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Structural and Biochemical Characterization of PGC-1a Coactivation of the Tumor Suppressor Protein P53
Background: p53 plays a critical role in preventing tumor formation and is the most commonly mutated gene in human cancer. It is induced in response to a variety of stress signals such as DNA-damaging agents, and induces cell-cycle arrest and/or apoptosis. Loss of p53 function results in genetic instability and contributes to tumor formation. In unstressed cells, p53 is maintained at low levels by targeted degradation mediated by MDM2. In response to various stress signals, the p53 pathway is activated and executed through interactions with various proteins. Recent work in our laboratory has shown that the transcriptional coregulator PGC-1a coactivates p53 via a physical interaction and facilitates the pro-apoptotic effects of p53 (unpublished data). PGC-1a levels are reduced in both colon and breast cancer, indicating a p53-dependent role for the coactivator in these cancers. However the molecular basis for this interaction remains unexplored.
Objectives/Hypothesis: The objective is to understand the functional interaction between PGC-1a and p53 through biochemical and structural approaches. The hypothesis is that mutations in the p53 gene affect the interaction and coactivation of p53 by PGC-1a, and hence its function. We also hypothesize that MDM2 influences the PGC-1a:p53 interaction.
Specific Aims: (1) Map the domains involved in the PGC-1a:p53 interaction. (2) Co-crystallize PGC-1a-p53 interaction domains. (3) Analyze coactivation and interaction of PGC-1a with several breast cancer related p53 mutants. (4) Analyze the effect of MDM2 on the PGC-1a - p53 interaction.
Study Design: (1) Generation of various PGC-1a and p53 alleles, biochemical analysis using in vitro assays. (2) Co-crystallization of the PGC-1a:p53 complexes. (3) Biochemical and functional analysis of the mutant PGC-1a:p53 interactions. (4) Biochemical analyses of MDM2 effect on the PGC-1a:p53 interaction complex.
Potential Outcomes and Benefits of the Research: A detailed structural analysis of the PGC-1a:p53 complexes would help characterize these interactions at a molecular level. The study will elaborate the stereo-chemical basis for PGC-1a function as a coactivator of p53 and provide a detailed molecular understanding of the defects of the breast cancer related p53 mutations. A study of the effect of MDM2 on the PGC-1a:p53 interaction could provide insights into the regulation of p53 function. The contact surfaces of these protein complexes are predicted to have unique structure and properties and represent prospective targets for rational structure-based drug design.
Inherited and acquired genetic changes are the basic cause for cancer and stem from a variety of complex factors. These factors cause mutations in our genetic material resulting in cells that divide anomalously. The tumor suppressor p53 plays an important role in maintaining genomic stability by eliminating damaged and potentially dangerous cells that might otherwise become cancerous. p53 is activated in response to various DNA-damaging signals and triggers the DNA repair mechanism or induces the cells to undergo programmed cell death. The p53 protein undergoes various chemical changes and interacts with multiple proteins in order to maintain genomic integrity. PGC-1alpha is a coactivator that binds to p53 and mediates its ability to induce apoptosis. p53 is activated in response to cellular stress and is negatively regulated by MDM2 in a normal, unstressed state. Mutations in p53 gene result in a mutant protein incapable of executing the DNA-repair or the apoptosis pathways and are found in most tumor types. It is also known that the levels of PGC-1alpha are reduced in colon and breast cancer, indicating a significant role for the PGC-1alpha:p53 complex in tumor suppression. Furthermore, PGC-1alpha and MDM2 appear to have opposing functions: PGC-1alpha coactivates p53 while MDM2 represses it. There is very little known about the molecular mechanisms governing the PGC-1alpha:p53 interaction. Our objective is to understand this interaction employing biochemical and structural methods. We will utilize biochemical techniques to determine the domains of interaction between PGC-1alpha and p53. The results of these studies will be used to determine the high-resolution structures of the complex using X-ray crystallography. Such structures will provide a better understanding of PGC-1alpha function as a p53 coactivator. Breast cancer related p53 mutants will be studied in terms of their interaction with PGC-1alpha and are expected to provide insights into the molecular defects that result in an aberrant behavior of the mutant p53. Studies will be performed to analyze whether PGC-1alpha and MDM2 mutually influence their interactions with p53. The structural studies are expected to provide a detailed understanding of the interaction surfaces in the PGC-1alpha:p53 complexes. These surfaces are unique to each complex and provide an exciting target for potential drugs to modulate these interactions. This study attempts to elucidate the molecular details of these interactions at an atomic level and provide a framework for rational structure-based drug design.