Final Activity Report Summary - HMWGAMMA (Structural characterization of the high molecular weight complex of gamma-secretase) Gamma-secretase, an aspartyl protease of the intramembrane cleaving proteases (iCLiPs) family, is responsible for the generation of beta-amyloid peptides (ABETA), which are the primary component of the senile plaques in the brains of Alzheimer disease (AD) patients. Gamma-secretase is a complex containing presenilin, nicastrin, Aph-1 and Pen-2 that cleaves various type I membrane proteins, such as the amyloid precursor protein (APP) and Notch. While these four components are necessary and sufficient for gamma-secretase activity, additional proteins might be involved in its regulation. We established a methodology to purify an active gamma-secretase complex from reconstituted presenilin deficient fibroblasts using tandem affinity purification (TAP) method. We biochemically identified proteins that were associated with active gamma-secretase, including the ones involved in complex maturation, membrane trafficking and the tetraspanin web. An elaborated functional analysis strengthened the hypothesis that various proteins involved in sub-cellular trafficking or complex assembly affected ABETA production. We further corroborated the novel association of gamma-secretase with the tetraspanin web in the cell membrane using co-precipitation and floating assays. In tetraspanin deficient cells several gamma-secretase substrates accumulated and the ratio of long to short ABETA generated by tetraspanin web-associated gamma-secretase was affected. Thus, association with tetraspanin domains tuned gamma-secretase proteolytic activity. Our data provided a first proteome of the gamma-secretase complex and, potentially, novel targets for medication for AD.