Investigation of secreted amyloid precursor protein-α binding partners
Alzheimer’s disease is characterised by progressive loss of memory and cognitive functions. The symptoms extend to severe dementia and, in extreme cases, death. The neurological damage arises from the accumulation of a neurotoxic peptide known as amyloid-β (Aβ). This peptide arises from the processing of a membrane bound protein, amyloid precursor protein (APP). Processing of APP can follow one of two mutually exclusive proteolytic pathways; the amyloidogenic pathway involves the actions of a β-secretase (BACE) and a γ-secretase and results in the generation of the neurotoxic Aβ fragment and the N-terminal secreted ectodomain, secreted amyloid precursor protein-β (sAPPβ). The non-amyloidogenic pathway requires the action of an α-secretase (ADAM9, 10 or 17) and the γ-secretase and results in an N-terminal fragment secreted amyloid precursor protein-α (sAPPα) that has 16 amino acids more than sAPPβ. sAPPα has been shown to be neuroprotective, neurotrophic, and has been shown to regulate long term potentiation and long term depression, models of mammalian memory. Moreover, these beneficial effects are thought to be receptor mediated. Identification of the receptor(s) with which sAPPα interacts could identify a potential therapeutic target and will assist in further understanding the biology of sAPPα.In light of these findings, the current study aimed to use a protein pull-down approach to isolate and characterise putative receptor(s) and any intracellular binding proteins. A GST-sAPPα fusion protein was used as bait and protein samples from rat hippocampus and a human neuroblastoma cell culture line used as prey. The proteins found to interact with the GST-fusion protein were purified on a glutathione column and identified by mass spectrometry to create a portfolio of binding proteins. To ensure that the proteins found were binding specifically to sAPPα, sAPPβ (the similar proteolytic fragment of APP that lacks the 16 C-terminal residues and has been found to be 100-fold less effective as a neuroprotective molecule) and GST alone were included as controls. Proteins that bound to either of these molecules as well were put aside in order to identify proteins that uniquely bound to sAPPα. Several novel sAPPα interacting proteins of interest were identified; HMGB1, prohibitin, hornerin, protein SET and several structural proteins were identified in the study. A previous interaction with albumin preproprotein was also confirmed. Prohibitin and HMGB1 provide the most likely candidates for transducing the effects of sAPPα. Functional studies on the proteins will need to be carried out to investigate the importance of these interactions.
Advisor: Tate, Warren; Williams, Joanna
Degree Name: Master of Science
Degree Discipline: Biochemsitry
Publisher: University of Otago
Research Type: Thesis