Genetic and Chemical Genetic Tools for Examining Context-Dependent Protein-Protein Interactions
Baruti, Omari
2019
Abstract
A vast network of transient protein-protein interactions (PPIs) underlies many cellular processes such as homeostasis, development, and disease. Because these networks have evolved over time to a high degree of complexity, characterization of transient PPIs is key to elucidation of the structure-function relationship of this complex network. Due to the role that these many PPIs play in maintaining a healthy cellular environment, dysregulation is associated with several diseases, including cancer, cardiovascular defects, and neurological disorders. Thus, there is significant interest in the development of tools to characterize the transient PPIs that underlie these diseases. In Chapter 2 of this dissertation we characterized the structure-function relationship of the transient PPIs of the mitogen activated protein kinase (MAPK) signaling pathway Ras-Raf. Ras is a small GTPase consisting of four isoforms that rapidly interact with the Ras binding domain (RBD) of the kinase Raf to promote signal transduction of the MAPK pathway. Characterization of this interaction is difficult due to the high degree of homology between isoforms and the fast rate of interaction between interacting partners. We have developed a photoaffinity probe that permits the covalent capture of interacting proteins through the site-specific genetic incorporation of an unnatural amino acid, benzoylphenylalanine (BPA), for quantitative detection of the transient PPIs in vivo. The development of this tool was made possible through optimization of protein expression conditions in E. coli and incorporation sites. The photoreactive probe was then utilized to irreversibly capture active Ras. We then analyzed the crosslinked product by Western Blot and observed a molecular weight shift indicating the effectiveness of our probe. Overall, we demonstrate that BPA-photocrosslinking in RBD represents a feasible strategy for characterizing Ras in its native context and should illuminate points of therapeutic intervention of Ras-related disease in future efforts. In Chapter 3 we continue our investigation of the structure and function of PPIs with a detailed analysis of the transcriptional coactivator-activator complexes CBP/p300 KIX-activator binding partners. CBP/p300 KIX is a highly conserved and intrinsically disordered coactivator involved in transcriptional regulation. The highly dynamic structure of KIX allows for the interaction of several transcriptional activators at the MLL or c-Myb binding sites. However, while there is extensive characterization of KIX-activator PPIs in vitro, there is little cellular data that demonstrates a role for the PPIs in a functional context. Here we used a uniquely selective natural product inhibitor of KIX-activator PPIs to demonstrate that these complexes play an important role in cell cycle regulation in the context of leukemia cells. Specifically, we have utilized the small molecule garcinolic acid to inhibit KIX-dependent transcription and its impact on cellular health. We evaluated its effect through phenotypic assays that analyzed viability and mechanistic assays that analyzed gene expression and DNA content. Inhibition of these PPIs with the natural product garcinolic acid leads to down-regulation of key cyclins, induction of apoptosis, and G1 arrest. Taken together, these data indicate that the KIX-activator PPIs may be a potential candidate for therapeutic intervention.Subjects
Genetic and Chemical Genetic Tools for Examining Context-Dependent Protein-Protein Interactions
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