Purification of Full-Length <i>β</i>-Secretase Involved in Alzheimer’s Disease, and Proteomic Identification of Binding Partners — Oak Academic Publishing
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Purification of Full-Length <i>β</i>-Secretase Involved in Alzheimer’s Disease, and Proteomic Identification of Binding Partners
Lipid Mediator Unit, William Harvey Research Institute, Barts and the London Medical Schools, London, UK
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Institute of Pharmaceutical Science, King’s College London, London, UK
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Department of Biomedical Science, St George’s University of London, London, UK
1 Lipid Mediator Unit, William Harvey Research Institute, Barts and the London Medical Schools, London, UK
2 Institute of Pharmaceutical Science, King’s College London, London, UK
3 Department of Biomedical Science, St George’s University of London, London, UK
β -Secretase (BACE1 or β -site APP cleaving enzyme) is an acid protease that releases the neurotoxic 40 - 42 residue peptides ( β -amyloid or A- β ) from its glycoprotein precursor, (APP or amyloid precursor protein) which when released in brain is thought to give rise to cognitive decline in patients with Alzheimer’s Disease. Most structural studies on β -secretase have previously been performed with recombinant forms of the protease, in which the transmembrane coding region has been deleted. However, interactions with proteins of the same species are best studied using the full-length β -secretase as interactions are likely to be influenced by the hydrophobic nature and localization of its transmembrane regions. Here we develop a multi-step purification procedure that isolates a complex containing BACE1 from recombinant human cells using mild detergents in a procedure that retains other proteins within the complex and remains active in its β -site APP cleaving activity. Some of these proteins, eg reticulon 4, are identified by proteomics, and are known by previous studies performed by others to regulate the activity of BACE1 against APP. These interactions may aid the development of small proteins and peptides that could inhibit the release of aggregated forms of β -amyloid, and thus be useful therapeutically.
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