␥-Secretase cleavage of ␤-amyloid precursor protein (APP) is crucial in the pathogenesis of Alzheimer disease, because it is the decisive step in the formation of the C terminus of ␤-amyloid protein (A␤). To better understand the molecular events involved in ␥-secretase cleavage of APP, in this study we report the identification of a new intracellular long A␤ species containing residues 1-46 (A␤ 46 ), which led to the identification of a novel -cleavage site between the known ␥-and ⑀-cleavage sites within the transmembrane domain of APP. Our data clearly demonstrate that the new -cleavage is a presenilin-dependent event. It is also noted that the new -cleavage site at A␤46 is the APP717 mutation site. Furthermore, we show that the new -cleavage is inhibited by ␥-secretase inhibitors known as transition state analogs but less affected by inhibitors known as non-transition state ␥-secretase inhibitors. Thus, the identification of A␤ 46 establishes a system to determine the specificity or the preference of the known ␥-secretase inhibitors by examining their effects on the formation or turnover of A␤ 46 .The amyloid deposits in the brain of Alzheimer disease (AD) 1 patients are principally composed of the 39 -43-amino acid residue amyloid ␤-peptide (A␤), which is derived from a large ␤-amyloid precursor protein (APP). In the amyloidogenic pathway, APP is first cleaved at the N terminus of A␤ sequence by ␤-secretase, to produce a soluble ectodomain, sAPP␤, and a membrane-anchored C-terminal fragment, CTF␤. CTF␤ is then subsequently cleaved within the transmembrane domain by ␥-secretase to produce the full-length A␤ and the intracellular domain (AICD) (1). ␤-Secretase has been identified as a type I membrane aspartyl protease (2, 3). The findings that knockout of presenilin 1 (PS1) and PS2 results in the abolishment of the ␥-secretase cleavage of APP and that two aspartate residues in two transmembrane domains of presenilin have been identified as critical for the ␥-secretase activity suggest that presenilin may be the ␥-secretase (4 -7). Recently, several other molecules, namely nicastrin, Aph-1, and Pen-2, have been identified as essential components of the ␥-secretase complex of which presenilin may function as the catalytic subunit (8).Most of the A␤ species contain 40 or 42 amino acids. Recently, sequence analysis revealed that the N terminus of AICD starts at residue 50 of the A␤ sequence, which is 7-9 amino acids away from the C termini of A␤ 40 and A␤ 42 . This led to the finding of the ⑀-cleavage site between A␤49 and A␤50 (9 -12). Now the cleavage at A␤40/42 has been specifically referred to as ␥-cleavage site (12). However, neither the intermediate A␤ peptide, which ends at the ⑀-cleavage site, nor the C-terminal fragment, which starts with an N terminus generated by ␥-cleavage, has ever been detected. One possibility is that ␥-and ⑀-cleavages occur simultaneously. The other possibility is that there may be additional cleavages(s) between ␥-and ⑀-cleavages. Here we report that, in our effort to determine th...

␤-Amyloid precursor protein apparently undergoes at least three major cleavages, ␥-, ⑀-, and the newly identified -cleavage, within its transmembrane domain to produce secreted ␤-amyloid protein (A␤). However, the roles of ⑀-and -cleavages in the formation of secreted A␤ and the relationship among these three cleavages, namely ⑀-, -, and ␥-cleavages, remain elusive. We investigated these issues by attempting to determine the formation and turnover of the intermediate products generated by these cleavages, in the presence or absence of known ␥-secretase inhibitors. By using a differential inhibition strategy, our data demonstrate that A␤ 46 is an intermediate precursor of secreted A␤. Our co-immunoprecipitation data also reveal that, as an intermediate, A␤ 46 is tightly associated with presenilin in intact cells. Furthermore, we identified a long A␤ species that is most likely the long sought after intermediate product, A␤ 49 , generated by ⑀-cleavage, and this A␤ 49 is further processed by -and ␥-cleavages to generate A␤ 46 and ultimately the secreted A␤ 40/42 . More interestingly, our data demonstrate that ␥-cleavage not only occurs last but also depends on -cleavage occurring prior to it, indicating that -cleavage is crucial for the formation of secreted A␤. Thus, we conclude that the C terminus of secreted A␤ is most likely generated by a series of sequential cleavages, namely first ⑀-cleavage which is then followed by -and ␥-cleavages, and that A␤ 46 produced by -cleavage is the precursor of secreted A␤ 40/42 .The mechanism of the formation of the ␤-amyloid protein (A␤) 2 is the central issue in Alzheimer disease research, not only because A␤ is the major constituent of senile plaques, one of the neuropathological hallmarks of Alzheimer disease, but also because A␤ formation may be a causative event in the disease (1). A␤ is proteolytically derived from a large single transmembrane protein, the ␤-amyloid precursor protein (APP), as a result of sequential cleavages by ␤-and ␥-secretases (1). ␤-Secretase has been identified as a type I membrane aspartyl protease (2, 3). Although the exact nature of ␥-secretase is still a matter of debate, accumulating evidence supports the idea that ␥-secretase is a multiple molecular complex composed of, at least, presenilins, nicastrin, Aph-1, and Pen-2 and that presenilin may function as the catalytic subunit (4).In understanding the mechanism by which the C termini of secreted A␤ are generated during the processing of APP, three major intramembranous cleavages have been established. The first one is the cleavage now specifically referred to as ␥-cleavage (5), which produces the C termini of most of the secreted A␤ species that end at amino acids 40 (A␤40) or 42 (A␤42) of the A␤ sequence. The second one is the ⑀-cleavage occurring between A␤ residues 49 and 50, which produces the N terminus of most of the APP intracellular domain (AICD) (5-8). The identification of this ⑀-cleavage site raises a question as to whether this ⑀-cleavage is obligatory for the generation of the...

Recent studies have suggested a possible role for presenilin proteins in apoptotic cell death observed in Alzheimer's disease. The mechanism by which presenilin proteins regulate apoptotic cell death is not well understood. Using the yeast two-hybrid system, we previously isolated a novel protein, presenilin-associated protein (PSAP) that specifically interacts with the C terminus of presenilin 1 (PS1), but not presenilin 2 (PS2). Here we report that PSAP is a mitochondrial resident protein sharing homology with mitochondrial carrier protein. PSAP was detected in a mitochondria-enriched fraction, and PSAP immunofluorescence was present in a punctate pattern that colocalized with a mitochondrial marker. More interestingly, overexpression of PSAP caused apoptotic death. PSAP-induced apoptosis was documented using multiple independent approaches, including membrane blebbing, chromosome condensation and fragmentation, DNA laddering, cleavage of the death substrate poly(ADP-ribose) polymerase, and flow cytometry. PSAP-induced cell death was accompanied by cytochrome c release from mitochondria and caspase-3 activation. Moreover, the general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, which blocked cell death, did not block the release of cytochrome c from mitochondria caused by overexpression of PSAP, indicating that PSAP-induced cytochrome c release was independent of caspase activity. The mitochondrial localization and proapoptotic activity of PSAP suggest that it is an important regulator of apoptosis.Alzheimer's disease (AD), 1 the most common form of senile dementia, is pathologically characterized by the deposition of amyloid ␤-peptide (A␤), the formation of neurofibrillary tangles, and massive neuronal cell loss in the brain (1).A subset of AD cases is familial AD (FAD), which occurs as an inherited autosomal dominant disease caused by defects in any of three genes: presenilin 1 (PS1) on chromosome 14, presenilin 2 (PS2) on chromosome 1, and the amyloid precursor protein (APP) on chromosome 21 (for review, see Ref.2). The majority of FAD cases have been associated with mutations in PS1 and PS2 (2). It has been shown that FAD-associated mutations in PS1 and PS2 affect the processing of APP, leading to an increased production of the more amyloidogenic A␤ peptide, both in vivo and in vitro (3-6). In addition to their roles in APP processing, roles for PS1 and PS2 in programmed cell death or apoptosis have also been reported in several studies. It has been shown that overexpression of a C-terminal fragment of PS2 protects neural cells against apoptosis (7). It was also reported that overexpression of a C-terminal fragment of PS1 delays anti-Fas-induced apoptosis in Jurkat cells (8). A role for PS1 and PS2 in apoptosis is also supported by studies demonstrating that overexpression of PS1 or PS2 bearing FAD mutations results in increased sensitivity to apoptotic insults (9 -13). These studies suggest that mutant PS1 and PS2 may be directly involved in neuronal cell death found in the AD brain...

It has been hypothesized that different C-terminus of bamyloid peptide (Ab) may be generated by different c-secretase activities. Recently, we have identified a new f-cleavage site at Ab46, leading to an important finding that the C-terminus of Ab is produced by a series of sequential cleavages. This finding prompted us to examine the effects of the known c-secretase inhibitors on different steps of the c-secretase-mediated sequential cleavages and specifically their effects on the formation and turnover of the intermediate Ab 46 . Our results demonstrate that some of the known inhibitors, such as L-685,458 and III-31C as well as inhibitors IV and V, inhibit the formation of secreted Ab 40/42 by inhibiting the formation of the intermediate Ab 46 . However, most of the other inhibitors show no inhibitory effect on the formation of the intermediate Ab 46 , but rather inhibit the turnover of Ab 46 , resulting in its accumulation. In addition, the non-steroidal anti-inflammatory drugs (NSAIDs) ibuprofen and sulindac sulfide have no effect on the formation and turnover of Ab 46 , but rather modulate the ratio of secreted Ab at a step after the formation of Ab 40 and Ab 42 . Thus, our data strongly suggest that the multi-sequential intramembrane cleavages of amyloid precursor protein C (APP) are likely catalyzed by the same c-secretase.

Background: DR6-induced apoptosis mechanism is unknown. Results: DR6-induced apoptosis is dependent on cytochrome c release and Bax translocation, but is independent of caspase-8 and Bid. Conclusion: DR6-induced apoptosis is mediated by a unique pathway, different from type I and type II pathways. Significance: This study will lead to a better understanding of the mechanism by which DR6 induces apoptosis.