Abstract 12207: NEDD9 Cysteinyl Thiol Oxidation Impairs NEDD9 Binding by Smad3 to Increase Pulmonary Endothelial Matrix Metalloproteinase-2 Proteolytic Activity

Abstract

In pulmonary arterial hypertension (PAH), endothelial dysfunction due to increased oxidant stress induces adverse vascular remodeling. In cancers that share overlap with PAH pathobiology, the CAS-L protein NEDD9 upregulates the remodeling enzyme MMP-2. Clearance of NEDD9 requires Smad3 binding to its protein-docking site, which includes Cys18. We hypothesized that NEDD9 Cys18 oxidation impairs NEDD9-Smad3 binding to increase NEDD9 and MMP-2 activity in pulmonary artery endothelial cells (PAECs). To test this hypothesis, cells were treated with hydrogen peroxide (H 2 O 2 ) (250 μM) for 2.5, 5.0, and 7.5 min and NEDD9 levels were assessed by immunoblot. Compared to time 0, H 2 O 2 increased NEDD9 levels by 2.4-, 2.5-, and 3.1-fold, respectively (P<0.05). Next, structural homology to NEDD9 was modeled after 1wyx, 2181, and 3t6g, and the pKa and accessible surface area (ASA) of 10 putative Cys sulfur atoms was analyzed. By this approach, Cys18 (pKa=6.65; ASA=17.5 Å 2 ) was predicted as the most reactive cysteinyl thiol. Mass spectrometry performed on in-gel trypsin digested samples of immunoprecipitated NEDD9 from PAECs treated with H 2 O 2 (500 μM) for 20 min confirmed Cys18 oxidation to cysteinyl sulfonic acid. To determine if Cys18 oxidation was associated with changes in NEDD9-Smad3 binding, PAECs were treated with vehicle (V) control, the pro-oxidant hormone aldosterone (ALDO) (10 -7 mol/L) for 24 hr, or H 2 O 2 (500 μM) for 20 min and immunoprecipitated using an antibody to the Cys18 region of NEDD9. Anti-cysteine sulfenic acid (R-SOH) immunoblotting showed that compared to V-treated cells, ALDO and H 2 O 2 increased NEDD9-SOH levels by 54% and 73%, respectively (P<0.04). In turn, increased NEDD9-SOH induced by ALDO or H 2 O 2 decreased NEDD9-Smad3 binding by 64% (P=0.08) and 87% (P<0.01), respectively. Compared to V-treated cells, ALDO also increased MMP-2 proteolysis by zymography (184 ± 4.9 vs. 248 ± 11 a.u., P<0.01), which was inhibited by 38% (P<0.05) in ALDO-treated cells transfected with si-NEDD9. Oxidative modification of NEDD9 Cys18 is a novel molecular mechanism regulating NEDD9 in PAECs in vitro. Identifying NEDD9 Cys18 as a novel target to inhibit endothelial remodeling proteins may have important therapeutic implications for patients with PAH.