Characterisation of the human gamma-secretase complex using an E. coli system

• Main Author: Amir, Sarah
• Published: Cardiff University 2005
• Subjects: 616.8
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.583719

Alzheimer's disease (AD) is a form of progressive dementia which affects many of the world's population. AD patients show a decrease in cognitive function, loss of memory and at a later stage, decreasing physical activity. So far, the only definite diagnosis of AD is still based on post-mortem demonstration of extensive cell loss, and the presence of amyloid plaques and neurofibrillary tangles in the brains of sufferers. Cleavage of the amyloid precursor protein (APP) by p-secretase results in the production of the C-terminal fragment (CTF) of APP, C99, which is in turn cleaved by the y-secretase. This cleavage event produces the Ap peptides, which aggregate to form the amyloid deposits seen in the brains of AD patients. y-Secretase has eluded identification for many years, however it has been shown in various eukaryotic systems that it is a highly stable protein complex of high relative molecular mass consisting of the membrane proteins PS-1, Nicastrin (NicA), Pen-2 and Aph-1. This thesis shows that E. coli represents a simple and efficient system that can be used to analyse human membrane proteins, and in this case to verify that PS-1, NicA, Pen-2 and Aph-1 are the minimal components required for y-secretase activity. Components of the y-secretase complex were successfully cloned and expressed in E. coli and their topologies in the bacterial membrane were studied in vivo by a genetic approach which involves generating hybrids of the target membrane proteins to alkaline phosphatase (PhoA). In addition, initial experiments carried out with the aim of reconstituting y-secretase activity in E. coli are described. Furthermore, the E. coli two hybrid system was successfully utilised to unveil interactions between the y-secretase components and to isolate candidate interaction partners of PS-1 and its gain of function mutant PS-lAexon9. Thus E. coli can be used as a model system to aid our understanding of the processes involved in AD.