Development and application of genus-specific peptide arrays for kinome analysis
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Phosphorylation represents a central mechanism to regulate cell function. Protein phosphorylation is catalyzed by a class of enzymes called kinases, the cellular complement of which is referred to as its kinome. The study of the kinome has led to the development of peptide arrays as a high throughput tool. Though the approach was effective, it was limited to genera with characterized phosphoproteomes, as well as by the practice of interpreting emerging data through platforms designed for gene expression data. Within this thesis, these pivotal roadblocks are addressed through the presentation of 1) an approach for the development of custom-designed genus-specific arrays based on proteomic information and 2) a data analysis pipeline, Platform for Integrated, Intelligent Kinome Analysis (PIIKA), developed specifically for peptide array kinome data. The utility of these advances is demonstrated through the creation of the customized peptide arrays and subsequent confirmation of PIIKA-enhanced data transformation and mining. These techniques (custom array and PIIKA) were then applied to two complex host-pathogen interactions: prion diseases and Mycobacterium avium subsp. paratuberculosis (MAP) infection. Prion diseases result from the misfolding of the widely expressed and highly conserved cellular prion protein (PrPC) into an infectious and pathological scrapie-like conformation (PrPSc). Little is known about the function of PrPC in either the normal or diseased states, although a role in signal transduction has been suggested. Two PrPC protein-specific ligands, PrP 106-126 prion fragment and the prion-specific monoclonal antibody 6H4, were used to induce signalling in neuronal cells. Kinome analysis revealed distinct signalling pathways and varied signalling responses for each PrPC ligand. This observation is consistent with the emerging concept that PrPC interacts with multiple cellular proteins and plays a multifunctional role in regulating cellular responses. MAP is the causative agent of Johne’s disease in cattle. MAP establishes a persistent infection and has the ability to evade immune responses while replicating inside macrophages. Kinome analysis indicated that MAP is able to inhibit the interferon gamma (IFNγ)-induced JAK-STAT signalling pathway, eliminating the activation of downstream effectors. Further analysis indicated that the JAK-STAT pathway was blocked at the level of the IFNγ receptor, and JAK-STAT suppressor molecules known as SOCS were activated, both novel findings in the field of MAP pathogenesis. Collectively, these investigations highlight the use of custom-designed and genus-specific peptide arrays to address complex biology in distinct genera.
DegreeDoctor of Philosophy (Ph.D.)
SupervisorNapper, Scott K.
CommitteeLee, Jeremy S.; Roesler, William J.; Misra, Vikram; Griebel, Philip J.; Pato, Mary D.
Copyright DateDecember 2011
Mycobacterium avium subsp. paratuberculosis