2004 Fiscal Year Final Research Report Summary
Design and development of tumor suppressor protein p53 variant that cannot form heterooligomers with the wild-type protein
| Project/Area Number |
14380290
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Bioorganic chemistry
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| Research Institution | HOKKAIDO UNIVERSITY (2003-2004)
Kyushu University (2002) |
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Principal Investigator |
SAKAGUCHI Kazuyasu Hokkaido Univ., Grad.School of Sci., Prof., 大学院・理学研究科, 教授 (00315053)
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| Co-Investigator(Kenkyū-buntansha) |
SHIMOHIGASHI Yasuyuki Kyushu Univ., Fac.of Sci., Prof., 大学院・理学研究院, 教授 (00211293)
CHUMAN Yoshiro Hokkaido Univ., Grad.School of Sci., Inst., 大学院・理学研究科, 助手 (40372263)
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| Project Period (FY) |
2002 – 2004
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| Keywords | heterooligomer / combinatorial library / peptide / dominant negative / tumor suppressor protein |
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| Research Abstract |
The tumor suppressor protein p53 is a 393 amino acid phosphoprotein that is a transcriptional enhancer in tetrameric form and suppresses cell cycle progression in response to genotoxic stress. The tetramerization domain of p53 is essential for efficient site-specific DNA binding and contributes to p53's ability to activate transcription from natural promoters. The p53 tetramerization domain is located at residues 326-356. More than 50% of human cancer is associated with p53 gene mutation, which is mostly located at sequence specific DNA binding domain. The missense mutant p53 inactivates a wild-type p53 in a dominant-negative manner via hetero-oligomerization.
In this study, we designed and synthesized two peptide libraries of the 40 amino acid p53 tetramerization domain to screen peptides that cannot form hetero-oligomer with the wild-type p53. For the first library, termed p53lib-4, four hydrophobic residues, which are located on the α-helix at the interface of two dimers, were randomized with seven hydrophobic amino acids including Tip, Tyr, Phe, Met, Lew, Ire, and Val. In the second library, p53lib-9, all nine residues that form the hydrophobic core of p53 tetramer were randomized in the same way. Two peptide libraries were screened for non-heterooligomerizing peptides with the wild-type sequence. The screened fraction showed the almost identical CD spectrum, indicating that peptides in the fraction could form a wild-type tetrameric structure. Second library was designed by characterization of the screened peptides on the first screening. The library was subjected for second screening. The results indicated that a peptide fraction isolated by this screening could contain peptides that form homo-tetramers by themselves and do not form hetero-oligomers with wild type. The results demonstrated that the strategy used in this study could be applied to screen peptide that does not form hetero-oligomers.
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