To investigate the potential contribution of the lysosomal compartment in the processing of amyloid precursor protein (APP) to amyloid beta-peptides (A beta s), we stably overexpressed a series of lysosomal proteases (the cysteine proteases, cathepsins B, L and S, and the aspartic protease, cathepsin D) in a human kidney epithelial cell line (293) transfected to express high levels of beta APP. Preliminary experiments indicated that 293 cells endogenously synthesize cathepsins B, L and D, but not cathepsin S. A beta secretion was assessed by immunoprecipitation and ELISA and found to be increased approximately 2-fold following cathepsin S expression, but to be unchanged (cathepsins B, L) or decreased (cathepsin D) in the other double transfectants. E-64d, an inhibitor of lysosomal cysteine proteases, significantly reduced A beta secretion by the cathepsin S transfectants, but had no effect on cells expressing the other proteases. Radiosequencing of A beta secreted by cathepsin S-expressing cells revealed that a previously unreported variant beginning at Met -1 (relative to the most common A beta N-terminus, Asp -1) accounted for most of the increase in A beta secretion. Immunostaining of human brain sections revealed cathepsin S in cortical neurons and glia in samples of brain from patients with Alzheimer's disease. These results provide evidence in living cells for a pathway in which cathepsin S generates A beta from amyloidogenic fragments of beta APP in the endosomal/lysosomal compartment. This pathway appears to be inducible, distinct from a constitutive pathway used by 293 and other cells to generate A beta, and may be relevant to the pathogenesis of Alzheimer's disease.

Previous studies have indicated that control and hemin-treated human erythroleukemia K-562 cells fail to produce adult-type beta-globin mRNA transcripts and to translate them into nascent beta-globin chains. Expression of the beta-globin DNA sequences in K-562 cells can occur, however, under certain conditions. To readdress this issue and to examine the possibility of whether these cells produce immature and untranslatable beta-globin RNA transcripts, we prepared total cytoplasmic RNA from control and inducer-treated cells and performed Northern blot hybridization analysis using 5' end-labeled fragments of the human beta-globin DNA rather than 3' end fragments as probes. Although hybridization of both cytoplasmic and nuclear K-562 RNA with a 32P-labeled 3' end fragment (1.6kb Bam H1 cut) coding for a large part of the first exon of beta-globin failed to detect beta-globin RNA transcripts, hybridization with a 5' end 32P-Labeled 2.0kb Bam H1 fragment (coding for the third exon and part of the second) revealed the presence of relatively small (less than 7S) RNA molecules both in nuclear and cytoplasmic fraction. S1 nuclease mapping of both cytoplasmic and nuclear RNA with the use of 5' end-labeled 2.0 kb Bam H1 fragment of human beta-globin DNA indicated protection of a small portion located 64 bp 5' upstream from the Bam H1 site of the second exon. The amount of protected portion was relatively higher in K-562 cells undergoing erythroid maturation.(ABSTRACT TRUNCATED AT 250 WORDS)

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