Khader, Rasha
(2023)
Protein Kinase C-Delta C2 Domain Interactions in Breast Cancer.
PhD thesis, University of Nottingham.
Abstract
The role of protein kinase C-δ (PKC-δ) in breast cancer has been a major focus for a while. PKC-δ expression correlates negatively with the prognosis of breast cancer, and high PKC-δ expression was associated with reduced relapse-free survival in patients. PKC-δ was implicated in breast tumours growth, size, and resistance to treatment. In the cells, PKC-δ regulates many aspects of the cell life including proliferation, apoptosis, gene transcription, and the cytoskeleton. The structure-function relationship in PKC-δ and its cellular localization are very important for its cellular responses. The C2 domain, the first domain in the kinase sequence, serves to regulate its sub-cellular translocation, its interactions, and its stability in the inactive state. This study aims to manipulate PKC-δ through its C2 domain to induce apoptosis in breast cancer cells.
To achieve this, the full-sequence structure of PKC-δ had to be explored. A biology-based homology modelling approach was used to create a three-dimensional (3D) structure of PKC-δ. The 3D structure and the modelling approach were validated using molecular dynamics simulations, and by modelling the closely related PKC isoforms, βII and θ. The structure was mapped to study the C2 domain interactions within the kinase in search for a targeting window to manipulate PKC-δ, biologically and chemically. To biologically manipulate PKC-δ, the C2 domain was overexpressed in the cells and different aspects of cell life were monitored to evaluate the biological effects of the C2 domain on the cell and on the native PKC-δ system. The 3D structure of PKC-δ was also scanned for possible binding pockets to selectively target the kinase chemically at the C2 domain. Virtual high throughput screening was used to screen one million compounds against the C2 domain to find potential HITs. The HITs were evaluated for their biological response and their selectivity towards PKC-δ C2 domain.
The 3D structure of PKC-δ represented the first functionally accurate complete structure of an inactive PKC. It unveiled the relative locations of the domains in the kinase, established essential interdomain interactions, and highlighted several structural features that were never seen in a PKC structure before. At the C2 domain, the 3D structure illustrated a striking interaction between the C2 and V5 domains, which is a novel non-biased finding of this study. This interaction controls the nuclear localization of the kinase in response to apoptotic stimuli. The interaction was used to manipulate the kinase through its C2 domain.
Overexpressing the C2 domain in a bid to manipulate this interaction caused the cells to suffer significant apoptosis, cell cycle arrest, and significant increase in cell size. This in turn caused significant reduction in cell count and viability, which persisted over time. As a ligand of the V5 domain, the overexpressed C2 domain also exhibited cyto-protective effects.
Surface analysis of PKC-δ 3D structure and its isolated C2 domain revealed several druggable pockets in the kinase. Most importantly, three pockets at the C2 domain with the potential to induce apoptosis through PKC-δ. Virtual screening uncovered five HITs, four of which have shown reduced cell viability and increased apoptosis through selective modulation of PKC-δ C2 domain.
This study established the C2 domain as a target to selectively manipulate PKC-δ, but also as a target for selective PKC-δ breast cancer treatments.
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