The amyloid-β precursor protein (APP) is known to play a role in the pathogenesis of Alzheimer’s disease (AD) as its proteolysis results in the generation of amyloid-β peptides (Aβ) and mutations in APP cause autosomal dominant early onset AD [1
]. Aβ can oligomerize and aggregate into extracellular deposits of amyloid plaques, a classic pathologic hallmark of AD associated with perturbed neuronal functions, disrupted cellular networks, cell death and tissue degeneration [2
]. The resulting lesions can have a significant impact and dire clinical consequences on cognitive functions in patients. Unfortunately, current therapies for AD remain largely symptomatic and disease-modifying drugs have yet to be discovered.
Production of Aβ from APP occurs by an amyloidogenic-processing pathway. First, APP is cleaved by β-secretase (also known as the β site APP cleaving enzyme (BACE1), or memapsin-2 [3
]) at the extracellular domain to release a secreted ectodomain fragment, sAPPβ, and a membrane-bound C-terminal fragment, CTFβ or C99. Then, CTFβ is cleaved by γ-secretase (a large multi-subunit complex comprised of presenilin (PS), nicastrin (Nct), anterior pharynx defective (Aph-1) and the presenilin enhancer (Pen-2) [5
]) via a process called regulated intramembrane proteolysis (RIP). This results in the production of Aβ and an intracellular domain fragment, AICD (APP intracellular domain), which can translocate to the nucleus and has been implicated in the regulation of gene expression [6
]. Depending on the location of the γ-secretase cleavage, Aβ peptides of 39 to 42 amino acids are made [7
]. In AD, Aβ40 and Aβ42 are of most concern, with Aβ42 being the most toxic to neurons and synapses. Alternatively, under non-amyloidogenic condition, APP is first cleaved by α-secretase (members include the A disintegrin and metalloproteinase proteins, ADAM9, ADAM10 and ADAM17/tumor necrosis factor a-converting enzyme (TACE) [9
]) to release a truncated ectodomain fragment, sAPPα. The remaining C-terminal fragment, CTFα or C83, is then cleaved by γ-secretase to release a p3 peptide and AICD. Most APP molecules undergo this non-amyloidogenic processing pathway.
APP is a single-pass transmembrane glycoprotein with a large extracellular N-terminal domain and a short intracellular C-terminal domain. Its structure resembles a cell-surface receptor [12
] that can bind ligands and participate in protein-protein interactions. Like many receptor proteins that dimerize upon ligand activation, APP can also form homodimers [12
], as well as heterodimers with Notch for cellular signaling [14
] and with its homologs the amyloid precursor like proteins 1 and 2 (APLP1 and 2) for cellular adhesion and cell-cell communication [17
]. The precise function of APP and the significance of its dimerization have yet to be determined. However, many biological activities have been linked to the protein, including that of platelet aggregation [19
], metal homeostasis [20
], gene transcription and cellular metabolism [6
], and various neuronal processes such as cellular growth, differentiation, migration, aborization, axonal transport, memory formation, and neuroprotection [24
APP has a few notable dimerization sites. First, the E1 growth factor like domain (GFLD) has 3 cysteine bridges where the bridge between Cys98 and Cys105 forms an important beta hairpin loop for dimerization [32
]. Secondly, the residues His147, His151, Tyr168, and Met170 at the E1 copper-binding domain (CuBD) form a tetrahedral structure that can coordinate metals such as copper and zinc that can regulate APP dimerization [33
]. The E2 collagen-binding site (CBD) can bind ligands such as Type I collagen and heparin for cellular adhesion and participate in both cis- and trans- APP dimerization [35
]. Additionally, binding of Aβ to the juxtamembrane region facilitates APP multimerization, which may be crucial for cell death signaling [37
]. Finally, 3 consecutive GxxxG motifs at the transmembrane domain (Aβ residues 25-37) [8
], and the GXXXA sequence that comes directly after [38
], are the most studied sites for APP dimerization.
Recent studies suggest that APP dimerization may influence Aβ production. For example, replacement of a lysine residue by cysteine at the juxtamembrane region of APP (K624C mutant) resulted in constitutive APP dimerization via a disulphide bond, and a concomitant increase in Aβ levels [13
]. Likewise, mutations that disrupted the GxxxG transmembrane dimerization motif of APP resulted in a corresponding decrease of toxic Aβ42 under a ToxR system. In particular, a G33I mutant abolished both dimerization and Aβ production [8
]. However, in a follow-up study on GxxxG mutations, it was found that increased APP dimerization at the C-terminal domain by these mutants lead to a decrease in Aβ [39
]. Also, when examining familial APP mutants that cause early onset AD, the mutations destabilized APP transmembrane dimerization and increased Aβ42/40 ratio in a way that negatively correlated with the mean age of onset of AD symptoms [38
]. Furthermore, when using an FKBP/rapamycin system to examine APP dimerization and its effects on Aβ generation in the absence of mutations, it was found that induction of dimerization decreased Aβ production [40
]. Regardless of whether APP dimerization increases or decreases Aβ, multiple groups report that it does have an effect on Aβ levels demonstrating the importance of APP dimerization in AD pathogenesis and that further evaluation of this process is warranted.
To better understand the relationship between APP dimerization and Aβ production, we developed an assay to identify small molecule APP dimerization inhibitors and subsequently tested their effects on Aβ generation. A high throughput screen (HTS) was conducted using the method of Firefly luciferase enzyme fragment complementation (FLuc EFC). This technique has successfully been used by others to study epidermal growth factor receptor dimerization [41
], chemokine receptor dimerization CXCR4 and 7 [42
], rapamycin induced FRB/FKBP association [43
], phospho-dependent association of Cdc25C/14-3-3ε in cell cycle division [43
], IFN-γ induced nuclear translocation of STAT1/STAT1 complexes [43
], cellular marconuclear delivery vehicles, [44
] as well as protein-protein interactions in plants [45
]. Applications of the method also allow visualization of luminescence in live cells and animals [43
]. Here, we applied the same principle with the goal of detecting APP-APP interactions. Two inactive deletion mutants of the FLuc enzyme were separately fused to APP. When APP is in monomeric form, the FLuc fragments remain inactive. However, upon APP-APP dimerization, the two inactive FLuc fragments are brought in close proximity to form a fully functional enzyme. Quantification of APP-APP interactions is then made possible by measuring chemiluminescence following the addition of luciferin. The FLuc EFC is an effective way to monitor APP-APP interactions that could be used for a better understanding of APP processing and Aβ production.