The Design and Application of Peptides and Synthetic Receptors to Chemical Biology
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Fayer, Effrat. The Design and Application of Peptides and Synthetic Receptors to Chemical Biology. 2016. https://doi.org/10.17615/58xe-3546APA
Fayer, E. (2016). The Design and Application of Peptides and Synthetic Receptors to Chemical Biology. https://doi.org/10.17615/58xe-3546Chicago
Fayer, Effrat. 2016. The Design and Application of Peptides and Synthetic Receptors to Chemical Biology. https://doi.org/10.17615/58xe-3546- Last Modified
- March 20, 2019
- Creator
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Fayer, Effrat
- Affiliation: College of Arts and Sciences, Department of Chemistry
- Abstract
- The need for chemical tools to probe biological process has become increasingly apparent in the last decade. The work presented here focused on developing such tools, taking on two different paths of development-one using supramolecular chemistry to aid in proteomics, a highly popular focus of research, and the other making use of peptides to create reporters with the ability to probe enzyme activity in cells. Posttranslational Modifications (PTMs) of proteins are implicated in a wide range of biological processes, including gene transcription, DNA replication and repair, mitosis, and meiosis.1 Consequently, their dysregulation is linked to various diseases, including cancer, asthma, and diabetes, among others, and can thus serve as valuable diagnostic indicators of disease progression.1,2 Due to these biological ramifications, there is great interest in mapping where, when, why, and how PTMs are installed and their subsequent downstream effects, though this is often hampered by their presence in complex mixtures, consisting of mostly un-modified proteins/peptides. Using synthetic supramolecular receptors developed in our lab,3 an affinity chromatography based method is described here that allows for the separation/enrichment of posttranslationally methylated peptides from such mixtures. This takes advantage of the receptor’s greater affinity towards methylated lysine over its non-methylated counterpart. When attached to a solid support, the receptors can be used to make a column, through which peptides will travel at different rates based on the methylation states of lysine residues. This allows for the separation of these peptides, drastically simplifying their detection and analysis. Just as important as PTMs are the enzymes that install them. Dysregulation of various enzymatic pathways is implicated in many diseases. The ability to monitor enzyme activity within cells is becoming increasingly important, for promoting further discovery, as well as to enable early detection and patient monitoring during treatment. The use of peptide substrates for such assays is extremely advantageous, as they are the best mimics of the enzymes’ natural substrates. Furthermore, the ease of synthesizing peptides allows them to be easily modified for specialized function and detection, making them applicable to multiple types of assays.4 While they work quite well for in vitro assays, they are incompatible with the cytosolic environment, as they are rapidly destroyed by cytosolic peptidases.5 In this dissertation, a variety of approaches towards increasing the lifetime of peptides in cytosolic environment were tested. Kinase substrates were selected as test peptides due to their role in a diverse set of vital processes, and their importance in current drug development efforts. For the most successful method, the rates of proteolytic degradation in cell lysates and in vitro phosphorylation were measured and analyzed using capillary electrophoresis paired with laser induced fluorescence (CE-LIF). Comparison to unmodified substrate peptides was used to assess the effect of dimerization on protease resistance and substrate efficacy. Finally, a dimerized Abl kinase substrate was used to monitor phosphorylation in living cells, demonstrating the utility of this method for intracellular assays. We find that N-terminal dimerization provides comparable half-lives to the best previously reported methods, with significantly greater synthetic accessibility, suggesting that this is a promising new method for developing peptide-based intracellular probes.
- Date of publication
- August 2016
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- Resource type
- Rights statement
- In Copyright
- Advisor
- Johnson, Jeffrey
- Waters, Marcey
- Weeks, Kevin
- Gagne, Michel
- Allbritton, Nancy
- Degree
- Doctor of Philosophy
- Degree granting institution
- University of North Carolina at Chapel Hill Graduate School
- Graduation year
- 2016
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