Structural and functional studies on APH-1 and the γ-secretase complex

The γ-secretase complex processes numerous Type I membrane proteins by cleavage within their TM domains. Among these are the amyloid precursor protein (APP) and Notch. γ-secretase-cleavage of APP produces AP peptides, which form amyloid plaques in Alzheimer\u27s disease (AD) brains, while cleavage of Notch is critical for signaling during development. The complex is composed of four proteins: presenilin (PS), Nicastrin, APH-1, and Pen-2. Studying how the complex associates and cleaves its substrates will provide insight that may eventually enable therapeutic targeting of specific pathological activities of the γ-secretase complex without interfering with its critical biological functions. In the work described in this thesis, we have investigated several fundamental aspects of one of the newly described members of the complex, APH-1, as well as a novel domain of PS that is functionally important. We first investigated the membrane topology of APH-1 and found that it is a seven-transmembrane domain protein that topologically resembles a G-protein coupled receptor. We found that APH-1 can be endoproteolyzed at a site just N-terminal to its fifth transmembrane domain, which is reminiscent of a well-known PS cleavage event that is critical for its activity. Next, we studied the association of APH-1 with other members of the complex. We found that APH-1 can bind to both Nicastrin and PS independently and very rapidly after synthesis, while its association with Pen-2 is delayed and probably indirect. We also found that APH-1 is retained in the endoplasmic reticulum (ER) until it associates with Nicastrin and PS, and that even though Pen-2 association is delayed, this protein probably also joins the complex in the ER since artificially ER-retained complex exhibits full activity. Finally, we identified a ‘GxxxG-like’ motif in the eighth transmembrane domain of PS. Mutagenesis studies revealed that this region of PS is functionally important and is required upstream of PS activation. Further studies of this domain will be aimed at determining whether it mediates PS homodimerization. The implications of PS homodimerization for y-secretase biology and for the mechanism of AD-causing mutations in PS are discussed in the concluding chapter