This proposal outlines a training program which will contribute to our understanding of the biochemical properties and biological functions of serine protease inhibitor (serpins) as well as provide me with a foundation for a career as an independent investigator in the molecular and cellular biology of the regulation of lung inflammation. We are interested in studying the interactions of serpins and neutrophils in maintaining or altering the protease antiprotease balance during inflammation. Neutrophil serine proteases, especially elastase (HNE), have been implicated as major mediators or acute lung injury. The serpins alpha-1-protease inhibitor (alpha1PI) and alpha-1- antichymotrypsin (ACT) are the predominant inhibitors of neutrophil serine proteases in the plasma and lung parenchyma. alpha1PI is the predominant HNE inhibitor. ACT and alpha1PI may inhibit neutrophil reactive oxygen species (ROS0 production by the NADPH oxidase system. The neutrophil, on the other hand, may form microenvironments where alpha1PIs inhibitory activity is attenuated by neutrophil ROS or cleavage by neutrophil metalloproteinases (NMPs) and non-target serine proteases. We have extensive experience producing recombinant variants of serpins with altered inhibitory activity and other altered biochemical characteristics. The studies described in this proposal use in vitro models of extracellular matrix degradation by activated neutrophils and recombinant variants of alpha1PI that are designed to resist ROS modification and/or NMP cleavage in order to examine the hypotheses (1) neutrophil derived ROS and (2) NMPs play a role in inflammation, in part, by decreasing alpha1PI activity at sites of inflammation. We also examine the hypotheses that alpha1PI and ACT regulate (1) neutrophil ROS production and (2) NMP activation thereby limiting the neutrophil's ability to decrease serpin activity at sites of inflammation. The role of HNE mediated cleavage of ACT on altering the activity of ACT at sites of inflammation will also be examined.
The Specific Aims based on these hypotheses include: (1) evaluate the effect of wild type and variant serpins on ROS production; (2) design oxidation resistant alpha1PI variants and compare them to wild type alpha1PI with regard to efficacy in protecting model extracellular matrix, and determine if serpin mediated ROS inhibition augments the efficacy of wild type alpha1PI; (3) evaluate the effect of wild type and variant serpins on NMP activation; (4) design NMP-resistant alpha1PI variants and compare them to NMP- sensitive alpha1PI variants with regard to efficacy in protecting model extracellular matrix, and determine if serpin mediated inhibition of NMP activation augments the efficacy of NMP-sensitive alph1PI variants; (5) design recombinant ACT variants that are relatively resistant to HNE cleavage and NMP cleavage and compare the effectiveness of these variants with wild type ACT in the augmentation studies.
| Plotnick, Michael I; Samakur, Madhurika; Wang, Zhi Mei et al. (2002) Heterogeneity in serpin-protease complexes as demonstrated by differences in the mechanism of complex breakdown. Biochemistry 41:334-42 |
| Plotnick, Michael I; Rubin, Harvey; Schechter, Norman M (2002) The effects of reactive site location on the inhibitory properties of the serpin alpha(1)-antichymotrypsin. J Biol Chem 277:29927-35 |
| Plotnick, M I; Schechter, N M; Wang, Z M et al. (1997) Role of the P6-P3' region of the serpin reactive loop in the formation and breakdown of the inhibitory complex. Biochemistry 36:14601-8 |
