S-Nitrosylation of Rap1 and Relationship to Rap1 Activity and Disease Status In SCD

Abstract 2662

Red cell adhesion appears to be a critical contributor to vaso-occlusion in sickle cell disease (SCD). We have been interested in intra-erythrocytic signaling events that can affect sickle red cell (SS RBC) adhesion and thereby stimulate vaso-occlusion. One such agonist-inducible signaling pathway is that of the β2 adrenergic receptor. β2 adrenergic receptor signaling pathways, involving both protein kinase A and the small guanosine triphosphatase Rap1, have been shown to affect multiple RBC adhesion receptors. Therefore, we sought to determine whether specific post-translational modifications of RBC membrane and intracellular proteins were associated with the signaling pathways affecting adhesion. Protein S-nitrosylation, a post-translational modification of cysteine thiol by nitric oxide (NO), is one of several post-translational protein modifications that can regulate diverse cell signaling pathways. Given the many reports of abnormal NO biology in SCD, we investigated how S-nitrosylation of erythrocyte proteins affects molecules involved in regulation of SS RBC adhesion. First, we identified RBC membrane proteins that underwent S-nitrosylation in RBCs from healthy donors (n=2) and SCD patients (n=6; three patients treated with hydroxyurea [HU] and three without HU treatment). Highly purified RBC membrane ghosts were pretreated with or without 0.05 mM CysNO (to promote S-nitrosocysteine formation) for 10 min, and S-nitrosylated proteins were isolated using the resin-assisted capture (SNO-RAC) method (Forrester et al. 2009). Captured S-nitrosylated proteins were visualized by SDS-PAGE, and protein identification was accomplished via an in-situ digestion of the bead-bound proteins, release of the residual bead-bound peptides, and analysis using nanoscale capillary liquid chromatography coupled to high resolution, accurate mass tandem mass spectrometry (LC/MS/MS). A number of proteins underwent S-nitrosylation in both normal and SS RBCs. Among these, MS analysis indicated that Rap1, which has previously been shown to affect SS RBC adhesion to laminin, underwent S-nitrosylation much more extensively in SS than in normal RBCs. These data were confirmed by SNO-RAC followed by immunoblotting. Using a RalGDS Ras-binding domain pull-down assay, we further quantitated the amount of detectable activated Rap1 in highly purified preparations of normal and SS RBCs (lacking either contaminating leukocytes or platelets), before and after RBC loading with NO. Active Rap1 was undetectable or minimally present in RBCs from healthy donors (n=6) but easily detectable in all SS RBCs (n=4). Loading of RBCs with NO in these same samples led to an increased amount of detectable activated Rap1 in both normal and SS RBCs. Finally, we found that patients admitted for pain episodes (n=3) had higher levels of Rap1 nitrosylation and activity than patients in steady state (n=5). In summary, we have found that Rap1, a protein that acts downstream of adrenergic signaling in RBCs, undergoes S-nitrosylation and that this modification is associated with increased Rap1 activity. Most importantly, SS RBCs contain significantly more S-nitrosylated Rap1 and activated Rap1 than do normal RBCs, and this difference in content of active Rap1 is more pronounced in patients with ongoing vaso-occlusion.