γ-Secretase is a multi-subunit protease that executes an extraordinary cleavage of substrates within the lipid bilayer. This process of target hydrolysis within the membrane environment is known as regulated intramembrane proteolysis (RIP) [1
] whereby cleavage by γ-secretase releases a protein fragment from its membrane tether that can then transmit its signal. γ-Secretase was originally identified as the enzyme responsible for cleavage of the amyloid precursor protein (APP) [2
]. Cleavage of APP generates β-amyloid peptides that are believed to play a causative role in the neuropathogenesis of Alzheimer's disease [3
] according to the "amyloid cascade hypothesis." Additionally, it has been determined that γ-secretase cleaves a multitude of other substrates that include the Notch receptors [4
], ErbB-4 [5
], CD44 [6
], as well as the Notch ligands Delta-1 and Jagged-2 [7
] amongst others. Deregulated Notch signaling has been associated with the development of various cancers, including T-cell Acute Lymphoblastic Leukemia (T-ALL) [9
]. Due to the central role of γ-secretase in these pathologies, considerable efforts have been made to characterize this unique protease.
In order to better understand γ-secretase, in vitro
assays using purified exogenous recombinant substrate [10
] or assays utilizing isolated membrane from systems overexpressing substrate have been developed and reported [11
]. Currently, there are two predominant options to study this protease in a cell line of interest: 1) stably transfect the cell line with plasmids encoding APP, Notch or other substrate fragments and conduct whole-cell based detection assays, or 2) obtain large quantities of the cell line and isolate the membrane fraction in a time-consuming process. This can then be examined using an in vitro
assay that employs exogenous recombinant substrate as mentioned previously. Due to these limitations, it is often an extremely challenging task to quickly characterize γ-secretase activity in multiple cell lines and primary cells. Furthermore, it is currently impossible to examine the real-time effect of various treatments on the status of γ-secretase in cell systems without stable transfection. For instance, treatment of a Notch-dependent cell line with γ-secretase inhibitors may have an anti-proliferative effect, but available methods cannot ascertain the extent of real-time γ-secretase inhibition in the system. Therefore, development of an assay that does not require transfection or membrane preparation and is applicable for any cell type has become an urgent issue for defining the relationship of γ-secretase inhibition and its biological responses. This is particularly critical to evaluate γ-secretase inhibitors being used in preclinical and clinical studies because assessment of target inhibition will facilitate the identification and establishment of effective therapies. Recently, we have determined that the use of biotinylated substrate greatly enhanced substrate activity and assay sensitivity over previous versions [13
]. This prompted us to apply a similar strategy to the development of a simplified γ-secretase assay capable of quantifying real-time activity in cell-based systems.
In this study we have developed a novel γ-secretase assay that does not require membrane preparation and/or substrate plasmid transfection. This γ-secretase assay that we refer to as an "exo-cell" assay applies a highly active, biotinylated recombinant substrate (Sb4) of γ-secretase exogenously to cells in the presence of a small quantity of CHAPSO detergent. We have found that this 96-well assay format can detect γ-secretase activity from as little as a few thousand cells. Furthermore, we can easily detect γ-secretase activity from primary B-cell Chronic Lymphocytic Leukemia (B-CLL) cells isolated from patients. More importantly, this assay can monitor the real-time γ-secretase activity in a 96-well format after inhibitor treatment and has allowed us to establish a correlation between the anti-proliferative effect of γ-secretase inhibitors against lymphoma cells and real-time reduction in γ-secretase activity. Taken together, the development of this novel assay allows for the characterization of real-time γ-secretase activity directly in cell lines as well as primary patient samples. This assay will simplify the study of γ-secretase and provide new tools in the characterization of this enzyme as well as facilitate the development of therapies against Alzheimer's disease and Notch-dependent neoplasms. Furthermore, the application of this simplified method will greatly enhance our ability to examine this unique enzyme and advance our understanding of γ-secretase biology.