Our goal is to elucidate mechanisms by which regulatory serine proteases are controlled at and near the cell surface by three protease nexins (PN-1, PN-2 and PN-3) which we identified in earlier studies. The PNs are protein protease inhibitors that are synthesized and released by a variety of cultured cells. They form covalent complexes with certain proteases in the extracellular environment; the complexes bind back to the cells and are rapidly internalized and degraded. With purified PN-1 and seven regulatory proteases it inactivates, we will measure second order association rate constants to provide a quantitative foundation for our studies. Then, based on our finding that the surface of fixed fibroblasts accelerates the reaction between PN-1 and thrombin, we will determine if the reactions between PN-1 and the other proteases are similarly accelerated. Some of the acceleration of the reaction between PN-1 and thrombin appears to involve cell surface/extracellular matrix glycosaminoglycans. We will check this by treating fibroblasts with glycosaminoglycan lyases, fixing the cells, and examining the effect of this on the ability of the cells to accelerate the reactions. In complimentary studies we will examine the effects of purified glycosaminoglycans, in the absence of cells, on the reactions between PN-1 and the proteases. About 50% of the acceleration of the PN-1 and thrombin reactions appears to involve the Mr=150,000 cell surface binding sites for thrombin. We will study the basis of this and determine if there are cell surface binding sites for the six other regulatory proteases that might similarly participate in their control. With PN-2 that we recently purified to homogeneity, we will screen for serine proteases it effectively inactivates, measure second order association rate constants with them, and begin studies on PN-2 along the lines of the above studies for PN-1. Finally, we will purify PN-3 from serum-free culture medium conditioned by fibroblasts.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Research Project (R01)
Project #
5R01AG010598-12
Application #
2051818
Study Section
Pathobiochemistry Study Section (PBC)
Project Start
1991-09-01
Project End
1996-08-31
Budget Start
1994-09-01
Budget End
1995-08-31
Support Year
12
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of California Irvine
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
161202122
City
Irvine
State
CA
Country
United States
Zip Code
92697
Guttridge, D C; Lau, A; Tran, L et al. (1997) Thrombin causes a marked delay in skeletal myogenesis that correlates with the delayed expression of myogenin and p21CIP1/WAF1. J Biol Chem 272:24117-20
Guttridge, D C; Cunningham, D D (1996) Characterization of the human protease nexin-1 promoter and its regulation by Sp1 through a G/C-rich activation domain. J Neurochem 67:498-507
Donovan, F M; Vaughan, P J; Cunningham, D D (1994) Regulation of protease nexin-1 target protease specificity by collagen type IV. J Biol Chem 269:17199-205
Vaughan, P J; Cunningham, D D (1993) Regulation of protease nexin-1 synthesis and secretion in cultured brain cells by injury-related factors. J Biol Chem 268:3720-7
Cunningham, D D; Pulliam, L; Vaughan, P J (1993) Protease nexin-1 and thrombin: injury-related processes in the brain. Thromb Haemost 70:168-71
Guttridge, D C; Lau, A L; Cunningham, D D (1993) Protease nexin-1, a thrombin inhibitor, is regulated by interleukin-1 and dexamethasone in normal human fibroblasts. J Biol Chem 268:18966-74