Riqiang Yan

Reticulon family members modulate BACE1 activity and amyloid-β peptide generation

Inhibiting the activity of the β-amyloid converting enzyme 1 (BACE1) or reducing levels of BACE1 in vivo decreases the production of amyloid-β. The reticulon family of proteins has four members, RTN1, RTN2, RTN3 and RTN4 (also known as Nogo), the last of which is well known for its role in inhibiting neuritic outgrowth after injury. Here we show that reticulon family members are binding partners of BACE1. In brain, BACE1 mainly colocalizes with RTN3 in neurons, whereas RTN4 is more enriched in oligodendrocytes. An increase in the expression of any reticulon protein substantially reduces the production of Aβ. Conversely, lowering the expression of RTN3 by RNA interference increases the secretion of Aβ, suggesting that reticulon proteins are negative modulators of BACE1 in cells. Our data support a mechanism by which reticulon proteins block access of BACE1 to amyloid precursor protein and reduce the cleavage of this protein. Thus, changes in the expression of reticulon proteins in the human brain are likely to affect cellular amyloid-β and the formation of amyloid plaques.

Employing a superior BACE1 cleavage sequence to probe cellular APP processing

The involvement of β‐secretase (BACE1; β‐site APP‐cleaving enzyme) in producing the β‐amyloid component of plaques found in the brains of Alzheimers patients, has fueled a major research effort to characterize this protease. Here, we describe work toward understanding the substrate specificity of BACE1 that began by considering the natural APP substrate and its Swedish mutant, APPSw, and proceeded on to include oxidized insulin B chain and ubiquitin substrates. From these findings, and the study of additional synthetic peptides, we determined that a decapeptide derived from APP in which the P3‐P2′ sequence, …VKM‐↓‐DA…, was replaced by …ISY‐↓‐EV… (‐↓‐ = β site of cleavage), yielded a substrate that was cleaved by BACE1 seven times faster than the corresponding APPSw peptide, SEVNL‐↓‐DAEFR. The expanded peptide, GLTNIKTEEISEISY‐↓‐EVEFRWKK, was cleaved an additional seven times faster than its decapeptide counterpart (boldface), and provides a substrate allowing assay of BACE1 at picomolar concentrations. Several APP mutants reflecting these β‐site amino acid changes were prepared as the basis for cellular assays. The APPISYEV mutant proved to be a cellular substrate that was superior to APPSw. The assay based on APPISYEV is highly specific for measuring BACE1 activity in cells; its homolog, BACE2, barely cleaved APPISYEV at the β‐site. Insertion of the optimized ISY‐↓‐EV motif at either the β‐site (Asp1) or β′‐site (Glu11) directs the rate of cellular processing of APP at these two accessible sites. Thus, we have identified optimal BACE1 substrates that will be useful to elucidate the cellular enzymatic actions of BACE1, and for design of inhibitors that might be of therapeutic benefit in Alzheimers disease.