Identification of β-Secretase (BACE1) Substrates Using Quantitative Proteomics

Abstract

β-site APP cleaving enzyme 1 (BACE1) is a transmembrane aspartyl protease with a lumenal active site that sheds the ectodomains of membrane proteins through juxtamembrane proteolysis. BACE1 has been studied principally for its role in Alzheimer's disease as the β-secretase responsible for generating the amyloid-β protein. Emerging evidence from mouse models has identified the importance of BACE1 in myelination and cognitive performance. However, the substrates that BACE1 processes to regulate these functions are unknown, and to date only a few β-secretase substrates have been identified through candidate-based studies. Using an unbiased approach to substrate identification, we performed quantitative proteomic analysis of two human epithelial cell lines stably expressing BACE1 and identified 68 putative β-secretase substrates, a number of which we validated in a cell culture system. The vast majority were of type I transmembrane topology, although one was type II and three were GPI-linked proteins. Intriguingly, a preponderance of these proteins are involved in contact-dependent intercellular communication or serve as receptors and have recognized roles in the nervous system and other organs. No consistent sequence motif predicting BACE1 cleavage was identified in substrates versus non-substrates. These findings expand our understanding of the proteins and cellular processes that BACE1 may regulate, and suggest possible mechanisms of toxicity arising from chronic BACE1 inhibition.

β-Site Amyloid Precursor Protein (APP)-cleaving Enzyme 1 (BACE1)-deficient Mice Exhibit a Close Homolog of L1 (CHL1) Loss-of-function Phenotype Involving Axon Guidance Defects

“…EphrinEph receptor signaling causes growth cone collapse and axon repulsion (36), so a short IPB would be consistent with increased signaling. We chose to examine ephrinB3 and EphA4 because of the following: 1) ephrinB3 Ϫ/Ϫ mice have abnormally long IPBs suggesting ephrinB3 decreases IPB length (38); 2) a gradient of EphA4 expression in dentate gyrus granule cells establishes a topographic map of mossy fiber projections to CA3 (37), and 3) EphA4 was recently identified as a candidate BACE1 substrate in vitro (27). Although EphA4 is cleaved by BACE1 when overexpressed in HEK293 cells, we found no evidence of physiological EphA4 processing by endogenous BACE1 in the hippocampus (Fig.…”

“…In addition, ephrinB3 plays a role in determining IPB length, such that ephrinB3 deficiency results in a long IPB (38). Recently, EphA4 was shown to be a potential BACE1 substrate in vitro (27), but in vivo validation was not obtained in that study.…”

“…Initially, a small number of BACE1 substrates were discovered through individual studies (26). However, the list of potential BACE1 substrates has recently grown considerably because of the use of traditional and novel proteomics screening methods (27)(28)(29). Recent reports suggest that molecules involved in axon guidance, neurite outgrowth, and synapse formation are BACE1 substrates.…”

“…Recent reports suggest that molecules involved in axon guidance, neurite outgrowth, and synapse formation are BACE1 substrates. For example, members of the ephrin and Eph receptor families of axon guidance molecules were identified as BACE1 substrates in a proteomics screen using nonneuronal cell lines (27). More recently, the neural cell adhesion molecules L1 and close homolog of L1 (CHL1) were shown to be BACE1 substrates in primary neuron cultures and in the brain (28,29).…”

Reversing Synapse Loss in Alzheimer's Disease: Rho-Guanosine Triphosphatases and Insights from Other Brain Disorders

“…Aberrant Rac1 activity in AD could also be caused by abnormal EphA4 signaling. Indeed, EphA4 has been identified as a substrate for both β-secretase (BACE1) and γ-secretase [111,112]. EphA4 processing by γ-secretase upon increased synaptic activity generates EphA4 intracellular domain fragments, which trigger the formation of dendritic spines through a Rac1-dependent pathway.…”