Alzheimer's disease is a neurodegenerative disorder of the central nervous system resulting in a progressive loss of memory and other intellectual functions beginning in middle to late life. The disease is characterized by the accumulation of certain neuropathological lesions in the brains of affected individuals. The identification of alpha1-antichymotrypsin (ACT) as a component of Alzheimer amyloid filaments and the ? that the beta- protein-the major amyloid protein-is a proteolytic fragment of a larger precursor protein, ? the subject of antiproteases and proteases into the biochemical discussion of Alzheimer's disease. The discovery of a Kunitz inhibitor domain in the beta-protein precursor, and our finding that the beta-protein itself ? a striking resemblance to the active site of serine proteases and can stably bind to ACT via a protease-antiprotease interaction further underscores the need to understand the biochemistry and physiology of protease and antiprotease function in Alzheimer brain. This application proposes to continue our study of two ? of the role of proteases and their inhibitors in Alzheimer's disease-in the structure of the amyloid ? and in the proteolytic processing of the beta-protein from its precursor. With respect to the structure of Alzheimer amyloid filaments, we will determine the specific amino acids in the beta-protein and in the active ? of ACT necessary for their interaction in vitro. We will also design peptides that should bind ACT better in the beta-protein and might serve to inhibit ACT-beta-protein binding in vitro and potentially in vivo. We have discovered two chymotrypsin-like proteases (CLIPs) with the substrate specificity to cleave the terminus of the beta- protein from its precursor. One (clipsin) has been purified to homogeneity. The enzyme ? been partially sequenced and is likely identical to rat mast cell protease I. It preferentially degrades the protein precursor in brain membrane extracts, is inhibited by ACT, and can make the correct cleavage in ? peptide substrates. We will establish whether clipsin can cleave the beta-protein from its native precursor and are in the process of cloning the clipsin gene from rat and human brain cDNA. While clipsin/RMCP I is the best beta-protein-generating candidate protease now known, we are using PCR identify and clone other brain CLIPs. These will be expressed in a baculovirus system and their encoded ? tested for their ability to cleave the beta-protein N-terminus. We will also test the level and distribution their expression in normal, Alzheimer, and Down syndrome brain, to determine why the beta-protein is differentially generated in the disease process.
