NMR is able to detect site-specific changes associated with posttranslational modifications (49, 50, 51)

NMR is able to detect site-specific changes associated with posttranslational modifications (49, 50, 51). is found in 97% of Caucasian individuals over the age of 50 and is the most common SH-4-54 form of localized amyloid. SH-4-54 It is found in close association with the internal elastic lamina, an elastin-rich layer that supports the endothelium and allows for arterial stretch (1, 2). The main constituent of AMA is a 50 amino acid polypeptide called medin (3). Experimental evidence suggests that prefibrillar intermediates of medin are toxic to aortic endothelial cells in vitro (4) and may underlie the pathogenesis of sporadic thoracic aortic aneurysm in vivo through weakening of the aortic wall (5). Molecular information about the mechanisms that trigger medin aggregation and the formation of insoluble fibrils is sparse. Posttranslational modifications occur in vivo in many proteins and SH-4-54 may represent a mechanism for modulating fibril formation. Oxidative or nitrative stress results when there is a loss of balance between oxidant formation and antioxidant removal through a defense mechanism (e. g., superoxide dismutase) (6). When this physiological balance is weakened, nitroxidative stress emerges and causes damage, including nitration of tyrosine residues. Superoxide is produced in endothelial cells, smooth muscle cells, and cardiomyocytes, and nitric oxide (NO) plays a role in maintaining cellular homeostasis in the cardiovascular system (7, 8, 9). Tyrosine nitration is a normal process, with low levels found in standard cells. However , elevated levels of 3-nitrotyrosine have been reported in a range of human pathologies and animal models of diseases, including atherosclerosis (10) and myocardial malfunction (11), and upon aging (12). Nitration has been shown to modulate the aggregation properties of proteins associated with neurodegenerative diseases, such as tau (13), -amyloid (14), and-synuclein (15, 16), and has been identified in disease-associated amyloid deposits (17, 18). Nitrotyrosine levels are significantly higher in human atherosclerotic lesions associated with coronary heart disease, and decrease after treatment with statin, an indirect antioxidant (19). In this work, we assess a possible driving factor in amyloid formation, specifically, whether nitration of medin alters its in vitro aggregation and toxicological properties. As medin is found in an environment rich with oxidative species, SH-4-54 we propose that nitration could play a role in mediating fibril formation of the protein, as has been shown for other amyloid proteins. We present data that suggest that medin could induce oxidative conditions in vivo and investigate the role that these conditions play in AMA pathogenicity. == Materials and Methods == == Expression of protein == Unlabeled and13C, 15N isotope-labeled medin was expressed using pOPINS-medin in Lemo 21 (DE3) cells (20). Cells were induced at OD6000. 81. 0 with isopropyl–D-thiogalacto-pyranoside (1 mM) for 16 h at 20C. Cells were harvested by centrifugation (3, 000 g, 20 min, 4C). Pellets were resuspended in 6 M guanidine hydrochloride (GdmCl), 0. 5 M sodium phosphate, 20 mM NaCl, pH 8. 0, and frozen at 20C. The cells were homogenized and cell debris Rabbit Polyclonal to NOC3L was removed by centrifugation (19, 000 g, 15 min, 4C). The supernatant was loaded onto a 5 mL Ni2+-NTA column and washed with 4 column volumes (CV) of 6 M GdmCl, pH 8, followed by 4 CV of 6 M GdmCl, pH 6. It was eluted with 3 CV of 6 M GdmCl, pH 2, and stored at 20C. Fusion protein was buffer exchanged into 20 mM Tris-Cl, 0. 5 M NaCl, pH 7. 4, and the His6-SUMO tag was removed by incubation with SUMO protease I at 4C for 3 h. The cleavage mixture was then passed through a 5 mL Ni2+-NTA column and the flow-through containing medin was collected and characterized by matrix-assisted laser desorption and ionization mass spectrometry. Medin was buffer exchanged for different applications as described below. == NO and superoxide production == Human umbilical vein endothelial cells (HUVECs; Lonza, Walkersville, MD; passages 410) were exposed to medin (0. 15M in 20 mM sodium phosphate, 20 mM NaCl, pH 7. 4) for 2024 h. NO gas was measured with the use.