The amyloid precursor protein (APP) undergoes constitutive shedding by a protease activity called a-secretase. This is considered an important mechanism preventing the generation of the Alzheimer's disease amyloid-b peptide (Ab). a-Secretase appears to be a metalloprotease of the ADAM family, but its identity remains to be established. Using a novel a-secretase-cleavage site-specific antibody, we found that RNAi-mediated knockdown of ADAM10, but surprisingly not of ADAM9 or 17, completely suppressed APP a-secretase cleavage in different cell lines and in primary murine neurons. Other proteases were not able to compensate for this loss of a-cleavage. This finding was further confirmed by mass-spectrometric detection of APPcleavage fragments. Surprisingly, in different cell lines, the reduction of a-secretase cleavage was not paralleled by a corresponding increase in the Ab-generating b-secretase cleavage, revealing that both proteases do not always compete for APP as a substrate. Instead, our data suggest a novel pathway for APP processing, in which ADAM10 can partially compete with c-secretase for the cleavage of a C-terminal APP fragment generated by b-secretase. We conclude that ADAM10 is the physiologically relevant, constitutive a-secretase of APP.

Cell surface proteolysis is essential for communication between cells and results in the shedding of membraneprotein ectodomains. However, physiological substrates of the contributing proteases are largely unknown. We developed the secretome protein enrichment with click sugars (SPECS) method, which allows proteome-wide identification of shedding substrates and secreted proteins from primary cells, even in the presence of serum proteins. SPECS combines metabolic glycan labelling and click chemistry-mediated biotinylation and distinguishes between cellular and serum proteins. SPECS identified 34, mostly novel substrates of the Alzheimer protease BACE1 in primary neurons, making BACE1 a major sheddase in the nervous system. Selected BACE1 substrates-seizureprotein 6, L1, CHL1 and contactin-2-were validated in brains of BACE1 inhibitor-treated and BACE1 knock-out mice. For some substrates, BACE1 was the major sheddase, whereas for other substrates additional proteases contributed to total substrate shedding. The new substrates point to a central function of BACE1 in neurite outgrowth and synapse formation. SPECS is also suitable for quantitative secretome analyses of primary cells and may be used for the discovery of biomarkers secreted from tumour or stem cells.

Survival of plasma cells is regulated by B-cell maturation antigen (BCMA), a membrane-bound receptor activated by its agonist ligands BAFF and APRIL. Here we report that γ-secretase directly cleaves BCMA, without prior truncation by another protease. This direct shedding is facilitated by the short length of BCMA's extracellular domain. In vitro, γ-secretase reduces BCMA-mediated NF-κB activation. In addition, γ-secretase releases soluble BCMA (sBCMA) that acts as a decoy neutralizing APRIL. In vivo, inhibition of γ-secretase enhances BCMA surface expression in plasma cells and increases their number in the bone marrow. Furthermore, in multiple sclerosis, sBCMA levels in spinal fluid are elevated and associated with intracerebral IgG production; in systemic lupus erythematosus, sBCMA levels in serum are elevated and correlate with disease activity. Together, shedding of BCMA by γ-secretase controls plasma cells in the bone marrow and yields a potential biomarker for B-cell involvement in human autoimmune diseases.