Presenilins Are Processed by Caspase-type Proteases

Abstract

Presenilin 1 (PS1) and presenilin 2 (PS2) are endoproteolytically processed in vivo and in cell transfectants to yield 27-35-kDa N-terminal and 15-24-kDa C-terminal fragments. We have studied the cleavage of PS1 and PS2 in transiently and stably transfected hamster kidney and mouse and human neuroblastoma cells by immunoblot and pulse-chase experiments. C-terminal fragments were isolated by affinity chromatography and SDS-polyacrylamide gel electrophoresis and sequenced. Mutant presenilin 1 and 2 (PS1, PS2)1 genes are linked to early-onset familial Alzheimer's disease (1-5). The PS gene products are integral membrane proteins localized in Golgi and ER compartments (6 -8). They share ϳ65% sequence identity and similar structural features, i.e. 6 -8 transmembrane domains, hydrophilic regions at their N and C termini, and a large hydrophilic loop C-terminal to transmembrane domain 6 (6, 9, 10). The loop and the N terminus are the regions of significant sequence divergence between PS1 and PS2. More than 30 mutations in PS1 and 2 mutations in PS2 have been genetically linked to early-onset FAD (3, 4). The biological functions of PS currently remain unknown. Studies on the structural homologue in Caenorhabditis elegans, SEL-12, suggest a role in protein trafficking and in the Notch signaling pathway (11). Human carriers of PS FAD mutations show a small but significant increase (approximately 1.5-3-fold) in plasma levels of A␤ 1-42 compared with normal controls, whereas levels of A␤ 1-40 are unchanged (12-14). A specific effect on ␥-secretase processing of ␤-amyloid precursor protein has been proposed, and this enhanced misprocessing of ␤-amyloid precursor protein is thought to be crucial for FAD pathogenesis. Recent experiments have indicated a regulatory role for PS2 in cell apoptosis. C-terminal fragment of mouse PS2 reportedly protected T-cells or PC12 cells from different apoptosis-inducing challenges, whereas the susceptibility to the same challenges was enhanced when full-length PS2 was overexpressed (15,16).Extensive proteolytic cleavage of PS1 resulting in a 27-28-kDa N-terminal fragment and a 16 -17-kDa C-terminal fragment was reported for PS1-transfected COS-1 cells, mice transgenic for human PS1, and endogenous PS1 in human and murine tissues (12,(17)(18)(19). Proteolytic processing has also been reported for human PS2 resulting in a 35-kDa N-terminal fragment and a 20-kDa C-terminal peptide (3,10,13). In the present study we have identified proteolytic processing sites in PS1 and PS2 and demonstrate that processing occurs through caspase-type protease(s). We also observed additional, caspaseindependent cleavage products suggesting alternative pathways of presenilin processing. EXPERIMENTAL PROCEDURESAntibodies-Amino acids 1-80 of human PS2 (N terminus) and 270 -387 of murine PS2 (large hydrophilic loop between transmembrane domains 6 and 7) were expressed in Escherichia coli as (NANP) 19 -fusion proteins and used for immunization of mice. PST20 is a monoclonal antibody specific for the hydrophilic N-termi...

Inhibition of p53-dependent apoptosis by the KIT tyrosine kinase: regulation of mitochondrial permeability transition and reactive oxygen species generation

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Expression of Alzheimer’s Disease-associated Presenilin-1 Is Controlled by Proteolytic Degradation and Complex Formation

“…In addition to processing by the presenilinase, both PS proteins are substrates for proteinases of the caspase superfamily (31)(32)(33)(34). In this pathway the 20-kDa CTF 20 serves as a substrate for caspase cleavage (33) which generates a smaller ϳ10-kDa C-terminal fragment (Fig.…”

Stress in the brain: novel cellular mechanisms of injury linked to Alzheimer's disease

“…The endoproteolytical cleavage of PS1 and PS2 yields 27−35 kDa N-terminal fragments (NTFs) and 15−24 kDa C-terminal fragments (CTFs). This cleavage of PS1 or PS2 can be prevented by blockade of caspase 1 or 3 activity or the mutation of caspase cleave sites, such as Asp 345 /Ser 346 for PS1 and Asp 329 / Ser 330 for PS2 [129]. It has been demonstrated that multiple caspases possess the ability to cleave presenilins.…”