SPLIT-PROTEIN REASSEMBLY METHODS FOR THE DETECTION AND INTERROGATION OF BIOMOLECULAR INTERACTIONS AND MODULATORS THEREOF
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azu_etd_10661_sip1_m.pdf
Issue Date
2009Keywords
Bcl-2High Throughput
In Vitro Translation
Protein-Nucleic Acid
Protein-Protein
Split-Protein Complementation
Advisor
Ghosh, IndraneelCommittee Chair
Ghosh, IndraneelMetadata
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The University of Arizona.Rights
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.Abstract
The interactions between protein-protein, protein-nucleic acid, and protein-small molecules are central to biological processes and are key for the design of new therapeutics. Rapid and easy to implement methodologies are needed that enable the interrogation of these interactions in a complex cellular context. Towards this goal, I have utilized the concept of split-protein reassembly, also called protein complementation, for the creation of a variety of sensor architectures that enable the interrogation of protein-nucleic acid, protein-protein, and protein-small molecule interactions. Utilizing the enzymatic split-reporter β-lactamase and existing zinc finger design strategies we applied our recently developed technology termed SEquence-Enabled Reassembly (SEER) towards the creation of a sensor capable of the specific detection of the CryIA transgene. Additionally, the split β-lactamase reporter was utilized for the sitespecific determination of DNA methylation at cytosine residues that is involved in epigenetic regulation. This method, dubbed mCpG-SEER, enabled the direct detection of femtomole levels of dsDNA methylation in sequence specific manner. In a separate endeavor, we have developed and optimized the first cell-free split-reporter systems for GFP, split β-lactamase, and firefly luciferase for the successful dsDNA-dependent reassembly of the various reporters. Our cell free in vitro translation systems eliminates previous bottlenecks encountered in split-reporter technologies such as laborious transfection/cell culture or protein purification. Capitalizing on the ease of use and speed afforded by this new technology we describe the sensitive detection of protein-protein, protein-nucleic acid, and protein-small molecule interactions and inhibitors thereof. In a related area, we have applied this rapid cell-free split-firefly luciferase assay to the specific interrogation of a large class of helix-receptor protein-protein interactions. We have built a panel consisting of the clinically relevant Bcl-2 family of proteins, hDM2, hDM4, and p53 and interrogated the specificity of helix-receptor interactions as well as the specificity of peptide and small-molecule inhibitors of these interactions. Finally, we describe the further applications of our cell-free technology to the development of a large number of general split-firefly luciferase sensors for the detection of ssRNA sequences, the detection of native proteins, the evaluation of protease activity, and interrogation of enzyme-inhibitor interactions. The new methodologies provided in this study provides a general and enabling methodology for the rapid interrogation of a wide variety of biomolecular interactions and their antagonists without the limitations imposed by current in vitro and in vivo approaches.Type
textElectronic Dissertation
Degree Name
Ph.D.Degree Level
doctoralDegree Program
ChemistryGraduate College
Degree Grantor
University of ArizonaCollections
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Methods for the Detection of Protein-Nucleic Acid and Protein-Protein Interactions(The University of Arizona., 2008)We describe the first general approach for the DNA templated reassembly of proteins, which we term SEquence-Enabled Reassembly or SEER. SEER makes use of dissected signaling domains which are each attached to separate, sequence specific DNA-binding proteins. Described herein is an embodiment of SEER in which DNA catalyzes the reassembly of the green fluorescent protein which leads to a direct fluorescence readout of the corresponding DNA sequence. This strategy has also been extended to the first direct method for the site specific detection of DNA methylation. This mCpG-SEER system is capable of discriminating between methylated versus nonmethylated DNA with a 40-fold increase in fluorescence signal.In a separate undertaking we tested the efficiency of disulfide bond formation within the context of the ribosome display in vitro selection methodology. We established conditions for the enrichment of a cyclic peptide, which is specific for Neutravidin, by 2 x 10^6-fold. Using the knowledge gained from the above experiments, we combined the rapid protein expression and folding benefits of cell-free translation systems with a sensitive split-luciferase reassembly assay to yield the most rapid method to date for the detection of protein-nucleic acid and protein-protein interactions. Furthermore, we have shown that these split-luciferase cell-free reassembly systems can be compartmentalized, allowing for future molecular evolution studies.Lastly, we have applied this rapid cell-free split-luciferase assay system to the direct detection of clinically relevant proteins. We have engineered a system for the rapid characterization of HIV-1 clades utilizing single-chain antibody specificities. We also demonstrate that this platform can be used to determine the relative amounts of HER2 expression in human breast cancer cells, using a homogeneous assay format in which cells and reagents are mixed and luminescence is monitored directly.We envision that the assay platforms described herein will find applications in the rapid detection of nucleic acid sequences, protein identities, and relative protein abundances in the laboratory and clinic.
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Contribution of Heat Shock Protein 27 and Retinol Binding Protein to 11-Deoxy-16, 16-Dimethyl Prostaglandin E2 Mediated Cytoprotection(The University of Arizona., 2008)11-Deoxy-16,16-dimethyl prostaglandin E2 (DDM-PGE2) protects renal proximal tubular epithelial cells (LLC-PK1) against oncotic cell death induced by 2,3,5-tris(glutathione-S-yl)hydroquinone (TGHQ). Cytoprotection is associated with the up-regulation of several proteins including actin, heat shock protein 27 (Hsp27) and retinol binding protein (RBP). This dissertation reveals the induction and phosphorylation of Hsp27 by TGHQ treatment and DDM-PGE2 pretreatment. Treatment with TGHQ results in a dose-dependent disruption of the actin cytoskeleton that correlates with a decrease in cell viability, increased generation of reactive oxygen species, and the induction of Hsp27 nuclear translocation and co-localization with actin. Moreover, DDM-PGE2 pretreatment, but not co-treatment, prevents both TGHQ generation of ROS and actin cytoskeletal damage. DDM-PGE2 results in the enhanced induction and phosphorylation of nuclear Hsp27 that likely contributes to the inhibition of early effects of TGHQ induced ROS generation on the actin cytoskeleton. In correlation, we identify site specific phosphorylation of Hsp27 (p-Hsp27) at human Ser82 that negatively regulates cell survival, and p-Hsp27 at Ser15 associated with cell survival. We provide evidence that the TP receptor dependent increase in RBP expression is based on the ability of DDM-PGE2 to recruit the retinoid signaling pathway through activation of the RAR/RXR nuclear receptor heterodimers. All-trans retinoic acid (AtRA) pretreatment recapitulates the protective effects of DDM-PGE2 through a mechanism independent of TP receptor activation and the cytoprotective effects of AtRA were also investigated using an in vivo model. Finally, the ability of DDM-PGE2 to increase the cells antioxidant response, important in its cytoprotection against ROS is described. Taken together, these studies contribute to the mechanism of protection and will give insight into the affects of novel therapeutics in the modulation of chemical induced nephrotoxicity.
