Peptide Based Biosensors And Mass Spectrometric Methods To Measure The Activation Of Tyrosine Kinases
Cell signaling is a complex web of protein-protein interactions, post translational modifications and gene transcription events working in concert to control all cellular events from cell proliferation to cell death. These signals may arise externally or from within the cell. Tight regulation of signaling events is critical to proper function as many interactions are used and re-used to control multiple cellular responses. Damage to cellular macromolecules such as DNA and protein can progress to dysregulated signaling. Prolonged exposure to toxins, radiation, UV light and aging; are known damage causative agents and risk factors to DNA and protein integrity. The consequences of dysregulated signaling may lead to diseases such as cancers and neurodegeneration. Protein phosphorylation is a post-translational modification mainly observed on Serine (S), Threonine (T), and Tyrosine (Y) amino acids. Tyrosine phosphorylation accounts for roughly 1% of the total phospho-amino acid modifications observed. Modification of specific tyrosine residues by the addition of a phosphate groups is catalyzed by protein tyrosine kinases. Substrate recognition by protein tyrosine kinases is controlled by the interaction of the amino acids of both the peptide sequence and kinase catalytic domain. Additional domains that mediate protein-protein interactions have been shown to influence the activation state of the kinase as well as enhance the interaction between the substrate and catalytic domain. However, multiple tyrosine kinases may yet modify the same tyrosine residue. To de-convolute the role of individual tyrosine kinases, we can use exogenous peptide-based biosensors with demonstrated kinase selectivity. This is done by identifying a tyrosine kinase substrate that demonstrates selectivity toward a target tyrosine kinase over other competing kinases. Exogenous substrates offer many benefits to the chemist including the addition of functionalities intended to improve kinase interaction, cellular uptake, and post-assay analysis. In addition, exogenous substrates give better control of the amount of substrate used both in the assay and the analysis. The nature of peptide-based biosensors makes them ideal target substrates, but they are vulnerable to native degradation mechanisms of the cell. In this work, we describe strategies for developing peptide-based biosensors, which demonstrate resistance to endogenous proteases, while remaining recognizable by the kinase responsible for their phosphorylation. The first strategy is a retro-inverso (RI) modification of an ABL kinase selective reporter module, Abltide. This describes the first retro-inverso tyrosine kinase substrate in the literature. In the second strategy, we selectively incorporated D-amino acids into the Abltide reporter module. Mass spectrometry is a field of analysis that can be applied to the identification of multiple proteins within a cell matrix and targeted quantitation of expression and modification of specific peptides and proteins. We applied mass spectrometry to analyze exogenous biosensors to study recombinant tyrosine kinase activation within a variety of immortalized cell lines. Both substrates were phosphorylated by ABL kinase and demonstrated protease resistance. In Chapter 3 we studied the phosphorylation of alpha-synuclein (aSyn) by recombinant tyrosine kinase families, ABL, SRC and SYK. We applied mass spectrometric analysis to measure the phosphorylation of aSyn on tyrosine residues Y39 and Y125. In addition, we quantified the expression of aSyn expression in SH-SY5Y immortalized cells. In this study we confirmed the site specificity of the tyrosine kinases studied and developed sample preparation techniques to quantify the expression of aSyn without enrichment. The successful application of the sample preparation technique in this work to patient samples may help improve the analysis of aSyn expression in Parkinson’s disease patients.
Rochet, Purdue University.
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