Alpha-1 protease inhibitor (alpha1-PI) is a member of the human plasma serine protease inhibitor (serpin) family, which includes alpha1- antichymotrypsin (alpha1-ACT, alpha2-antiplasmin, antithrombin III and C1-inhibitor. Alpha1-PI contributes to maintaining the protease- antiprotease balance by limiting the activity of many serine proteases which contribute to emphysema, adult respiratory distress syndrome (ARDS), atherosclerosis, arthritis, ischaemia reperfusion injury, certain breast cancers, and many other inflammatory conditions. Both alpha1-PI-and alpha1-ACT are members of acute-phase reactants and contain a single conserved cysteinyl residue in their amino acid sequences, respectively. The functional role of the single thiol in alpha1-PI, its mutational variant, and other serpin is important for its activity during oxidative stress associated with infection and at sites of inflammation where neutrophils secrete oxiradicals. This single thiol may also play a similar role in variants of alpha-1-PI and in alpha1-antichymotrypsin. The effect of the formation of the mixed disulfide with cysteine-232 on protein conformation will be measured in native alpha1-PI as well as in variants of alpha1-PI such as preoxidized methionine (Met358 to METs=O), and methionine substituted by valine (Met358) to Val) at the active site in alpha1-PI by employing intrinsic tryptophan fluorescence. The effect of mixed disulfide formation on the structure of the constrained loop in alpha1-PI will be studied by employing circular dichroism (CD) spectroscopy and fluorescence kinetic methods. Inhibition of elastinolysis by alpha1-PI in which the thiol is free or complexed in a mixed disulfide will be explored. Kinetics of disulfide exchange in alpha1-PI will be measured by using glutathione and Ellman's reagent. Presteady state microscopic transient steps in the interaction of neutrophil elastase and alpha1-PI will be measured by employing rapid stopped flow kinetic approaches. Identification of free thiol group(s) in the neutrophil elastase/alpha1-PI complex will be carried out by employing fluorescence probes and amino acid analysis. Binding of alpha1-PI to elastin will be measured by using fluorescence and isoelectric focussing gels. To characterize the general role of the single thiol in other serpins, alpha1-ACT will be examined. The interactions of alpha1-ACT with human neutrophil cathepsin G, the second most abundant protease released in inflammation, will be studied by employing fluorescence and kinetic methods. Studies on the formation of the mixed disulfide in alpha1-PI and other serpins will have implications for understanding the effects of oxidative stresses on the function of these inhibitors, and will help in development of anti- oxidant drugs which can shift the equilibrium between reduced and mixed disulfide alpha1-PI in favor of reduced alpha1-PI.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29GM048595-04
Application #
2186089
Study Section
Pathobiochemistry Study Section (PBC)
Project Start
1992-05-01
Project End
1996-06-30
Budget Start
1995-05-01
Budget End
1996-06-30
Support Year
4
Fiscal Year
1995
Total Cost
Indirect Cost
Name
University of Missouri-Columbia
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
112205955
City
Columbia
State
MO
Country
United States
Zip Code
65211
Tyagi, Suresh C; Hayden, Melvin R (2003) Role of nitric oxide in matrix remodeling in diabetes and heart failure. Heart Fail Rev 8:23-8
Shastry, Suresh; Hayden, Melvin R; Lucchesi, Pamela A et al. (2003) Matrix metalloproteinase in left ventricular remodeling and heart failure. Curr Cardiol Rep 5:200-4
Sood, Harpreet S; Hunt, Matthew J; Tyagi, Suresh C (2003) Peroxisome proliferator ameliorates endothelial dysfunction in a murine model of hyperhomocysteinemia. Am J Physiol Lung Cell Mol Physiol 284:L333-41
Camp, T M; Tyagi, S C; Senior, R M et al. (2003) Gelatinase B(MMP-9) an apoptotic factor in diabetic transgenic mice. Diabetologia 46:1438-45
Mujumdar, Vibhas S; Tummalapalli, Chandra M; Aru, Giorgio M et al. (2002) Mechanism of constrictive vascular remodeling by homocysteine: role of PPAR. Am J Physiol Cell Physiol 282:C1009-15
Cox, Michael J; Sood, Harpreet S; Hunt, Matthew J et al. (2002) Apoptosis in the left ventricle of chronic volume overload causes endocardial endothelial dysfunction in rats. Am J Physiol Heart Circ Physiol 282:H1197-205
Hunt, Matthew J; Tyagi, Suresh C (2002) Peroxisome proliferators compete and ameliorate Hcy-mediated endocardial endothelial cell activation. Am J Physiol Cell Physiol 283:C1073-9
Tyagi, Suresh C; Hoit, Brian D (2002) Metalloproteinase in myocardial adaptation and maladaptation. J Cardiovasc Pharmacol Ther 7:241-6
Sood, Harpreet S; Cox, Michael J; Tyagi, Suresh C (2002) Generation of nitrotyrosine precedes activation of metalloproteinase in myocardium of hyperhomocysteinemic rats. Antioxid Redox Signal 4:799-804
Miller, Amanda D; Tyagi, Suresh C (2002) Mutation in collagen gene induces cardiomyopathy in transgenic mice. J Cell Biochem 85:259-67

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