In recent studies we demonstrated that systemic levels of protein-bound nitrotyrosine (NO 2 Tyr) and myeloperoxidase (MPO), a protein that catalyzes generation of nitrating oxidants, serve as independent predictors of atherosclerotic risk, burden, and incident cardiac events. We now show both that apolipoprotein A-I (apoA-I), the primary protein constituent of HDL, is a selective target for MPO-catalyzed nitration and chlorination in vivo and that MPO-catalyzed oxidation of HDL and apoA-I results in selective inhibition in ABCA1-dependent cholesterol efflux from macrophages. Dramatic selective enrichment in NO 2 Tyr and chlorotyrosine (ClTyr) content within apoA-I recovered from serum and human atherosclerotic lesions is noted, and analysis of serum from sequential subjects demonstrates that the NO 2 Tyr and ClTyr contents of apoA-I are markedly higher in individuals with cardiovascular disease (CVD). Analysis of circulating HDL further reveals that higher NO 2 Tyr and ClTyr contents of the lipoprotein are each significantly associated with diminished ABCA1-dependent cholesterol efflux capacity of the lipoprotein. MPO as a likely mechanism for oxidative modification of apoA-I in vivo is apparently facilitated by MPO binding to apoA-I, as revealed by cross-immunoprecipitation studies in plasma, recovery of MPO within HDL-like particles isolated from human atheroma, and identification of a probable contact site between the apoA-I moiety of HDL and MPO. To our knowledge, the present results provide the first direct evidence for apoA-I as a selective target for MPO-catalyzed oxidative modification in human atheroma. They also suggest a potential mechanism for MPO-dependent generation of a proatherogenic dysfunctional form of HDL in vivo.that promote oxidative damage, cell injury, and conversion of LDL, the major carrier of cholesterol in plasma, into an atherogenic form (9, 14). Protein-bound nitrotyrosine (NO 2 Tyr), a posttranslational modification specific for protein oxidation by , is markedly enriched within human atheroma (8, 21). Further, recent clinical studies demonstrate that systemic levels of protein-bound NO 2 Tyr serve as an independent predictor of atherosclerotic risk and burden in subjects and are modulated by known CVD risk-reducing therapies such as statins (10,22). Few studies to date have focused on defining the molecular targets of nitration in subjects with CVD, the attendant functional alterations, and the enzymatic participants in nitration.One potential enzymatic source for generation of NO-derived oxidants within human atheroma is the heme protein myeloperoxidase (MPO). MPO utilizes hydrogen peroxide (H 2 O 2 ) and a variety of low-molecular weight organic and inorganic substances as substrates to form reactive oxidant species capable of promoting protein halogenation, nitration, and oxidative cross-linking (4, 5). For example, MPO directly utilizes both NO (23) and the NO metabolite nitrite (NO 2 − ) as substrates in vitro (17-19, 24) and participates
In recent studies we demonstrated that systemic levels of protein-bound nitrotyrosine (NO(2)Tyr) and myeloperoxidase (MPO), a protein that catalyzes generation of nitrating oxidants, serve as independent predictors of atherosclerotic risk, burden, and incident cardiac events. We now show both that apolipoprotein A-I (apoA-I), the primary protein constituent of HDL, is a selective target for MPO-catalyzed nitration and chlorination in vivo and that MPO-catalyzed oxidation of HDL and apoA-I results in selective inhibition in ABCA1-dependent cholesterol efflux from macrophages. Dramatic selective enrichment in NO(2)Tyr and chlorotyrosine (ClTyr) content within apoA-I recovered from serum and human atherosclerotic lesions is noted, and analysis of serum from sequential subjects demonstrates that the NO(2)Tyr and ClTyr contents of apoA-I are markedly higher in individuals with cardiovascular disease (CVD). Analysis of circulating HDL further reveals that higher NO(2)Tyr and ClTyr contents of the lipoprotein are each significantly associated with diminished ABCA1-dependent cholesterol efflux capacity of the lipoprotein. MPO as a likely mechanism for oxidative modification of apoA-I in vivo is apparently facilitated by MPO binding to apoA-I, as revealed by cross-immunoprecipitation studies in plasma, recovery of MPO within HDL-like particles isolated from human atheroma, and identification of a probable contact site between the apoA-I moiety of HDL and MPO. To our knowledge, the present results provide the first direct evidence for apoA-I as a selective target for MPO-catalyzed oxidative modification in human atheroma. They also suggest a potential mechanism for MPO-dependent generation of a proatherogenic dysfunctional form of HDL in vivo.
Hydroxyl radical reacts with the aliphatic C-H bonds of amino acids by H atom abstraction. Under anaerobic conditions inclusion of a (2)H atom donor results in (1)H/(2)H exchange into these C-H bonds [Goshe et al. Biochemistry 2000, 39, 1761--1770]. The site of (1)H/(2)H exchange can be detected and quantified by (2)H NMR. Integration of the (2)H NMR resonances within a single spectrum permits the relative rate of H atom abstraction from each position to be determined. Analysis of the aliphatic amino acid spectra indicates that the methine and methylene positions were more reactive than the methyl positions. The (2)H NMR spectra of isoleucine and leucine show that H-atom abstraction distal to the alpha-carbon occurs preferentially. Significant (1)H/(2)H exchange was observed into the delta positions of proline and arginine and into the epsilon-methylene of lysine, indicating that a positive charge on a geminal N does not inhibit the (1)H/(2)H exchange. Comparisons of (2)H NMR integrations between amino acid spectra indicated that (1)H/(2)H exchange occurred in the following descending order: L > I > V > R > K > Y > P > H > F >M> T > A > [C, S, D, N, E, Q, G, W]. The extent of (1)H/(2)H exchange into methionine, N-glycyl-methionine, and methionine sulfoxide suggests that a prominent solvent exchange pathway involving hydroxyl radical mediated oxidation of methionine exists to account for the large (2)H incorporation into the gamma-methylene of methionine sulfoxide that is absent for N-glycyl-methionine. Analysis of the (1)H NMR spectra of the reactions with phenylalanine and tyrosine indicated that hydroxyl radical addition to the phenyl ring under the anaerobic reductive reaction conditions did not result in either exchange or hydroxylation.
Plasminogen activator inhibitor type 1 (PAI-1) plays key regulatory roles in fibrinolysis, cell migration, and tissue remodeling. A regulatory protein without known catalytic activity, PAI-1 modulates plasminogen activators through protein-protein interactions. Although global conformational alterations that occur in PAI-1 determine its regulatory activity, comprehensive assessments of concurrent dynamic, structural, and functional alterations of this critical regulatory protein have not yet been clearly defined. X-ray crystallographic studies have described four distinct PAI-1 conformational states: active, latent, reactive center loop peptide-annealed (RCL-PA), and cleaved mutant. In this study, backbone amide hydrogen-deuterium exchange detected by mass spectrometry was used to characterize dynamic and structural alterations of human PAI-1 (hPAI-1) in relation to its function. hPAI-1 conformers were defined by surface mapping the solvent-accessible sites for strategic secondary structural components of the protein. We observed a global protection from solvent for a majority of peptides in the latent conformer relative to the active conformer. Significant differences were observed in the RCL, helix A, helix D, and sheet 1C, and these regions were markedly more dynamic or solvent-exposed in the active conformation. The RCL-PA form adopts an intermediate conformational state between the active and the latent conformers. Our results demonstrate that the most dynamic regions of PAI-1 (the RCL, helices D and A, and sheet 5A) are flexible in the transition toward latency. They also show that the dynamic surface structures of the active, latent, and peptide-annealed conformers of PAI-1 are underestimated by theoretical solvent accessibility calculations derived from crystallographic data. Plasminogen activator inhibitor type 1 (PAI-1)1 is a key regulator of fibrinolysis, cell migration, and tissue remodeling (1-3). As a member of the serine protease inhibitor family, PAI-1 serves as a major inhibitor of the proteases tissue plasminogen activator and urokinase plasminogen activator (uPA) (4, 5). Recent clinical studies suggest important links between PAI-1 and accelerated development of atherosclerosis and a prothrombotic state, with attendant enhanced risks for development of myocardial infarction, stroke, and thromboembolic events (6 -9). Animal model studies further demonstrate that PAI-1 plays a critical role in ventricular remodeling following an acute myocardial infarction, a process important in the development of heart failure (10). The many links between PAI-1, cardiovascular disease, and thrombosis have thus made this regulatory protein a recent focus of intense research interest. Despite this, the structural and dynamic conformational alterations that underlie the regulatory functions of PAI-1 are incompletely understood.In vivo, synthesized PAI-1 is secreted and bound to an adhesive, stabilizing glycoprotein, vitronectin, in a noncovalent complex (11). PAI-1 bound to vitronectin remains folded in an "activ...
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