Protein Engineering Methods to Understand Kinase Signaling and Protein-Protein Interactions
Protein tyrosine kinases
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PublisherThe University of Arizona.
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EmbargoRelease after 05/10/2024
AbstractTyrosine phosphorylation is one of the key covalent post-translational modifications through which multicellular organisms communicate. Phosphorylation of tyrosine residues on proteins can modulate enzymatic activity and can create binding sites for the recruitment of downstream signaling proteins. Conservation of enzyme structure across the human kinome makes it difficult to design small molecules to selectively modulate kinase activity, which can help elucidate their roles in signaling pathways and in diseases. To circumvent this limitation, we have developed a split-protein method to control the activity of individual kinases by utilizing chemical inducer of dimerization (CID) proteins. The split-protein approach relies on the identification of viable fragmentation sites in a protein that can be used to generate ligand-gated control of protein activity. The major focus of this dissertation is the identification of new kinase and firefly luciferase fragmentation sites for temporal control of a specific kinase and for the study of protein-protein interactions, respectively. With a sequence dissimilarity-based approach and structure-guided analysis, we successfully identified new split-Src sites, the first split-Syk site, the first split-Abl-1aFL, and new firefly luciferase fragmentation sites utilizing the CIDs of rapamycin and/or abscisic acid. Temporal control of split-Abl-1a coupled with quantitative phosphoproteomics analysis aided in the understanding of Abl-1 cellular signaling. Both known and novel substrates were identified, and validation of novel phosphotyrosine targets like AFDN, AMOT, DDX3X, NCAPH present opportunities for studying and understanding unanticipated functions of Abl-1. In summary, this work describes the split-protein approach to selectively control and dissect the complex signaling processes of specific protein tyrosine kinases (PTKs) and for monitoring PPIs. The new tools developed could potentially be used to rewire signaling pathways and aid in the development of novel therapeutic targets.
Degree ProgramGraduate College
Degree GrantorUniversity of Arizona
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Methods for the Detection of Protein-Nucleic Acid and Protein-Protein InteractionsGhosh, Indraneel; Stains, Cliff; Ghosh, Indraneel; Hruby, Victor J.; McGrath, Dominic V.; Montfort, William R. (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.
Protein stickiness, rather than number of functional protein-protein interactions, predicts expression noise and plasticity in yeastBrettner, Leandra M.; Masel, Joanna; Present address: Ecology & Evolutionary Biology, University of Arizona, 1041 E Lowell St, Tucson, AZ, 85721, USA; Present address: Bioengineering, University of Washington, 3720 15th Ave NE, Seattle, WA, 98195, USA (BioMed Central, 2012)BACKGROUND:A hub protein is one that interacts with many functional partners. The annotation of hub proteins, or more generally the protein-protein interaction "degree" of each gene, requires quality genome-wide data. Data obtained using yeast two-hybrid methods contain many false positive interactions between proteins that rarely encounter each other in living cells, and such data have fallen out of favor.RESULTS:We find that protein "stickiness", measured as network degree in ostensibly low quality yeast two-hybrid data, is a more predictive genomic metric than the number of functional protein-protein interactions, as assessed by supposedly higher quality high throughput affinity capture mass spectrometry data. In the yeast Saccharomyces cerevisiae, a protein's high stickiness, but not its high number of functional interactions, predicts low stochastic noise in gene expression, low plasticity of gene expression across different environments, and high probability of forming a homo-oligomer. Our results are robust to a multiple regression analysis correcting for other known predictors including protein abundance, presence of a TATA box and whether a gene is essential. Once the higher stickiness of homo-oligomers is controlled for, we find that homo-oligomers have noisier and more plastic gene expression than other proteins, consistent with a role for homo-oligomerization in mediating robustness.CONCLUSIONS:Our work validates use of the number of yeast two-hybrid interactions as a metric for protein stickiness. Sticky proteins exhibit low stochastic noise in gene expression, and low plasticity in expression across different environments.
INITIATION MECHANISM OF PROTEIN-LINKED DNA REPLICATION: THE FUNCTION OF BACILLUS SUBTILIS PHAGE PHI-29 TERMINAL PROTEIN.SHIH, MENG-FU. (The University of Arizona., 1983)An in vitro initiation of a DNA replication system was developed to study the function of Ø29 terminal protein. Cell free extracts prepared from Bacillus phage Ø29-infected cells catalyzed the formation of a complex between a 30,000 dalton protein and dAMP in the presence of MgCl₂, Ø29 DNA-protein template and α-³²P dATP. Uninfected cell extracts did not support this reaction. The molecular weight of this complex, the nature of linkage between dAMP and 30,000 dalton protein as well as nucleotide specificity for this reaction suggest that the protein moiety of this complex is the terminal protein of Ø29. Similar results were obtained with phages GA-1 and M2Y infected cell extracts. The similar requirements for complex formation and DNA replication in vitro implies that the complex formation is an initiation step in DNA replication. This notion was supported by characterizing the elongation product which formed in the presence of ddCTP. Two distinct antibodies were prepared for analyse the function of the terminal protein in Ø29 DNA replication. These antibodies react with native terminal protein as assayed by immunoprecipitation and ELISA methods. The inhibition of complex formation by these antibodies provides strong evidence that the terminal protein is involved in complex formation. The notion that complex formation is an initial step of DNA replication was demonstrated conclusively by inhibition of anti-TP on DNA replication in vitro. The results presented in this dissertation provide evidence supporting the protein-priming mode of linear Ø29 DNA replication.