Differential effects of proteases involved in intracellular degradation of amyloid beta-protein between detergent-soluble and -insoluble pools in CHO-695 cells.
The deposition of amyloid beta-protein (A beta or beta A4) is a key feature of Alzheimer's disease. Most studies have focused on the generation of A beta, but little is known about the degradation of A beta. Recent reports suggest that insulin-degrading enzyme (IDE) and neutral endopeptidase (NEP) are involved in the extracellular degradation of A beta. To date, however, far less is known about the degradation of intracellular A beta. To elucidate the protease(s) responsible for the degradation of intracellular A beta, we investigated the effect of various protease inhibitors on A beta in two distinct intracellular pools (i.e., nonionic detergent-soluble and detergent-insoluble pools) in Chinese hamster ovary cells. Treatment with thiol and metal inhibitors resulted in the accumulation of intracellular A beta and oligomers in detergent-soluble and -insoluble fractions. The overexpression of thiol-metalloprotease IDE resulted in a marked reduction in levels of detergent-soluble intracellular A beta as well as extracellular A beta 40 and A beta 42. Moreover, intracellular A beta in the detergent-insoluble fraction extracted with 70% formic acid or 6 M guanidine hydrochloride decreased markedly in the cells overexpressing IDE. In contrast, expression of NEP degraded the A beta in the detergent-insoluble fraction markedly and partially degraded extracellular A beta 40 and A beta 42, but not intracellular soluble A beta. Thiorphan, an inhibitor of NEP, accumulated, albeit to a lesser extent, in insoluble A beta but not in soluble A beta. Thus, IDE appears to degrade intracellular A beta more effectively than does NEP in both the detergent-soluble and -insoluble fractions.
Sudoh
Shinji
S
Department of Pathology and Laboratory Medicine, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104-6082, USA.
Frosch
Matthew P
MP
Wolf
Bryan A
BA
D016229:Amyloid beta-Peptides / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D016466:CHO Cells; D002614:Chelating Agents / Q000737:chemistry; D006224:Cricetinae; D017931:DNA Primers; D003902:Detergents / Q000737:chemistry*; D010450:Endopeptidases / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D006868:Hydrolysis; D010618:Phenanthrolines / Q000737:chemistry; D012995:Solubility; D015032:Zinc / Q000737:chemistry
D016229:Amyloid beta-Peptides; D002614:Chelating Agents; D017931:DNA Primers; D003902:Detergents; D010618:Phenanthrolines; D010450:Endopeptidases; D015032:Zinc; C025205:1,10-phenanthroline