We propose to investigate via NMR spectroscopy the structure and function of genomic protein modules and multimodular arrays related to plasminogen (Pgn) and its activators urokinase (uPA) and tissue Pgnactivator (tPA). These proteins are found both in blood plasma and in the extracellular matrix (ECM) where they play crucial roles in the fibrinolytic dissolution of blood clots, cell proliferation and migration, embryogenesis, tissue remodeling metastasis, etc. Pgn, tPA and uPA contain kringle (K) domains that mediate their binding to specific substrates. The Pgn kringles interact with the inhibitor a2-antiplasmin C- and the Pgn N-terminal peptide domains. Interestingly, the Pgn KI, K2, K3, K5, their tandem arrays K123, K1234 (angiostatin), and K12345, as well as the uPA peptide KPSSPPEE 143 inhibit endothelial cell growth and migration, thus tissue and tumor vascularization. Antiangiogenic activities also are displayed by the blood plasma/ECM proteins kininogen D5 (a Zn2+-binding domain) and thrombospondin 2nd type-1 domain. Implicated in tissue remodeling is the matrix metalloproteinase 2, which digests denatured collagen (gelatin) via adhesion through three fibronectin type II domains. Activation of Pgn by uPA, a key step in metastatic cell propagation, involves a membrane-anchored receptor (uPAR) which contains three snake neurotoxin-type modules. Related to uPA activity is the receptor associated protein (RAP), a chaperon that stabilizes newly synthesized low density lipoprotein receptor-related protein and the very low density lipoprotein receptor, presumably via its C-terminal domain (ctRAP). Pgn and tPA also are found in brain where their presence correlates with memory processes. In addition, in brain is neurotrypsin, a novel kringle containing proteinase. Of related neurological interest is the SEA module of agrin, a protein produced by motoneurons that induces the aggregation of nicotinic acetylcholine receptors. Functional in nerve tissue extension are various transmembrane protein tyrosine kinase receptors which contain kringle and frizzled (cysteine-rich) domains, likely to be involved in ligand binding. A main thrust of the project will be the development of CLOUDS, a relaxation matrix approach that avoids the assignment bottleneck and aims at high throughput structural analysis of protein NMR data.
Showing the most recent 10 out of 63 publications
