The tumor suppressor p53 is mutated in about half of all human malignancies. In most of the remaining cancers, the function of p53 is compromised due to alterations in signaling pathways that regulate p53 activity. Despite recent advances, the overall 5-year survival rate for cancer patients remains very low and there is an urgent need for the development of more efficient anti-cancer therapies. p53 is an attractive therapeutic target and understanding the molecular mechanisms of its regulation is essential for the design of new treatment strategies. Our preliminary data reveal that p53 activity can be modulated by protein effectors 53BP (p53-binding protein 1) and L3MBT (lethal 3 malignant brain tumor) that recognize methylated lysine marks on p53. The molecular mechanisms underlying these novel interactions of p53 are unknown and will be elucidated in the proposed studies. Detailed knowledge of these mechanisms is of fundamental importance for understanding the physiological and tumorigenic activities of p53. We hypothesize that binding of the MBT domain of L3MBT to monomethylated p53 (p53K382me1) represses p53 transactivation, whereas binding of the 53BP Tudor domain to dimethylated p53 (p53K382me2) facilitates p53 accumulation at DNA damage sites and promotes DNA repair. Furthermore, we propose that nearby posttranslational modifications (PTMs) of p53 alter the binding properties of these effectors and fine-tune p53 functions. We seek to define the structural basis and functional consequences of the interactions of 53BP and L3MBT with methylated p53 (p53me).
The specific aims of this project are: (1) to determine the molecular basis of p53 recognition by 53BP and (2) to elucidate the molecular mechanism of p53 targeting by L3MBT. The three-dimensional structures of 53BP Tudor and L3MBT MBT in complex with singly and doubly modified p53 peptides will be determined by X-ray crystallography or NMR. The specificities, binding affinities and the crosstalk between PTMs of p53 will be examined. The binding site residues will be mutated and the mutant 53BP and L3MBT proteins will be tested in vitro by fluorescence spectroscopy, isothermal titration calorimetry and NMR and in vivo by immunoprecipitation (IP), chromatin IP, PCR and DNA damage assays. In-depth biochemical, structural and functional characterization of the p53me effectors will provide a comprehensive understanding of the mechanistic principles underlying physiological and oncogenic activities of p53 and may facilitate the development of novel anti-cancer therapies.
The major tumor suppressor p53 is mutated in about half of all human cancers. The proposed studies will greatly enhance our knowledge of how the biological functions of p53 are regulated in normal cells and during tumorigenesis and may help to identify new targets for therapeutic interventions.
|Klein, Brianna J; Piao, Lianhua; Xi, Yuanxin et al. (2014) The histone-H3K4-specific demethylase KDM5B binds to its substrate and product through distinct PHD fingers. Cell Rep 6:325-35|
|Klein, Brianna J; Lalonde, Marie-Eve; Côté, Jacques et al. (2014) Crosstalk between epigenetic readers regulates the MOZ/MORF HAT complexes. Epigenetics 9:186-93|
|Ezhov, Roman N; Metzel, Greg A; Mukhina, Olga A et al. (2014) Photoactive Spatial Proximity Probes for Binding Pairs with Epigenetic Marks. J Photochem Photobiol A Chem 290:101-108|
|Allen, Hillary F; Daze, Kevin D; Shimbo, Takashi et al. (2014) Inhibition of histone binding by supramolecular hosts. Biochem J 459:505-12|
|Ali, Muzaffar; Hom, Robert A; Blakeslee, Weston et al. (2014) Diverse functions of PHD fingers of the MLL/KMT2 subfamily. Biochim Biophys Acta 1843:366-71|
|Musselman, Catherine A; Khorasanizadeh, Sepideh; Kutateladze, Tatiana G (2014) Towards understanding methyllysine readout. Biochim Biophys Acta 1839:686-93|
|Lalonde, Marie-Eve; Avvakumov, Nikita; Glass, Karen C et al. (2013) Exchange of associated factors directs a switch in HBO1 acetyltransferase histone tail specificity. Genes Dev 27:2009-24|
|Ali, Muzaffar; Rincon-Arano, Hector; Zhao, Wei et al. (2013) Molecular basis for chromatin binding and regulation of MLL5. Proc Natl Acad Sci U S A 110:11296-301|
|Gatchalian, Jovylyn; Futterer, Agnes; Rothbart, Scott B et al. (2013) Dido3 PHD modulates cell differentiation and division. Cell Rep 4:148-58|