Distinct Secretases, a Cysteine Protease and a Serine Protease, Generate the C Termini of Amyloid β‐Proteins Aβ_1‐40 and Aβ_1‐42, Respectively

Abstract

Abstract:The carboxy-terminal ends of the 40-and 42-amino acids amyloid ␤-protein (A␤) may be generated by the action of at least two different proteases termed ␥(40)-and ␥(42)-secretase, respectively. To examine the cleavage specificity of the two proteases, we treated amyloid precursor protein (APP)-transfected cell cultures with several dipeptidyl aldehydes including N-benzyloxycarbonyl-Leu-leucinal (Z-LL-CHO) and the newly synthesized N-benzyloxycarbonyl-Val-leucinal (Z-VL-CHO). All dipeptidyl aldehydes tested inhibited production of both A␤ 1-40 and A␤ . Changes in the P 1 and P 2 residues of these aldehydes, however, indicated that the amino acids occupying these positions are important for the efficient inhibition of ␥-secretases. Peptidyl aldehydes inhibit both cysteine and serine proteases, suggesting that the two ␥-secretases belong to one of these mechanistic classes. To differentiate between the two classes of proteases, we treated our cultures with the specific cysteine protease inhibitor E-64d. This agent inhibited production of secreted A␤ 1-40 , with a concomitant accumulation of its cellular precursor indicating that ␥(40)-secretase is a cysteine protease. In contrast, this treatment increased production of secreted A␤ . No inhibition of A␤ production was observed with the potent calpain inhibitor I (acetyl-Leu-Leu-norleucinal), suggesting that calpain is not involved. Together, these results indicate that ␥(40)-secretase is a cysteine protease distinct from calpain, whereas ␥(42)-secretase may be a serine protease. In addition, the two secretases may compete for the same substrate. Dipeptidyl aldehyde treatment of cultures transfected with APP carrying the Swedish mutation resulted in the accumulation of the ␤-secretase C-terminal APP fragment and a decrease of the ␣-secretase C-terminal APP fragment, indicating that this mutation shifts APP cleavage from the ␣-secretase site to the ␤-secretase site. Key Words: Alzheimer's disease-Amyloid precursor protein-Amyloid peptide-Proteolysis-Protease inhibitors-␥-Secretase. J. Neurochem. 72, 1417-1422 (1999).Alzheimer's disease is a neurodegenerative disorder characterized by the presence of insoluble protein aggregates that accumulate in pathological lesions, such as neuritic amyloid plaques, cerebrovascular amyloid, and neurofibrillary tangles. The amyloid fibers of the neuritic amyloid plaque cores and cerebrovascular amyloid consist mainly of the 40 -42-amino acids amyloid ␤-protein (A␤), derived from the proteolytic processing of the amyloid precursor protein (APP) (for review, see Robakis, 1994). APP is a transmembrane glycoprotein processed through amyloidogenic and nonamyloidogenic pathways. In a nonamyloidogenic pathway, cleavage by ␣-secretase between A␤ sequence Lys 16 and Leu 17 of APP (Anderson et al., 1991) results in the production of an 8-kDa C-terminal APP fragment that is considered the precursor of the p3 peptides (Higaki et al., 1995;Citron et al., 1996). In the amyloidogenic pathway, proteolytic cleavage by ␤-secretase at the N t...

Cathepsins B and L Differentially Regulate Amyloid Precursor Protein Processing

“…Finally, it cannot be ruled out that catL also modulates APP through ␥-secretase. A similar specific rise in A␤42, accompanied by a reduction in A␤40, was observed in CHO-APP751 cells treated with E64D (Figueiredo-Pereira et al, 1999). Although we did not observe any accumulation of APP CTFs after catL inhibition, catL may also modulate, rather than inhibit, ␥-secretase.…”

The Calpain System

“…Ca 2ϩ -binding properties of the complete calpain molecules are discussed in section IVC2. Comparison of structures obtained in the presence (Ca 2ϩ in EF-1Ј, EF-2Ј, and EF-3Ј) or in the absence of Ca 2ϩ indicated that the Ca 2ϩ -induced structural changes in domain VI crystals are very small. The largest Ca 2ϩ -induced structural changes occur in the EF-1Ј region of the molecule (residues [98][99][100][101][102][103][104][105][106][107][108][109][110][111][112][113][114][115]Ref. 39).…”

“…These proteolytic cleavages are mediated by a class of proteases called secretases: 1) ␣-secretase, which removes the large extracellular NH 2 terminus of APP; 2) ␤-secretase, which also removes the extracellular domain of APP; and 3) ␥-secretase, which removes the COOH-terminal 60 or 58 amino acids of APP and together with the ␤-secretase cleavage, produces the A␤40 or A␤42 amyloid peptides. The ␣-, ␤-, and ␥-secretases are not related to the calpains, so the calpains are not involved in A␤ peptide production (115). On the other hand, Ca 2ϩ concentrations are elevated in AD brains (240), immunohistochemical studies indicate that m-calpain levels are increased in brains from AD patients (145,464), and autolysis of -calpain to its 76-and 78-kDa forms is enhanced in brains from AD patients (380).…”

Formation and participation of nano-amyloids in pathogenesis of Alzheimer's disease and other amyloidogenic diseases

“…В результате посттрансляционного процессинга АРР С99 протеолизом b-секретазой, а затем g 40 -и g 42 -секретазами образуются пептидные фрагменты Аb и Аb [6]. Было показано, что АРР может расщепляться каспазами.…”