OP1 Jagged1 redox signalling regulates endothelial cell cycle, adhesion and extracellular matrix pathways

Jagged1 has essential roles in cardiovascular development and maintenance by regulating angiogenesis, which is tightly controlled by redox signalling in endothelial cells (EC). Proteins sense redox signals through certain cysteine residues forming oxidative post translational modifications (ox-PTM) which alter protein function. Iodoacetyl-tandem mass tag (Iodo-TMT) redox proteomics was performed to identify the range of redox-sensitive proteins involved in angiogenesis. To induce changes in redox modifications we used overexpression of Glutaredoxin-1 (Glrx1), which catalyses the removal of S-glutathionylation, a common ox-PTM. Human microvascular ECs (HCMVEC) were transduced with adenoviral Glrx1 or adLacZ (control) and treated with VEGF (50 ng/ml, 24h) or hypoxia (0.5% O2, 24h). IodoTMT thiol labelling revealed 113 cysteine modifications across 78 proteins which were altered by treatment, including Jagged1, a pivotal angiogenic regulator which has yet to be observed under oxidative control. To assess the effects of redox signalling on Jagged1 (Jag1) function redox-insensitive variants were generated using cysteine to serine substitutions. Quantitative proteomics revealed changes in adhesion, extracellular matrix and cell cycle related proteins in endothelial cells overexpressing a 'redox dead' (RD) Jag1 variant. Investigation into localisation showed impaired trafficking of redox-insensitive variants to the plasma membrane. Additionally, the formation of a Jag1 intracellular domain (ICD) fragment recently shown to have direct transcriptional activity is decreased in redox-insensitive variants. Importantly, oxidative treatment also alters the abundance of this ICD fragment in WT Jag1 only. Together, this evidence suggests redox signalling regulates Jag1 ICD fragment formation, altering important EC processes including cell cycle, adhesion, and extracellular matrix.