A reverse genetics-based deubiquitinase (DUB) screen for VEGFR2 stability and turnover in angiogenesis

The biological phenomenon of angiogenesis is the sprouting of new blood vessels from pre-existing ones. This process is required for vascular homeostasis, revascularisation and wound healing. This physiological phenomenon can be subverted in pathological states such as cancer, age-related macular degeneration and cardiovascular disease. Vascular endothelial growth factor A (VEGF-A) is a multitasking growth factor that controls various aspects of vascular function with vital roles in animal health and disease. VEGF-A transmits proangiogenic signals upon binding vascular endothelial growth factor receptor 2 (VEGFR2) and activating downstream signal transduction pathways. Different studies indicate that VEGFR2 undergoes steady-state and VEGF-regulated ubiquitination, which impacts VEGFR2 trafficking and proteolysis, thus influencing signalling and endothelial cell responses. The central hypothesis of this thesis is that deubiquinases (DUBs) which target VEGFR2 affect both steady-state and VEGF-A-stimulated endothelial responses. To identify such DUBs, an unbiased screen of the human DUB family was carried out to assess their effects on VEGFR2. A reverse genetics approach using small inhibitory RNA (siRNA) pools combined with a high throughput microscopy-based assay was used to deplete specific DUB levels and assess effects on steady-state or activated VEGFR2. The results of this study show that VEGFR2 levels were either unchanged, elevated or decreased upon specific DUB knockdown. Strikingly, the data suggest that multiple DUBs have distinct and compartmentalised roles in regulating VEGFR2 trafficking, turnover or both. One such DUB, USP2, is implicated in biosynthetic VEGFR2 trafficking, whereas USP38 is involved in the distal plasma membrane and endosomal trafficking. These findings suggest that targeting USP2 or USP38 could provide new anti-angiogenic strategies in disease therapy.