Employing Chemical Biology Tools for Selective Control of Acetyltransferases and Interrogation of Signal Transduction Pathways of Kinases
Authorde Silva, Chandi Sagarika
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PublisherThe University of Arizona.
RightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction, presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
EmbargoRelease after 12/17/2020
AbstractThe reversible post translational modifications (PTMs) of proteins are often integral for the cell to respond to external stimuli. The cellular pathways involve molecular interactions between proteins and other molecules and/or enzymatic activity. The molecular interactions, trafficking between cellular compartments and enzymatic activity are often regulated by PTMs. Reversible PTMs allow cells to respond and subsequently return to a resting state. Human diseases often arise from defects in these signaling networks sometimes from perturbations in PTMs. Clearly ascribing a particular PTM on a particular protein in a signaling network to a particular enzyme has been very challenging. Advances in understanding may help in an improved understanding of signaling and thus impact our battle against the disease. The primary focus of this dissertation is to discuss the fragment complementation approach which was employed to regulate the function of proteins belong to two major classes that govern two classes of post translational modifications, phosphorylation and acetylation. The >500 protein kinases in humans catalyze the phosphorylation of Tyr, Ser and Thr residues on proteins and thus modulate their function. The structural similarity of kinases makes it challenging to selectively turn-on or turn-off desired kinases using small molecule inhibitors or activators. Genetic approaches are powerful, but cells and organisms respond and adapt to long-term changes in gene expression levels, thus making it challenging to understand the exact role of any enzyme in time and space. Towards a potential solution to this problem, we have developed a method to control the activity of individual enzymes utilizing small molecules. This approach was successfully applied to protein kinases and phosphatases, leading to the control of their activity in vitro and in cells. The strategy entails sequence alignment and the identification of regions that harbor significant dissimilarities to eventually generate ligand-gated split proteins. The well-studied chemical inducer of dimerization (CID), rapamycin dependent heterodimerization of FKBP/FRB was used as the first test of a successful ligand-gated split-enzyme. Moreover, orthogonal CIDs rapamycin and abscisic acid were successfully used for regulating the activity of Kinases in mammalian cells. Src is the first identified proto-oncogene and is often considered as the quintessential member of a Src kinase family proteins. We generated two stable cell lines each expressing constitutively active split Src kinase that can be conditionally regulated by two chemical inducers rapamycin and abscisic acid. Subsequently, those stable cell lines were used for phosphoproteomic studies to understand Src signaling. Rapid activation of stably expressing systems enabled identification of numerous potential direct or indirect Src substrates and specific phosphosites. These downstream targets may be implicated in many different cellular networks thus, provides a general understanding of the role of Src and Src family kinases in cell signaling. We have also used the split-protein approach for Lysine Acetyl Transferases (KATs). There are numerous KATs, and currently available small molecule-based inhibition methods are not uniquely specific while RNAi based gene knockdown studies can fail to provide details related to the true role of any enzyme as compensatory acetylation may occur. To address this problem, the first generation of ligand-inducible split-KATs, GCN5 and PCAF were successfully tested in bacterial expression systems. In this work, we validated a series of full-length split acetyltransferases and developed in cellulo approaches for their study. However, we were not successful in developing robust methods for observing ligand inducible full-length split-KATs in cells. Finally, the last section of this dissertation focuses on developing an approach for selective DNA methylation studies that would improve upon methods to target specific DNA sequences using designed zinc fingers. Transcription activator-like effectors (TALEs) were designed, cloned and tested as the DNA binding domain for targeting mir 200c promoter regions, which are known to demonstrate hypermethylation activity. We discovered that all the designed TALEs displayed higher binding specificity to the desired targets of the mir 200c promoter than zinc finger-based designs. This work sets the stage for the further design of selective reagents for targeting DNA. In summary, this work describes approaches for selectively targeting and studying protein phosphorylation, protein acetylation as well as DNA methylation.
Degree ProgramGraduate College