Proteolytic processing of amyloid precursor protein generates ␤-amyloid (A␤) peptides that are deposited in senile plaques in brains of aged individuals and patients with Alzheimer's disease. Presenilins (PS1 and PS2) facilitate the final step in A␤ production, the intramembranous ␥-secretase cleavage of amyloid precursor protein. Biochemical and pharmacological evidence support a catalytic or accessory role for PS1 in ␥-secretase cleavage, as well as a regulatory role in select membrane protein trafficking. In this report, we demonstrate that PS1 is required for maturation and cell surface accumulation of nicastrin, an integral component of the multimeric ␥-secretase complex. Using kinetic labeling studies we show that in PS1 ؊/؊ /PS2؊/؊ cells nicastrin fails to reach the medial Golgi compartment, and as a consequence, is incompletely glycosylated. Stable expression of human PS1 restores these deficiencies in PS1 ؊/؊ fibroblasts. Moreover, membrane fractionation studies show co-localization of PS1 fragments with mature nicastrin. These results indicate a novel chaperone-type role for PS1 and PS2 in facilitating nicastrin maturation and transport in the early biosynthetic compartments. Our findings are consistent with PS1 influencing ␥-secretase processing at multiple steps, including maturation and intracellular trafficking of substrates and component(s) of the ␥-secretase complex.Alzheimer's disease is pathologically characterized by the cerebral deposition of 39 -42 amino acid peptides, termed ␤-amyloid (A␤), 1 which are generated by proteolytic processing of amyloid precursor protein (APP) (1). Mutations in the genes encoding presenilin 1 (PS1) and presenilin 2 (PS2) account for the majority of the cases of familial early-onset Alzheimer's disease (2). Biochemical and genetic evidence demonstrate that PS1 and PS2 facilitate the final step in A␤ production, the intramembranous ␥-secretase cleavage of APP (3-5). Moreover, it has been proposed that PS1 deficiency alters the trafficking of select membrane proteins (4). Familial Alzheimer's diseaselinked mutant PS1 and PS2 elevate the levels of highly amyloidogenic A␤42 peptides, thus promoting A␤ deposition in senile plaques (6, 7). However, the specific mechanism(s) involved in the selective elevation of A␤42 production by familial Alzheimer's disease-linked PS variants has not been clearly understood.The precise role played by PS1 and PS2 in ␥-secretase cleavage of APP and Notch has been under intense scrutiny. Biochemical fractionation and ␥-secretase inhibitor studies have suggested a catalytic role for PS1 in the intramembraneous cleavage of substrates (5, 8 -10). On the contrary, biochemical evidence also suggests an indirect role for PS1 in facilitating ␥-secretase cleavage of substrates (11). It is unlikely that this apparent controversy can be resolved by biochemical analysis, since PS-derived N-terminal (NTF) and C-terminal (CTF) fragments are components of high molecular weight multimeric protein complexes (12). One of the more recent members of the g...

CD95 is a member of the family of the death receptors that initiate apoptosis by recruiting Fas-associated death domain protein (FADD), procaspase-8, procaspase-10, and cellular FLICE-like inhibitory protein to the death-inducing signaling complex (DISC), which forms after binding of the cognate ligand (CD95L) (1). CD95 type I and II cells differ in their dependence on mitochondria for the execution of apoptosis in that type II cells require mitochondrial amplification to die (2). Hence, overexpression of antiapoptotic Bcl-2 family members only inhibits CD95-mediated apoptosis in type II cells. We proposed this CD95 two-pathway model based on a study of four tumor cell lines (2, 3), and a number of transgenic and knockout mice have demonstrated that this distinction also applies to normal tissues (e.g., liver cells are type II, and T cells are type I) (reviewed in ref. 4). One of the most striking differences between type I and II cells lies in the way the CD95 signal is generated at the receptor level. An efficient formation of the DISC is observed only in type I cells, whereas in type II cells it is difficult to detect by Western blotting (2). We recently demonstrated that formation of the DISC in type I cells involves F-actin (5) and that the receptor internalizes in an actin-and caspase-8-dependent fashion only in type I cells (6). These data suggest that type I cells differ from type II cells in the way the CD95 signal is initiated. This difference in signal initiation could result in a difference in sensitivity to CD95 stimuli.The cognate CD95L is expressed as both a membrane-bound (mCD95L) and soluble (sCD95L) form that is generated by metalloprotease cleavage of mCD95L (7,8). As yet, no clear specific separate function has been assigned to sCD95L or mCD95L. Here we uncover a striking difference in the response of type I and II tumor cell lines to different forms of CD95L. We found that a preparation of soluble sCD95L (S2) (9) efficiently kills type II cells. In contrast, type I cells are resistant to this cytotoxic activity. S2 therefore represents a tool for identifying type I and II cells. We applied this tool to a collection of 58 tumor cell lines of various histologic origin [of the Developmental Therapeutics Program of the National Cancer Institute (NCI)] (10). These cell lines have been subjected to a comprehensive microarray analysis to determine their patterns of gene expression and were found to cluster into two very distinct classes of cells (epithelium-like and mesenchymal-like) that share expression of similar sets of genes (11). We have determined that 22 of these 58 cell lines are CD95 apoptosis-sensitive and have classified half of these sensitive cells as type I and half as type II based on their sensitivity to S2 stimulation and their ability to form a DISC. Ten of 11 of the cell lines that we classified as type I cells were found in the mesenchymal branch, whereas 9 of 11 of the type II cells were found in the epithelial branch. The type I͞type II status of the cells was used to query t...

Mutations in the death domain of the death receptor CD95 (APO-1/ Fas) cause lymphoproliferation and autoimmune disease in both lpr cg mice and in patients with autoimmune lymphoproliferative syndrome (ALPS) type Ia. By testing lymphocytes from ALPS type Ia patients, comparing heterozygous with homozygous lpr cg mice and coexpressing wild-type and mutant CD95 receptors, we demonstrate that induction of apoptosis requires two wild-type alleles of CD95. By contrast, nuclear factor-jB (NF-jB) can be fully activated in cells expressing both a mutant and a wild-type CD95 allele, suggesting different thresholds to activate the two signalling pathways. This was confirmed by testing lymphocytes from heterozygous lpr mice, which showed reduced sensitivity to CD95-mediated apoptosis but normal activation of NF-jB when compared with wild-type mice. Mutations in CD95 may eliminate the tumour-suppressive function of CD95, at the same time allowing induction of survival or proliferative pathways, which could contribute to the increased risk for lymphoma seen in ALPS type Ia patients.