␥-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...

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.

Aims:  To search for novel Vip3A proteins for controlling insect pests. Methods and Results:  A pair of universal primers was designed based on the conserved regions of five vip3A genes. Amplified products were digested with the HindIII and EcoR enzymes so as to confirm different restriction fragment length polymorphism (RFLP) patterns used to identify vip3A‐type genes. The vip3A gene types of 606 Bacillus thuringiensis strains were screened and three patterns of RFLP were successfully identified. Two novel vip3A genes were found and one of these, vip3Aa19, was further characterized and its product was confirmed toxic to Spodoptera exigua, Helicoverpa armigera and Plutella xylostella larvae. Partial sequences of another novel vip3A‐type gene were obtained that shared 83% homology with that of the vip3Af1 gene. Conclusions:  A polymerase chain reaction (PCR)‐RFLP system we developed could be used for identifying novel vip3A‐genes from B. thuringiensis strains. A novel Vip3A protein was found to have a broader insecticidal spectrum. Significance and Impact of the Study:  The reported method is a powerful tool to find novel Vip3A proteins from large‐scale B. thuringiensis strains. The novel Vip3A protein may be used to control insect pests or resistant insect pests by constructing genetically engineered strains or transgenic plants.

Overwhelming evidence supports the amyloid hypothesis of Alzheimer’s disease that stipulates that the relative level of the 42 amino acid β-amyloid peptide (Aβ42) in relationship to Aβ40 is critical to the pathogenesis of the disease. While it is clear that the multi-subunit gamma secretase is responsible for cleavage of the amyloid precursor protein (APP) into Aβ42 and Aβ40, the exact molecular mechanisms regulating the production of the various Aβ species remain elusive. To elucidate the underlying mechanisms, we replaced individual amino acid residues from positions 43 to 52 of Aβ with phenylalanine to examine the effects on the production of Aβ40 and Aβ42. All mutants, except for V50F, resulted in a decrease in total Aβ with a more prominent reduction in Aβ for residues 45, 48, and 51, following an every three residue repetition pattern. In addition, the mutations with the strongest reductions in total Aβ had the largest increases in the ratio of Aβ42/Aβ40. Curiously, the T43F, V44F, and T48F mutations caused a striking decrease in the accumulation of membrane bound Aβ46, albeit by a different mechanism. Our data suggest that initial cleavage of APP at the ε site is crucial in the generation of Aβ. The implicated sequential cleavage and an α-helical model may lead to a better understanding of the γ-secretase-mediated APP processing and may also provide useful information for therapy and drug design aimed at altering Aβ production.