👤 Paul Saftig

The beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) is the predominant β-secretase, cleaving the amyloid precursor protein (APP) via the amyloidogenic pathway. In addition, BACE1 as an amyloid degrading enzyme (ADE), cleaves Aβ to produce the C-terminally truncated non-toxic Aβ fragment Aβ34 which is an indicator of amyloid clearance. Here, we analyzed the effects of BACE1 inhibitors on its opposing enzymatic functions, i.e., amyloidogenic (Aβ producing) and amyloidolytic (Aβ degrading) activities, using cell culture models with varying BACE1/APP ratios. Under high-level BACE1 expression, low-dose inhibition unexpectedly yielded a two-fold increase in Aβ42 and Aβ40 levels. The concomitant decrease in Aβ34 and secreted APPβ levels suggested that the elevated Aβ42 and Aβ40 levels were due to the attenuated Aβ degrading activity of BACE1. Notably, the amyloidolytic activity of BACE1 was impeded at lower BACE1 inhibitor concentrations compared to its amyloidogenic activity, thereby suggesting that the Aβ degrading activity of BACE1 was more sensitive to inhibition than its Aβ producing activity. Under endogenous BACE1 and APP levels, "low-dose" BACE1 inhibition affected both the Aβ producing and degrading activities of BACE1, i.e., significantly increased Aβ42/Aβ40 ratio and decreased Aβ34 levels, respectively. Further, we incubated recombinant BACE1 with synthetic Aβ peptides and found that BACE1 has a higher affinity for Aβ substrates over APP. In summary, our results suggest that stimulating BACE1's ADE activity and halting Aβ production without decreasing Aβ clearance could still be a promising therapeutic approach with new, yet to be developed, BACE1 modulators. Show less

The beta‑site amyloid precursor protein (APP) cleaving enzyme (BACE1) was discovered due to its "amyloidogenic" activity which contributes to the production of amyloid-beta (Aβ) peptides. However, BACE1 also possesses an "amyloidolytic" activity, whereby it degrades longer Aβ peptides into a non‑toxic Aβ34 intermediate. Here, we examine conditions that shift the equilibrium between BACE1 amyloidogenic and amyloidolytic activities by altering BACE1/APP ratios. In Alzheimer disease brain tissue, we found an association between elevated levels of BACE1 and Aβ34. In mice, the deletion of one BACE1 gene copy reduced BACE1 amyloidolytic activity by ~ 50%. In cells, a stepwise increase of BACE1 but not APP expression promoted amyloidolytic cleavage resulting in dose-dependently increased Aβ34 levels. At the cellular level, a mislocalization of surplus BACE1 caused a reduction in Aβ34 levels. To align the role of γ-secretase in this pathway, we silenced Presenilin (PS) expression and identified PS2-γ-secretase as the main γ-secretase that generates Aβ40 and Aβ42 peptides serving as substrates for BACE1's amyloidolytic cleavage to generate Aβ34. Show less

Skeletal pathologies are frequently observed in lysosomal storage disorders, yet the relevance of specific lysosomal enzymes in bone remodeling cell types is poorly defined. Two lysosomal enzymes, ie, cathepsin K (Ctsk) and Acp5 (also known as tartrate-resistant acid phosphatase), have long been known as molecular marker proteins of differentiated osteoclasts. However, whereas the cysteine protease Ctsk is directly involved in the degradation of bone matrix proteins, the molecular function of Acp5 in osteoclasts is still unknown. Here we show that Acp5, in concert with Acp2 (lysosomal acid phosphatase), is required for dephosphorylation of the lysosomal mannose 6-phosphate targeting signal to promote the activity of specific lysosomal enzymes. Using an unbiased approach we identified the glycosaminoglycan-degrading enzyme arylsulfatase B (Arsb), mutated in mucopolysaccharidosis type VI (MPS-VI), as an osteoclast marker, whose activity depends on dephosphorylation by Acp2 and Acp5. Similar to Acp2/Acp5 Show less

Mannose 6-phosphate (Man6P) residues represent a recognition signal required for efficient receptor-dependent transport of soluble lysosomal proteins to lysosomes. Upon arrival, the proteins are rapidly dephosphorylated. We used mice deficient for the lysosomal acid phosphatase Acp2 or Acp5 or lacking both phosphatases (Acp2/Acp5(-/-)) to examine their role in dephosphorylation of Man6P-containing proteins. Two-dimensional (2D) Man6P immunoblot analyses of tyloxapol-purified lysosomal fractions revealed an important role of Acp5 acting in concert with Acp2 for complete dephosphorylation of lysosomal proteins. The most abundant lysosomal substrates of Acp2 and Acp5 were identified by Man6P affinity chromatography and mass spectrometry. Depending on the presence of Acp2 or Acp5, the isoelectric point of the lysosomal cholesterol-binding protein Npc2 ranged between 7.0 and 5.4 and may thus regulate its interaction with negatively charged lysosomal membranes at acidic pH. Correspondingly, unesterified cholesterol was found to accumulate in lysosomes of cultured hepatocytes of Acp2/Acp5(-/-) mice. The data demonstrate that dephosphorylation of Man6P-containing lysosomal proteins requires the concerted action of Acp2 and Acp5 and is needed for hydrolysis and removal of degradation products. Show less