The serpins are a homologous family of blood proteins that regulate the proteolytic cascades of inflammation, coagulation and fibrinolysis. They do this by forming tight, equimolar complexes with specific target proteinases, and the resulting complexes are rapidly removed from circulation. Congenital or acquired serpin deficiencies, particularly those of alpha/1 proteinase inhibitor (alpha/1 PI) and antithrombin III, lead to connective tissue and coagulation diseases, due to unregulated target enzyme activity. Recently, supplemental therapy of these serpins, purified from blood, has been used in attempts to ameliorate the diseases and the proteins have acquired orphan drug status. Though the biological roles of these serpins are fairly well established, the mechanism they use to inhibit their target enzymes is far from clear. This lack of knowledge has important clinical implications, for it remains unknown whether long- term administration is without consequence, and whether these specific inhibitors are the best reagents for controlling the unregulated target enzymes. A far more complete description of the serpin inhibitory mechanism is essential before we can understand why the body employs serpins as its primary defense against its own proteinases, and whether their clinical use can be extended. Much of our understanding of proteinase inhibitory mechanisms comes from X-ray analyses of the small """"""""standard mechanism inhibitors"""""""" that inhibit proteinases by positioning a relatively rigid reactive site loop (RSL) in a conformation complementary to the proteinase substrate binding pocket. Though X-ray structures of serpins are available none of them are in an inhibitory conformation, so other techniques are required to investigate the mechanism. This proposal is based on the hypothesis that serpins act as standard mechanism inhibitors, but that their RSLs are more mobile than most because of their ability to interact with units of secondary structure special to serpins. We will examine this hypothesis by concentrating on the following three serpins: alpha/1 PI, alpha/1 antichymotrypsin (alpha/1 ACT), and hen ovalbumin. These are serpins whose structures are most well documented, and who show the most deviation in length and structure of their RSLs. Specifically, we will perform site-directed mutagenesis on residues at the junctions of specific units of secondary structure. We will test the constructs for inhibition of target proteinases, for thermodynamic stability and structural integrity.
| Fuentes-Prior, Pablo; Salvesen, Guy S (2004) The protein structures that shape caspase activity, specificity, activation and inhibition. Biochem J 384:201-32 |
| Boatright, Kelly M; Salvesen, Guy S (2003) Caspase activation. Biochem Soc Symp :233-42 |
| Boatright, Kelly M; Salvesen, Guy S (2003) Mechanisms of caspase activation. Curr Opin Cell Biol 15:725-31 |
| Turk, Boris; Turk, Dusan; Salvesen, Guy S (2002) Regulating cysteine protease activity: essential role of protease inhibitors as guardians and regulators. Curr Pharm Des 8:1623-37 |
| Stennicke, Henning R; Ryan, Ciara A; Salvesen, Guy S (2002) Reprieval from execution: the molecular basis of caspase inhibition. Trends Biochem Sci 27:94-101 |
| Riedl, S J; Renatus, M; Snipas, S J et al. (2001) Mechanism-based inactivation of caspases by the apoptotic suppressor p35. Biochemistry 40:13274-80 |
| Snipas, S J; Stennicke, H R; Riedl, S et al. (2001) Inhibition of distant caspase homologues by natural caspase inhibitors. Biochem J 357:575-80 |
| Renatus, M; Zhou, Q; Stennicke, H R et al. (2000) Crystal structure of the apoptotic suppressor CrmA in its cleaved form. Structure 8:789-97 |
| Deveraux, Q L; Stennicke, H R; Salvesen, G S et al. (1999) Endogenous inhibitors of caspases. J Clin Immunol 19:388-98 |
| Yang, X; Stennicke, H R; Wang, B et al. (1998) Granzyme B mimics apical caspases. Description of a unified pathway for trans-activation of executioner caspase-3 and -7. J Biol Chem 273:34278-83 |
Showing the most recent 10 out of 18 publications
