Purification and Cloning of Brain Proteases Capable of Degrading the β‐Amyloid Precursor Protein^a

Familial Alzheimer's Disease Cells Abnormally Accumulate β‐Amyloid‐Harbouring Peptides Preferentially in Cytosol but not in Extracellular Fluid

“…In human lymphoid cells, the 16-kDa and 15-kDa peptides, which are cleaved at the 30-th and 15th amino acids, respectively, N-terminal to PA4, and the 14-kDa peptide with the PA4 N-terminus are the three major sources for PA4 accumulation in the cytosol. Peptides N-terminal to the PA4 N-terminus are possibly cleaved by chymotrypsin-type serine proteases, including the cathepsin-G-like protease prepared from brains of Alzheimer's disease patients (Abraham et al, 1992) andor the 68-kDa protease of familial Alzheimer's disease lymphoblastoid cells (Matsumoto and Fujiwara, 1994). The resulting peptides with PA4 N-terminal ends may be further processed at the PA4 C-terminus and around the C-terminus of the transmembrane domain, respectively, by an elastaselike protease or a metalloprotease and a trypsin-like protease (Nelson et a]., 1993).…”

Ca2+-Dependent 68-Kilodalton Protease in Familial Alzheimer's Disease Cells Cleaves the N-Terminus of .beta.-Amyloid

“…Abraham et al (1991) reported that a 68-kDa, Ca2+-dependent serine protease that is increased in AD and aged normal brain cells cleaves each bond in the Lys_2-Met_1-Asp^A4_1-Ala2 sequence of the /3/A4 N-terminal and is inhibited by the serine protease inhibitors protease nexin-2 and ai-antichymotrypsin. They now have reported that this proteolytic activity is the action of three different proteases: a 28-kDa Ca2+-dependent serine protease which preferentially cleaves the Lys-Met bond of the oligopeptide substrate at the N-terminus of /3/A4; a cysteine-dependent metalloprotease that requires dithiothreitol for its proteolysis at the Met-Asp bond of the same oligopeptide; and another cysteine-dependent protease that cleaves the Lys-Met bond of the same substrate (Abraham et al, 1993). This 28-kDa protease appears to have cathepsin G-like epitopes, and the first cysteine-dependent protease to be homologous to rat endopeptidase (EC 3.4.24.15) (Abraham et al, 1993).…”

“…They now have reported that this proteolytic activity is the action of three different proteases: a 28-kDa Ca2+-dependent serine protease which preferentially cleaves the Lys-Met bond of the oligopeptide substrate at the N-terminus of /3/A4; a cysteine-dependent metalloprotease that requires dithiothreitol for its proteolysis at the Met-Asp bond of the same oligopeptide; and another cysteine-dependent protease that cleaves the Lys-Met bond of the same substrate (Abraham et al, 1993). This 28-kDa protease appears to have cathepsin G-like epitopes, and the first cysteine-dependent protease to be homologous to rat endopeptidase (EC 3.4.24.15) (Abraham et al, 1993). The 68-kDa protease from lymphoblastoid cells does not show cross-reactivity to the anticathepsin G antibody (unpublished observation) and does not have a subunit structure because its size in SDS-PAGE is unaltered in the presence of reducing agents.…”

Dysregulation of signal transduction pathways as a potential mechanism of nervous system alterations in HIV-1 gp120 transgenic mice and humans with HIV-1 encephalitis.

“…Heterozygous tg B6 ϫ SJL mice expressing HIV LAV -gp120, E. coli ␤ -galactosidase (lacZ) or human ␣ 1-antichymotrypsin (ACT) in astrocytes were from the previously established GFAP-gp120 lines 2, 10, and 16 (8), the GFAP-lacZ line C-445-16 (18), and the GFAP-ACT line 13 (8,19). Transgene expression in these models is directed by a modified GFAP gene.…”

“…Regular media consisted of DME containing 10% fetal bovine serum, 2 mM L -glutamine, penicillin (100 U/ml), and streptomycin (100 g/ml); selection media consisted of regular media plus Geneticin (600 g/ml) (GIBCO-BRL, Gaithersburg, MD). Cells were transfected at 30-50% confluency with 10 g GFAP-gp120 (8), GFAPlacZ (C-445) (18), or GFAP-ACT (8,19), in a modified pGEMEX-2 plasmid, in combination with 1 g pSV2-neo (CLONTECH, Palo Alto, CA) using Lipofectin (GIBCO-BRL) per manufacturer's recommendations. Similarly, C6 cells were transfected with gp120-coding sequences (NotI segment of GFAP-gp120 [8]) placed downstream of the cytomegalovirus (CMV) promoter of pCMV ␤ (CLONTECH); the lacZ element of the latter plasmid was deleted.…”