proteinase, even around the putative “active-site” Asp-257 and Asp-385 residues, and more direct evidence is needed in support of this concept.
β-Secretase: Rate-Limiting Enzyme for Aβ Production. The London mutations in APP and the missense mutations in PS-1 that lead to Alzheimer’s disease have in common their alteration of the relative cleavage at the –40 and –42 sites in the TM domain of APP. The specificity of these γ-secretase cleavages were analyzed further by sequentially replacing amino acids 35–48 in the TM domain with Phe (42), akin to the “Ala scan” used for other scanning mutagenesis approaches. The production of Aβ and the relative ratios of x-40 vs. x-42 forms were then analyzed in the CM of cells transfected with these mutant forms. Although position 45 was identified as being critical for –42 cleavage, there was little specificity at the γ-cleavage sites; although there were alterations in the relative ratios, total Aβ formation was relatively unaffected by the scanning mutagenesis, suggesting that the precise identity of the amino acid residues at or near the γ-cleavage sites was not critical to total cleavage.
In sharp contrast, site-directed mutagenesis at the Met-Asp cleavage site on the β-end leads to dramatic effects on Aβ production (43). Although the substitution of Leu for Met at the P1 position (akin to the Swedish mutation) leads to enhancement of Aβ formation, substitution at this site by most other amino acids leads to a suppression of Aβ release in the extracellular medium, presumably by inhibition of β-secretase cleavage. Effective β-secretase cleavage is thus a prerequisite for formation and secretion of Aβ. In the case of some of the mutants, the fact that shorter Aβ peptides are secreted at a lower rate may represent the effect of an alternate cleavage site exposed as a result of conformational change in the mutated protein.
In conjunction with the results obtained with bafilomycin, it seems that β-secretase cleavage is a rate-limiting event for the formation of the “substrate” for γ-secretase. The latter enzymatic process is quite capable of turning over even the 5- to 6-fold excess β-CTFs generated in APP Swedish-transfected HEK293 cells, over that produced with Wt alone. Further, the Swedish mutation, unfortunately for the pedigree, causes disease by presenting a preferred β-cleavage site to the cellular enzyme.
β-Secretase: Isolation and Characterization. The search for enzymes that specifically cleave at the β-cleavage site in APP was initiated long before there was any cellular evidence for the presence of such a metabolic pathway. Although enzymes such as the metalloendopeptidase (EC 188.8.131.52) and cathepsin D were proposed to be candidate β-secretases, primarily as a result of cleavage specificity shown by using short peptide substrates (44), neither enzyme has passed the tests of being able to cleave full-length APP specifically, generating both the N- and C-terminal fragments. Cotransfection of these enzymes along with APP into cells such as HEK293 did not lead to the overproduction of either Aβ or β-sAPP (45).
The existence of the β-secretase pathway of APP cleavage, enriched in neuronal cells, leads to specific cleavage of APP at the N terminus of the Aβ peptide sequence. This cleavage leads to the formation of the soluble β-sAPP, as well as the membrane-associated β-CTF, the immediate precursor to Aβ. The compilation of the cellular results obtained by studying APP processing thus suggests that a true candidate β-secretase should have, at a minimum, the following characteristics. (i) It should specifically cleave APP at the Met-Asp site to generate the corresponding β-sAPP and β-CTF fragments, (ii) A true candidate β-secretase should show preferential cleavage toward Swedish over Wt sequence at the cleavage site. (iii) A true candidate β-secretase should function optimally at an acidic pH. (iv) A true candidate β-secretase also would be enriched in brain and neuronal tissue but present in cell lines such as HEK293 as well. The isolation and enzymatic characterization of a membrane-bound protease from human brain that meets these criteria (46) has been made possible by using APP-based fusion proteins incorporating both Wt and Swedish sequences, as well as the development of very specific ELISA-based quantitative assays for measuring cleavage at the β-cleavage site(s) in these fusion proteins. Although the identity of this enzymatic activity is not yet published, recombinant expression and cotransfection with APP would establish whether such an enzyme fulfills the additional cellular criteria of showing enhanced, specific cleavage in APP proteins at the β-cleavage sites.
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