Cell cycle dysfunction, increased oxidant production and altered metabolism are widely associated with hyper-proliferative vascular diseases. Understanding the link is crucial for developing therapeutics to halt vascular remodelling or promote its regression. Cell-cycle CDKs have recently been linked to metabolism, so the project will test if CDKs’ oxidative modifications can cause cellular metabolism and/or proliferation changes. We identified a novel mechanism of CDK4-Cyclin D regulation through oxidation at a critical allosteric site that is essential for activity, causing cells stably expressing ‘redox-null’ CDK4 to proliferate at slower rates. We also characterised thiol oxidation sites in the functionally relevant CDK2-cyclin A domain; however, the metabolic role of these novel observations and their biological importance in disease are to be determined.
Aim 1. Generate CDK2 and CDK4 knockout cell lines, followed by wild-type or cysteine mutant protein reintroduction using lentiviruses to make stable cell lines. These wild-types or ‘redox-null’ cells will undergo characterisation, including assessment of kinases’ redox state and activity, substrate phosphorylation, proliferation and cell cycle progression assays in response to oxidants or growth factor stimulation.
Aim 2. Identify specific genes involved in oxidant-induced cell cycle arrest by RNA-sequencing in the above cells, validate and characterise differentially expressed genes and pathways of interest identified by using qPCR, immunoblotting or confocal imaging.
Aim 3. Compare metabolic profiling of wild-type or ‘redox-null’ cells at different cell cycle phases.
Aim 4. Validate in vitro findings in studies using a novel ‘redox-null’ transgenic mouse and/or vascular cells from donors/patients with Group 1 PH.