformational domain of wild type p53 regulate the DNA-binding activity of p53, thus providing a biochemical basis for the alterations in its function induced by point mutations.
Insights
Mutations in the p53 gene are common in human cancers. This study shows that cellular proteins interacting with the p53 conformational domain regulate its DNA-binding activity, offering insight into cancer development.
Area of Science:
- Molecular Biology
- Cancer Genetics
- Protein Biochemistry
Background:
- Mutational inactivation of the p53 tumor suppressor gene is a frequent event in human cancers.
- The p53 protein regulates cell growth by controlling gene expression.
- Missense mutations in p53 are concentrated in its central conformational domain (residues 100-300).
Purpose of the Study:
- To investigate the DNA-binding activity of the p53 conformational domain.
- To identify cellular proteins that interact with the p53 conformational domain.
- To determine how these interactions modulate the DNA-binding activity of wild-type p53.
Main Methods:
- A fusion protein of the p53 conformational domain and protein A was used.
- Zinc-dependent, sequence-specific DNA-binding assays were performed.
- Interactions with cellular proteins and SV40 large T antigen were analyzed.
- Modulatory effects of interacting proteins on p53 DNA-binding were assessed using selective elution.
Main Results:
- The p53 conformational domain exhibits Zn+2-dependent, sequence-specific DNA-binding activity.
- This domain interacts with at least five cellular proteins (30-90 kDa) and SV40 large T antigen.
- A mixture of p53-binding proteins enhanced p53 DNA-binding activity over two-fold.
- A 35 kDa protein was identified as a key activator, increasing p53 DNA-binding activity over four-fold.
- A tumor-derived p53 mutant (amino acid 175) failed to bind this 35 kDa protein.
Conclusions:
- Cellular proteins interacting with the p53 conformational domain play a crucial role in regulating its DNA-binding activity.
- The 35 kDa protein is a significant modulator of p53 function.
- Disruption of these interactions by p53 mutations may contribute to cancer development.
- This provides a biochemical basis for understanding how p53 point mutations alter its function in cancer.
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