Trimming of Amyloid Peptides by Gamma-Secretase

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DESCRIPTION (provided by applicant): The amyloid -protein (A¿), strongly implicated in the pathogenesis of Alzheimer's disease (AD), is formed by the sequential cleavage of the amyloid -protein precursor (APP) by ¿- and ?-secretases. ?-Secretases cleaves the APP transmembrane domain at the ? site, producing the C-terminus of A¿, and at the ? site, producing the N-terminus of the APP intracellular domain. The goal of this project is to understand the process by which the ?--secretase complex trims long A¿ peptide intermediates into shorter forms and how disease- causing PS mutations alter activity. With these goals in mind, the following specific questions will be addressed: (1) Which shorter A¿ peptides are produced via the trimming of specific long A¿ peptides? We will identify and quantify all A¿ peptides formed from normal ? cleavage products A¿49 and A¿48. We will also determine if A¿50, which is not a ? normal cleavage product, leads to the formation of the otherwise unobserved A¿47, A¿44 and A¿41. The result will help answer the question of whether trimming by every 3 residues is a general rule, and if so, how rigid is this rule. (2) How does ?-secretase accomplish C-terminal tripeptide trimming of long A¿ peptide intermediates? Evidence supports initial ? proteolysis to produce long A¿ peptides and then cleavage every 3 residues, but the mechanism by which this occurs is unknown. We hypothesize that the newly formed carboxy-terminus of long A¿ produced upon ? cleavage by ?-secretase is critical for trimming and with apparent precision by every 3 residues. To test this hypothesis, we will examine the ability of C-terminal amides of long A¿ peptides to serve as substrates. (3) What are the effects of Alzheimer-causing PS1 mutations on the trimming of long A¿ peptides? We have shown that such PS1-mutant ?-secretases complexes can increase the proportion of long-to-short A¿ peptides from recombinant APP substrate. Here, we will test the conversion of A¿49 and A¿48 by these mutant protease complexes, examining the A¿ products that are formed, the proportion of these products, and their rates of formation relative to the wild-type complex. We hypothesize that the disease-causing PS1 mutations increase the proportion of long A¿ peptides by slowing down the trimming process in general.